WO2013055898A1 - Methods for enhancing the efficacy of microrna mediated interference - Google Patents

Methods for enhancing the efficacy of microrna mediated interference Download PDF

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WO2013055898A1
WO2013055898A1 PCT/US2012/059721 US2012059721W WO2013055898A1 WO 2013055898 A1 WO2013055898 A1 WO 2013055898A1 US 2012059721 W US2012059721 W US 2012059721W WO 2013055898 A1 WO2013055898 A1 WO 2013055898A1
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nbr
acid sequence
expression
nucleic acid
mirna
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PCT/US2012/059721
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French (fr)
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Nancy M. BONINI
Sara Cherry
Nan Liu
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The Trustees Of The University Of Pennsylvania
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/50—Methods for regulating/modulating their activity

Definitions

  • the invention relates to methods for enhancing the efficacy of microRNA (miRNA) mediated interference. Specifically, the invention relates to inhibiting the expression or function of Nibbler (Nbr) to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA-mediated interference.
  • miRNA microRNA
  • Nbr Nibbler
  • miRNAs are endogenous non-coding small RNAs with important roles in many biological pathways, and as such, their generation and activity are under precise regulation.
  • miRNAs play critical roles in processes as diverse as normal development and cellular homeostasis. Moreover, recent evidence suggests that they can function as oncogenes or tumor suppressors.
  • RISC RNA-induced silencing complex
  • the invention relates to a method for enhancing the efficacy of microRNA (miRNA) mediated interference, the method comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the target specificity and/or efficacy of miRNA- mediated interference.
  • miRNA microRNA
  • the invention relates to a method for enhancing the efficacy of miRNA mediated interference or expression control, the method comprising: enhancing the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of one or more isoforms of miRNA, thereby enhancing the efficacy of miRNA mediated interference or expression control.
  • the invention relates to a method for modulating the efficacy of miRNA mediated interference, the method comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby modulate the target specificity and/or efficacy of miRNA-mediated interference.
  • the invention in another embodiment, relates to a method for regulating the production of an miRNA in a cell, the method comprising: regulating the expression or function of Nbr, a variant, or a homolog thereof in said cell, thereby regulating the production of said miRNA in said cell.
  • the invention relates to a method for enhancing silencing of a target RNA, the method comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA mediated interference of said target RNA, and thereby enhancing silencing of said target RNA.
  • the invention relates to a method for identifying an RNAi modulatory compound, comprising, contacting a cell expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
  • the invention relates to a method for identifying an RNAi modulatory compound, comprising, contacting a composition comprising Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
  • the invention relates to a method for identifying an RNAi modulatory compound, comprising, contacting an organism expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
  • the invention in another embodiment, relates to an assay for detecting modulation of RNA interference, comprising, contacting a reaction mixture comprising Nbr with a test compound, and evaluating the effect of the test compound on an indicator of RNA interference, such that modulation of RNA interference is detected.
  • the invention relates to a method for identifying a molecule that regulates Nbr mediated processing of miRNA in a subject, the method comprising: screening a library of molecules; identifying a molecule that regulates the expression or function of Nbr, a variant, or a homolog thereof, thereby identifying said molecule that regulates Nbr mediated processing of miRNA in said subject.
  • FIG. 1 Fly miR-34 Shows Multiple Isoforms Whose Generation Appears Dependent on 3' End Trimming.
  • A miR-34 has multiple forms in adult flies. Left, miR-34 precursor, mature 24-nt sequence in red. Right, Northern for miR-34. Isoforms of 24, 22 and 21nt, labeled a, b, c, respectively.
  • B miR-34 isoforms from a deep sequencing fly S2 cell dataset [21]. In red, 24nt isoform a. In blue, isoforms b and c. These are 99.1% of the miR-34 reads.
  • C Northern blot analysis of miR-34 isoform accumulation in vivo.
  • FIG. 3 Nbr Interacts with Agol-RISC.
  • A Small RNA Northern blot analysis of mir-34 isoforms. Depletion of Agol phenocopies Nbr knockdown.
  • B Agol and Nbr interact by co-IP. Cells were untreated or transfected with HA-Nbr and Flag- Agol or Flag-Ran (control). Following IP, interacting proteins were probed by immunoblot. Input, 10% of Flag- IP.
  • C All miR-34 isoforms co-IP with Agol. Cells were treated with dsRNA to control (LacZ), Nbr or Agol, and IPs were performed anti-GFP (control) or Agol antibodies. Input and IP-ed RNA were analyzed by Northern blotting for miR-34.
  • FIG. 4 nbr is Required in vivo to Process Select miRNAs and Silence Target mRNAs.
  • A Genomic map of the nbr locus. Coding region in red, with transposon insertion highlighted.
  • B Northern blot for nbr. The nbr ⁇ 02257 mutant shows complete mRNA loss.
  • C Shorter isoforms of miR-34 are abolished in the nbr mutant. Arrow, isoform a.
  • D Northern blot of single-isoform miR-277, which is not altered in nbr '7' .
  • E Comparison of multiple- isoform miRNAs from control and nbr 102257 flies.
  • nbr red arrowheads
  • F The ratio of the most frequent form of the miRNA in wild type, compared to the sum of all other forms, was generated for nbr and control. The ratios were compared ⁇ nbr ratio/control ratio), and plotted. The ratio was excessively large or low when isoform biogenesis is defective. Red boxes, miRNAs with extreme ratios that were further analyzed. Red symbols are confirmed Nbr-targets (Fig. 8).
  • G Scatterplot of microarray data from cells treated with dsRNA against Nbr or Renilla control. Highlighted, all of the genes >1.5 fold changed in either direction.
  • H Realtime PCR for mRNAs from nbr and logs mutant flies. (4-6 experiments; */? ⁇ 0.05, **/? ⁇ 0.01, ***/? ⁇ 0.001).
  • FIG. 5 (related to Figure 2). Reduction of nbr affects biogenesis of miR-34 shorter isoforms.
  • A mRNA Northern shows that treating cells with two independent dsRNAs directed to different regions of the nbr gene depleted nbr mRNA levels. Loading control, 18S rRNA.
  • B Upon reduction of nbr, biogenesis of miR-34 shorter isoforms was affected. Loading control, 2S rRNA.
  • C Loss of nbr has minimal effects on the level or pattern of miR-
  • FIG. 6 (related to Figure 3).
  • the interaction between Nbr and Agol is not dependent on RNA.
  • Agol and Nbr interact by co-immunoprecipitation in a manner that is RNA- independent.
  • Cells were either left untreated or were transfected with HA-Nbr and either Flag- Ago 1 or Flag-Ran (control), in the presence or absence of added RNase.
  • Flag-tagged protein was then immunoprecipitated, and interacting proteins were probed by Western immunoblot. Protein input is 10% of Flag-IP.
  • Figure 7 (related to Figure 4). New n&r-dependent candidate miRNAs. (A and B) Distribution of (A) overall reads and (B) reads mapping to the miRNA stemloops in control f02257
  • C and D The left panels are the distribution plots of read lengths for these miRNAs, from which the ratio plot in Fig. 4F was generated.
  • the right panels are small RNA Northerns from adult flies of control and nb mutants.
  • C candidate miRNAs from the low ratio end of the plot in Fig. 4F where the most abundant isoform in wild type is trimmed. The mutant shift in the distribution is observed by Northern blot analysis.
  • D are two miRNAs are from the high ratio side of the plot in Fig. 4F.
  • the invention encompasses methods for enhancing or modulating the efficacy of microRNA (miRNA) mediated interference. Specifically, the invention encompasses modulating the expression or function of Nibbler (Nbr) to modulate the accumulation of one or more forms of miRNA, thereby enhancing or modulating the efficacy of miRNA-mediated interference.
  • a method for enhancing the efficacy of miRNA-mediated interference comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the target specificity and/or efficacy of miRNA-mediated interference.
  • a method for enhancing the efficacy of miRNA mediated interference or expression control comprising: enhancing the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of one or more isoforms of miRNA, thereby enhancing the efficacy of miRNA mediated interference or expression control.
  • a method for modulating the efficacy of miRNA mediated interference comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby modulate the target specificity and/or efficacy of miRNA-mediated interference.
  • a method for regulating the production of an miRNA in a cell comprising: regulating the expression or function of Nbr, a variant, or a homolog thereof in said cell, thereby regulating the production of said miRNA in said cell.
  • a method for enhancing silencing of a target RNA comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA mediated interference of said target RNA, and thereby enhancing silencing of said target RNA.
  • RNAi modulatory compound comprising, contacting a cell expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
  • a method for identifying an RNAi modulatory compound comprising, contacting a composition comprising Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
  • RNAi modulatory compound in another embodiment, provided herein is a method for identifying an RNAi modulatory compound, comprising, contacting an organism expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
  • an assay for detecting modulation of RNA interference comprising, contacting a reaction mixture comprising Nbr with a test compound, and evaluating the effect of the test compound on an indicator of RNA interference, such that modulation of RNA interference is detected.
  • a method for identifying a molecule that regulates Nbr mediated processing of miRNA in a subject comprising: screening a library of molecules; identifying a molecule that regulates the expression or function of Nbr, a variant, or a homolog thereof, thereby identifying said molecule that regulates Nbr mediated processing of miRNA in said subject.
  • Drosophila miR-34 displays multiple isoforms that differ at the 3 'end, indicating a novel biogenesis mechanism involving 3 'end processing.
  • the inventors of the instant application performed an RNAi screen and identified a putative 3' ⁇ 5' exoribonuclease Nbr essential for the generation of the smaller isoforms of miR-34.
  • Nbr may refer to all isoforms, homologs, or variants of Nbr.
  • the term “Nbr” is used interchangeably with the terms “Trimmer” or “Tmr.”
  • Nbr has the amino acid sequence as set forth in GenBank accession number NP_610094.1 (SEQ ID NO: 2).
  • Nbr protein of the invention comprises the amino acid sequence:
  • Nbr is a homologue, a variant, an isomer, or a functional fragment of SEQ ID NO: 2.
  • Nbr is a homologue, a variant, an isomer, or a functional fragment of SEQ ID NO: 2 having an exoribonuclease domain.
  • Nbr is homologous to human Exonuclease 3 '-5' domain- containing protein 3 (EXD3). Each possibility represents a separate embodiment of the present invention.
  • Nbr includes polypeptides with amino acid sequences substantially similar to the amino acid sequence of SEQ ID NO.: 2.
  • Substantially similar amino acid sequence may refer to a sequence with at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to a compared amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA ⁇ 5:2444- 2448 (1988).
  • Nbr is encoded by the nucleic acid sequence as set forth in GenBank accession number NM_136250.3 (SEQ ID NO: 1).
  • Nbr protein of the invention is encoded by the nucleic acid sequence:
  • Nbr protein of the invention is encoded by a homologue, a variant, an isomer, or a functional fragment of SEQ ID NO: 1.
  • SEQ ID NO: 1 Each possibility represents a separate embodiment of the present invention.
  • Nbr is encoded by nucleic acid sequences substantially similar to the nucleic acid sequence of SEQ ID NO.: 1.
  • Substantially similar nucleic acid sequence may refer to a sequence with at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to a compared nucleic acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA ⁇ 5:2444-2448 (1988).
  • the expression or function of Nbr, a variant, or a homolog thereof may be inhibited by any suitable Nbr antagonist, known to one of skilled in the art.
  • the expression of Nbr can be inhibited, for example, by antisense inhibition, by inactivation of an activator or agonist, by activation of an inhibitor or antagonist, by inhibition through adding inhibitory antibodies, by adding active compounds, or by introducing negative dominant mutants, etc.
  • the step of regulating or modulating the expression or function may include enhancing the expression or function of Nbr, a variant, or a homolog thereof.
  • the expression or function of Nbr, said variant, or said homolog thereof may be enhanced by any suitable Nbr agonist, known to one of skilled in the art
  • a method for identifying a molecule that modulates the efficacy of miRNA mediated interference in a subject comprising: screening a library of molecules; identifying a molecule that regulates (e.g., upregulate or downregulate) the expression of Nbr, thereby identifying said molecule that modulates the efficacy of miRNA mediated interference in said subject.
  • a library of oligo nucleic acid molecules can be screened to identify a molecule that regulates the expression of Nbr.
  • a library of peptide or protein molecules can be screened to identify a molecule that regulates the expression of Nbr.
  • a library of small molecule compounds can be screened to identify a compound that regulates the expression of Nbr.
  • the screening can be accomplished by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring decon volution; the "one-bead one- compound” library method; and synthetic library methods using affinity chromatography selection.
  • the invention provides a method for screening or testing a library of molecules, the method comprising the steps of: contacting a plurality of test samples with said library of molecules; measuring the expression of Nbr; and identifying a molecule that is effective in regulating the expression of Nbr, thereby regulating miRNA production or processing.
  • Such molecules may include, but are not limited to, peptides made of D- and/or L- configuration amino acids (for example, the form of random peptide libraries; see e.g., Lam, K. S. et al., 1991, Nature 354:82-84), phosphopeptides (m, for example, the form of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778), antibodies, and small organic or inorganic molecules.
  • Molecules identified may be useful, for example, in modulating the activity of target gene proteins, preferably mutant target gene proteins, may be useful in elaborating the biological function of the target gene protein, may be utilized in screens for identifying molecules.
  • the invention relates to identifying a molecule that modulates the interaction of Nbr with its interactor or binding molecule.
  • modulators may be useful in regulation of: processing of miRNA precursors; mediating mRNA cleavage; mediating assembly of RNA induced silencing complex (RISC); directing translation repression (e.g., via miRNAs); a ribonuclease activity (e.g., cleavage of dsRNA); and initiation of RNAi.
  • RISC RNA induced silencing complex
  • a cell free assay known to one of skilled in the art, can be used to identify a modulator.
  • a test compound can contact with a composition comprising an assay reagent (e.g., Nbr), thereby the test compound's ability to modulate the interaction can be determined.
  • an assay reagent e.g., Nbr
  • the test compounds or polypeptides can be labeled with a radioisotope, for example, but are not limited to, 125 1, 35 S, 33 P, 32 P, 14 C, or 3 H, either directly or indirectly.
  • the radioisotope can be detected by radioemmission counting or by scintillation counting.
  • the test compounds or polypeptides can be enzymatically labeled with, for example, but are not limited to, horseradish peroxidase, alkaline phosphatase, or luciferase. The enzymatic label can be detected, for example, by determining the conversion of an appropriate protein to product.
  • Binding of reagents can be determined using any suitable methods or techniques known to one of skilled in the art.
  • a real-time Biomolecular Interaction Analysis can be used. See Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
  • Nbr can be used as bait proteins in a two-hybrid assay or three- hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with Nbr or Nbr interactor.
  • a two-hybrid assay or three- hybrid assay see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054
  • an assay is a cell based assay.
  • a cell based assay a cell expressing an Nbr is contacted with a test compound and the ability of the test compound to modulate the expression of Nbr is determined.
  • the cell for example, can be of mammalian origin, yeast, or others known to one skilled in the art.
  • an assay is an organism based assay.
  • an organism based assay an organism based assay, an organism expressing an Nbr is contacted with a test compound and the ability of the test compound to modulate the expression of Nbr is determined.
  • an anti-Nbr antibody or fragments thereof such as F(ab) 2 fragments, Fv fragments, single chain antibodies and other forms of "antibodies” can be used as modulators.
  • the antibody is a monoclonal antibody.
  • the antibody is a humanized form of the murine monoclonal antibody.
  • the antibody is a human antibody.
  • the activity of Nbr in the assays, can be determined by measuring the expression of its nucleic acids or amino acids. In other embodiments, the activity of Nbr can be determined by measuring a molecule in Nbr mediated pathway.
  • Nbr-mediated processing of regulated precursor microRNAs can also be measured in vitro or in vivo through the measurement of the levels of miRNA duplexes or mature miRNAs.
  • the levels of mature longer miRNA are expected to be higher in cells with reduced or inhibited Nbr activity.
  • the levels of the miRNA or various precursors can be measured using techniques known in the art, including, but not limited to, PCR (e.g., quantitative RT-PCR), Northern blots, and microRNA microarrays (see, for example,
  • the level of Nbr or its activity can be indirectly measured by monitoring the expression of a target polynucleotide (either the target mRNA or polypeptide encoded thereby) that is known to be regulated by the mature miRNA using techniques known in the art.
  • PCR e.g., quantitative RT-PCR
  • Northern blots or microarrays can be used to measure the level of target mRNAs
  • Western blots or an ELISA, for example, can be used to measure the level of target polypeptides.
  • assays that measure the activity of target polypeptides can be used as an indirect measure of miRNA production.
  • the levels of miRNA and the expression of target polynucleotides can also be affected by various downstream mechanisms, including, but not limited to other post- transcriptional and translational regulatory mechanisms.
  • the levels of pre-miRNA can be measured in a cell.
  • the invention also relates, in part, to a method of using the nucleic acids of the invention to reduce expression of a target gene in a cell, tissue or organ.
  • Expression of the target gene may be reduced by expressing a nucleic acid of the invention that comprises a sequence substantially complementary to one or more binding sites of the target mRNA.
  • the nucleic acid may be a miRNA or a variant thereof.
  • the nucleic acid may also be pri-miRNA, pre-miRNA, or a variant thereof, which may be processed to yield an miRNA.
  • the expressed miRNA may hybridize to a substantially complementary binding site on the target mRNA, which may lead to translational repression or activation of RISC-mediated gene silencing.
  • the target of gene silencing may also be a gene or protein that causes the silencing of a second gene or protein. By repressing expression of the target gene, expression of the second protein may be increased. Examples for efficient suppression of miRNA expression are the studies by Esau et al 2004 JBC 275-52361; and Cheng et al 2005 Nucleic Acids Res. 33-1290, which are incorporated herein by reference.
  • the present invention also relates to a method of using the nucleic acids of the invention to increase expression of a target gene in a cell, tissue or organ.
  • Expression of the target gene may be increased by expressing a nucleic acid of the invention that comprises a sequence substantially complementary to a pri-miRNA, pre-miRNA, miRNA or a variant thereof.
  • the nucleic acid may be an anti-miRNA.
  • the anti-miRNA may hybridize with a pri- miRNA, pre-miRNA or miRNA, thereby reducing its gene repression activity.
  • Expression of the target gene may also be increased by expressing a nucleic acid of the invention that is substantially complementary to a portion of the binding site in the target gene, such that binding of the nucleic acid to the binding site may prevent miRNA binding.
  • an miRNA may be used to increase gene expression. Recent evidence has shown that an miRNA may bind to a gene promoter and induce transcription (Place et al. PNAS. 2008 vol. 105 no. 5 1608-1613).
  • nucleic acid sequences coding for the above-described molecules can be obtained using recombinant methods known in the art, such as, for example, by screening cDNA and genomic libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.
  • the gene of interest can be produced synthetically, rather than cloned.
  • a DNA of the present invention can be prepared from a cDNA library from cells which express a protein of the present invention by conducting hybridization using a partial sequence of a DNA of the present invention as a probe.
  • a cDNA library can be prepared, for example, by the method described by Sambrook J. et al. (Sambrook J. et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press), or by using commercially available cDNA libraries.
  • a cDNA library can be also prepared by extracting RNA from cells expressing a protein of the present invention, synthesizing cDNA using reverse transcriptase, synthesizing an oligo DNA base on the sequence of the DNA of the present invention, conducting PCR using these as primers, and amplifying cDNA encoding the protein of the present invention.
  • a translation region encoded by the cDNA can be determined, and the amino acid sequence of a protein of the present invention can be obtained.
  • genomic DNA library using the obtained cDNA as a probe genomic DNA can be isolated.
  • mRNAs may first be prepared from a cell, tissue, or organ (for example, testis, brain, heart, liver, and kidney) in which a protein of the invention is expressed.
  • a cell, tissue, or organ for example, testis, brain, heart, liver, and kidney
  • Known methods can be used to isolate mRNAs. For instance, total RNA is prepared by the guanidine ultracentrifugation method (Chirgwin J M. et al. (1979) Biochemistry 18, 5294-5299) or by the AGPC method (Chomczynski P. and Sacchi N. (1987) Anal. Biochem. 162, 156-159), and mRNA is purified from total RNA using mRNA Purification Kit (Pharmacia).
  • mRNA may be directly purified by the QuickPrep mRNA Purification Kit (Pharmacia).
  • the obtained mRNA is used to synthesize cDNA using reverse transcriptase.
  • a cDNA may be synthesized using kits, such as the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).
  • kits such as the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).
  • a cDNA may be synthesized and amplified according to the 5'-RACE method (Frohman M A. et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8998-9002; Belyavsky A. et al. (1989) Nucleic Acids Res. 17:2919-2932) wherein primers, described herein, the 5'-Ampli FINDER RACE Kit (Clontech
  • a desired DNA fragment is prepared from the PCR products and ligated with a vector DNA.
  • the recombinant vectors are used to transform E. coli and a desired recombinant vector is prepared from a selected colony.
  • the nucleotide sequence of the desired DNA can be verified by conventional methods, such as, the dideoxynucleotide chain termination method.
  • a DNA of the invention may be also designed to have a sequence that is expressed more efficiently by taking into account the frequency of codon usage in the host to be used for expression (Grantham R. et al. (1981) Nucleic Acids Res. 9, 43-74).
  • a DNA of the present invention may be altered by a commercially available kit or a conventional method. For instance, a DNA may be altered by digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, or insertion of the initiation codon (ATG) and/or the stop codon (TAA, TGA, or TAG).
  • the DNAs of this invention include a DNA that (a) hybridizes with a DNA consisting of the nucleotide sequence of Nbr; and (b) encodes a protein that is functionally equivalent to a protein of this invention mentioned above.
  • Conditions for hybridization can be selected appropriately by those skilled in the art, and those conditions specifically mentioned above may be used. Under these conditions, DNA having higher homology is obtained as the temperature is raised.
  • the above-mentioned DNA to be hybridized is preferably a naturally occurring DNA, for example, a cDNA or chromosomal DNA.
  • the present invention also provides vectors into which a DNA of the present invention is inserted.
  • the vectors of the present invention are useful to retain a DNA of the present invention in host cell, or to express a protein of the present invention
  • a particular embodiment of the invention encompasses a polypeptide comprising all or a portion of the amino acid sequence of Nbr. Where a portion of the Nbr is used, the portion most preferably retains function so as to regulate miRNA processing.
  • Nbr protein may contain one or more mutations or deletions, so long as the product functions to regulate miRNA processing.
  • polypeptides of the invention can be prepared by methods known in the art. For example, chemical synthesis, such as the solid phase procedure described by Houghton et al., 1985, Proc. Natl. Acad. Sci. U.S.A., 82: 5131-5, can be used. A preferred method involves the recombinant production of protein in host cells transfected within a vector containing polynucleotide sequence(s) encoding Nbr, described above.
  • the invention includes an expression vector comprising a nucleic acid sequence containing an open reading frame (ORF) that encodes the Nbr, described herein.
  • the vector further includes regulatory sequences effective to express the ORF in a host cell; such sequences may be endogenous or heterologous (such as a secretory signals recognized in yeast, mammalian cells, insect cells, tissue culture or bacterial expression systems).
  • regulatory sequences may also include, 5' to the nucleic acid sequence, a promoter region and an ATG start codon in-frame with the hybrid fusion polypeptide coding sequence (chimeric nucleic acid molecule), and 3' to the coding sequence, a translation termination signal followed by a transcription termination signal.
  • the invention includes a method of recombinantly producing a transcriptional regulating hybrid fusion polypeptide using an expression vector.
  • Expression vectors are usually plasmids, but the invention includes other vector forms, such as viral vectors, including recombinant viral vectors known and used by those skilled in the art, as well as vectors that serve equivalent functions and become known in the art subsequently hereto.
  • the polynucleotide sequences encoding Nbr proteins can be stably integrated into the chromosome of an appropriate host cell using direct DNA introduction methods as practiced in the art.
  • Suitable expression vectors include, but are not limited to, mammalian cell expression vectors, such as pcDNA3 (available from Invitrogen), bacterial cell expression vectors, such as pET-30 (available from Novagen or Promega) or yeast expression vectors. Preferred are mammalian cell expression vectors.
  • Expression vectors typically contain regulatory elements capable of affecting expression of the Nbr protein.
  • a vector contains an origin of replication, a promoter, and a transcription termination sequence.
  • the vector may also include other regulatory sequences, including mRNA stability sequences, which provide for stability of the expression product; secretory leader sequences, which provide for secretion of the expression product; environmental feedback sequences, which allow expression of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium); marking sequences, which are capable of providing phenotypic selection in transformed host cells; restriction sites, which provide sites for cleavage by restriction endonucleases; and sequences which allow expression in various types of host cells, including prokaryotic cells, yeast, fungi, algae, plant cells, insect cells, mammalian cells, including human cells and non-human animal cells, cells of non-human primates, and cells of higher eukaryotes.
  • expression vectors comprise a nucleic acid sequence encoding Nbr, or their combinations, operably linked to at least one regulatory sequence or element.
  • Operably linked is intended to mean that the nucleic acid sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
  • Regulatory sequences are known in the art and are selected to direct expression of the desired protein in an appropriate host cell. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements (see, D. V. Goeddel, 1990, Methods Enzymol., 185:3-7). It will be appreciated by the skilled practitioner that the design of the expression vector can depend on such factors as the choice of the host cell to be transfected and/or the type of protein to be expressed.
  • Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and/or one or more expression cassettes.
  • the inserted coding sequences can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements (e.g., promoters, enhancers, and/or insulators) and/or to other amino acid encoding sequences can be carried out using established methods.
  • Host cells containing an expression vector that comprises a nucleic acid sequence encoding the Nbr fusion proteins of the present invention can be cultured under conditions suitable for the expression and recovery of the expressed protein, e.g., from cell membranes or cell lysates, using methods known and practiced by those in the art.
  • the host cells can contain an expression vector which comprises all or a portion of the DNA sequence encoding Nbr.
  • Suitable host cells include both prokaryotic cells (e.g., without limitation, E. coli strains HB 101, DH5a, XL1 Blue, Y1090 and JM101), plant cells, fungal cells, and eukaryotic cells.
  • Eukaryotic recombinant host cells are preferred.
  • Examples of eukaryotic host cells include, but are not limited to, yeast, e.g., S. cerevisiae cells, cell lines of human, bovine, porcine, monkey, and rodent origin, as well as insect cells, including but not limited to, Spodoptera frugiperda insect cells and Drosophila-derived insect cells.
  • Mammalian species- derived cell lines suitable for use and commercially available include, but are not limited to, L cells, CV-1 cells, CHO cells, (CHO-K1, ATCC CCL 61), COS-1 cells (ATCC CRL 1650), COS-7 cells (ATCC CRL 1651), HEK 293 cells, human skin fibroblasts, 3T3 cells (ATCC CCL 92), HeLa cells (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • the expression construct of the invention is delivered to a cell.
  • a wide variety of methods can be used to deliver the expression constructs to cells. Such methods include, for example, but are not limited to, DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyomithine-mediated transfection, electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery, or microinjection.
  • the expression constructs are packaged in liposomes prior to delivery to the cells.
  • Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • Liposomal preparations for use with the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, known in the art.
  • the molecules are produced by growing host cells transformed by an expression vector described above whereby the protein is expressed.
  • the expressed protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the product can be purified directly from the media. If it is not secreted, it can be isolated from cell lysates.
  • the selection of the appropriate growth conditions and recovery methods are within the skill of the art.
  • the product may be isolated and purified by any number of techniques, well known in the art.
  • a protein of the present invention obtained as above may be isolated from the interior or exterior (e.g., medium) of the cells or hosts, and purified as a substantially pure homogeneous protein.
  • the method for protein isolation and purification is not limited to any specific method. In fact, any standard method may be used. For instance, column chromatography, filtration, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization may be appropriately selected and combined to isolate and purify the protein.
  • chromatography for example, affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and such may be used (ed. Daniel R. Marshak et al. (1996) Strategies for Protein Purification and Characterization: A Laboratory Course Manual., Cold Spring Harbor Laboratory Press). These chromatographies may be performed by liquid chromatography, such as, HPLC and FPLC.
  • the expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties.
  • the invention provides a gene therepy vector comprising a nucleic acid sequence encoding Nbr protein.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems are known in the art.
  • adenovirus vectors are used.
  • a number of adenovirus vectors are known in the art.
  • the expression constructs of the present invention are delivered without a viral vector.
  • the construct can be delivered directly, or packaged in liposomes prior to delivery to the subject or to cells derived therefrom, as described above.
  • the expression constructs are encapsulated, adsorbed to, or associated with, particulate carriers.
  • particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG.
  • biolistic delivery systems employing particulate carriers, such as gold and tungsten, are used for delivering the expression constructs of the present invention.
  • the particles are coated with the construct to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a gene gun.
  • a gun powder discharge from a gene gun For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744.
  • the invention provides a transgenic stem cell having up-regulated or down- regulated Nbr.
  • the invention provides transgenic animals having up- regulated or down-regulated Nbr.
  • Transgenic animals can be farm animals (pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, and mice), non-human primates (for example, baboons, monkeys, and chimpanzees), and domestic animals (for example, dogs and cats).
  • Invertebrates such as Caenorhabditis elegans or Drosophila can be used as well as non-mammalian vertebrates such as fish (e.g., zebrafish) or birds (e.g., chickens).
  • compositions comprising agonists or antagonists of Nbr can be formulated as compositions, preferably physiologically acceptable compositions, according to known methods, such as by admixture with a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions may also include compounds which activate cellular interferon stimulated genes (ISGs).
  • ISGs interferon stimulated genes
  • the compositions can comprise Nbr agonists or antagonists.
  • compositions comprising the same can be, in another embodiment, administered to a subject by any method known to a person skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra-peritonealy, intra- ventricularly, intra- cranially, intra- vaginally or intra-tumorally.
  • the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation.
  • Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like.
  • Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the active ingredient is formulated in a capsule.
  • the compositions of the present invention comprise, in addition to the active compound (e.g. the mimetic compound, peptide or nucleotide molecule) and the inert carrier or diluent, a hard gelating capsule.
  • the pharmaceutical compositions are administered by intravenous, intra- arterial, or intra-muscular injection of a liquid preparation.
  • suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like.
  • the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration.
  • the pharmaceutical compositions are administered intra- arterially and are thus formulated in a form suitable for intra- arterial administration.
  • the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration.
  • compositions are administered topically to body surfaces and are thus formulated in a form suitable for topical administration.
  • Topical formulations include, in another embodiment, gels, ointments, creams, lotions, drops and the like.
  • the pharmaceutical composition is administered as a suppository, for example a rectal suppository or a urethral suppository.
  • the pharmaceutical composition is administered by subcutaneous implantation of a pellet.
  • the pellet provides for controlled release of active agent over a period of time.
  • the active compound is delivered in a vesicle, e.g. a liposome.
  • carriers or diluents used in methods of the present invention include, but are not limited to, a gum, a starch (e.g. corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
  • a gum e.g. corn starch, pregeletanized starch
  • a sugar e.g., lactose, mannitol, sucrose, dextrose
  • a cellulosic material e.g. microcrystalline cellulose
  • an acrylate e.g. polymethylacrylate
  • pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • parenteral vehicles for subcutaneous, intravenous, intraarterial, or intramuscular injection
  • parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like.
  • sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants.
  • water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
  • oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
  • compositions further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g.
  • binders e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone
  • disintegrating agents e.g.
  • cornstarch potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.
  • sodium lauryl sulfate sodium lauryl sulfate
  • permeation enhancers solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g.
  • stearic acid magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants.
  • plasticizers e.g. diethyl phthalate, triethyl citrate
  • emulsifiers e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate
  • polymer coatings e.g., poloxamers or poloxamines
  • coating and film forming agents e.g. ethyl cellulose
  • the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the active compound is released over a period of time after administration.
  • Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils).
  • the composition is an immediate-release composition, i.e. a composition in which of the active compound is released immediately after administration.
  • the pharmaceutical composition is delivered in a controlled release system.
  • the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
  • a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989).
  • polymeric materials are used; e.g. in microspheres in or an implant.
  • a controlled release system is placed in proximity to the target cell, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984); and Langer R, Science 249: 1527-1533 (1990).
  • compositions also include, in another embodiment, incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.)
  • polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.
  • particulate compositions coated with polymers e.g. poloxamers or poloxamines
  • polymers e.g. poloxamers or poloxamines
  • Also comprehended by the invention are compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline.
  • the modified compounds are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981 ; Newmark et al., 1982; and Katre et al., 1987).
  • Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound.
  • the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.
  • the methods of the present invention comprise administering an active compound as the sole active ingredient.
  • methods for treating diseases and disorders that comprise administering the active compound in combination with one or more therapeutic agents.
  • the methods described herein comprise in one embodiment, using a combination therapy.
  • the term "combination" is used in its broadest sense and means that a subject is treated with at least two therapeutic regimens.
  • subject refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae.
  • the subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans.
  • subject does not exclude an individual that is normal in all respects.
  • the term "treatment,” as used herein, refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of improving the subject's condition, directly or indirectly.
  • the term “treating” refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combination thereof in other embodiments. "Treating” embraces in another embodiment, the amelioration of an existing condition. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. Treatment also embraces palliative effects: that is, those that reduce the likelihood of a subsequent medical condition. The alleviation of a condition that results in a more serious condition is encompassed by this term
  • polypeptide and protein refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full- length proteins and fragments thereof are encompassed by the definition.
  • the terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like.
  • a "polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be serendipitous, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • nucleic acid can include both double- and single-stranded sequences and refers to, but not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral (e.g. DNA viruses and retroviruses) or procaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.
  • isolated refers to a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • the exoribonuclease Nibbler controls 3' end processin2 of microRNAs in Drosophila
  • miRNAs are typically annotated and observed as a single species, we found that miR-34 showed a pattern of three major isoforms of 24, 22 and 21 nt in Northern blots from adult Drosophila (Fig. 1A). Deep sequencing analysis also showed that miR-34 is present in multiple forms that all bear the same 5' terminus but differ at their 3' ends, presenting a nested series (Fig. IB). To assess the relationship among these, we designed a pulse-chase experiment to follow miR-34 biogenesis. Heat shock driven primary miR-34 was tightly induced for 30 min, then monitored over time in adult flies.
  • isoform a The longest isoform, isoform a (24nt), was predominant initially, while the accumulation of the shorter isoforms was delayed, but then increased over time (Fig. 1C). Moreover, as the 21 nt isoform accumulated, the 24 nt form was lost in a seemingly reciprocal manner, suggesting that the 24-mer may be converted into the 21-mer.
  • RNAi screen against the predicted 3' ⁇ 5'exonucleases in Drosophila, including components of the RNA exosome (Table 1).
  • This identified one gene, CG9247 (which we named nibbler/nbr), with a striking effect: depletion of nbr led to a dramatic accumulation of the miR-34 large isoform with a concomitant loss of the shorter isoforms (Fig. 2B; Fig. 5).
  • loss of nbr did not appear to alter the sizes or levels of miRNAs that normally show a single isoform by Northern, such as miR-14 and miR-277 (Fig. 2C).
  • the Nbr exoribonuclease domain shows closest sequence homology to human EXD3, falling within the E. coli RNAse D protein family; this includes the Werner exoribonuclease and C. elegans Mut-7 involved in transposon silencing (Fig. 5). Nbr, however, showed no predicted RNA binding domain, suggesting that it may function with a partner with RNA binding capacity, to bring Nbr activity to RNA substrates. To define these, we then performed a second RNAi screen genes known to bind RNA or associate with small RNA silencing pathways, including the two somatic RISC-associated Argonautes (Table 1).
  • nbr '1' flies were semi-lethal, and sterile, indicating that nbr function is critical.
  • RNA profiling data from cells confirmed that two of these ⁇ miR-263a, miR-305) had significant levels of multiple forms that differed at the 3'end (Table 2); miR-3, miR-12, miR- 281, and miR-274 were too low for analysis.
  • miR-279 displays a series of isoforms that do differ at the 3 'end; since miR-279 processing is nZ?r-independent, nbr may be one member of a larger set of genes or mechanisms responsible for 3 'end diversity. miR-79 isoforms differed at the 5 'end, suggesting that mechanisms also exist for 5' end diversity of miRNAs.
  • miRNAs where the most common length isoform of the miRNA was present at a much higher or much lower level in nbr ' than in wild type, reflecting an altered pattern of isoform distribution or relative abundance for these miRNAs in the absence of nbr.
  • miRNAs we had defined as trimmed and modulated by nbr (miR-34, miR-263a, miR-263b), with additional candidates (Fig. 4F, red boxes).
  • Northern blots were performed on the top and bottom 8 miRNAs that we had not tested; we confirmed 7 new n/?r-dependent miRNAs (miR- 7, miR-10, miR-11, miR-31b, miR-100, miR-190, miR-317; Fig. 7, Table 3, 4).
  • nbr promotes the diversity of some miRNAs (miR-34, miR-7, miR-317), and alters the relative abundance of the most prominent isoform of others (miR-190 and miR-10; Fig. 7C-F, Table 3).
  • transcriptional profiling of cells This would allow identification of mRNA targets whose stability was altered by miRNA trimming, but not targets primarily controlled by translational repression [24]. This identified 12 genes whose levels were affected by nbr depletion by >1.5-fold (Fig.
  • Nbr may impact strand selection within RISC because strand selection is influenced by the extent of 3' overhang and degree of pairing for any miRNA-miRNA duplex. Nbr may impact miRNA stability, as previous studies have demonstrated that tailing and trimming of mature Drosophila miRNAs influences their turnover. Trimming may also impact mRNA silencing by favoring alterative miRNA sites within mRNA targets. Although canonical miRNA-target specificity is thought to be driven largely by complementarity within the seed, non-canonical interactions can depend more heavily on 3' compensatory sites. Therefore, differences in the length of the 3' end of miRNAs may influence both target selection, as well as silencing efficiency of targets that require extensive 3 'end pairing.
  • Dcr-1 miRNA biogenesis TAATACGACTCACTATAGGCGCAACACGGTGACAATATC TAATACGACTCACTATAGGCGGAACACGATTATT
  • Dcr-2 siRNA biogenesis TAATACGACTCACTATAGGAAGAGCAAGTGCTCACGGTT TAATACGACTCACTATAGGTCTTGCAGTTTTTGC
  • Agol miRNA biogenesis TAATACGACTCACTATAGGCGCATGAAATTGCGCTAC TAATACGACTCACTATAGGCTGCTGGG
  • Ago2 siRNA biogenesis TAATACGACTCACTATAGGAGGATGGAGCAACTCAGGT TAATACGACTCACTATAGGATTCTAAACTGAGGA
  • rrp42 3' ⁇ S exonuclease activity TAATACGACTCACTATAGGTGCCACGCCGGA TAATACGACTCACTATAGGACCCGAAAGGAATG
  • ski6 3' ⁇ S exonuclease activity TAATACGACTCACTATAGGTACATGGAGCAG TAATACGACTCACTATAGGCAAGTGATCGATGT
  • ski6 3' ⁇ 5 exonuclease activity TAATACGACTCACTATAGGAAACTGGGAGTC TAATACGACTCACTATAGGCTCGATGACGGTTTC
  • Mrell exonuclease activity TAATACGACTCACTATAGGTTCCATCTGTTAG TAATACGACTCACTATAGGCCAAACCGAATCGCA
  • Mrell exonuclease activity TAATACGACTCACTATAGGTTCCATCTGTTAG TAATACGACTCACTATAGGATTAAACATGCAGG
  • RNA-induced silencing complex TAA TAC GAC TCA CTA TAG GCA ACA GCC TAA TAC GAC TCA CTA TAG GGT TCT TTT CGT
  • nito mRNA binding 14 TAA TAC GAC TCA CTA TAG GAC ACT GTT TAA TAC GAC TCA CTA TAG GGG CTT TGA GCT
  • GCG GAT ACT TC GTG ATT CTT Table 2 miRNA reads from deep sequencing data miR-263a Read count Percentage
  • Red are those miRNAs that were previously known or validated by Northern.
  • miRNAs that could not be confirmed included those that were not detectable by small RNA northerns, those with more complex isoforms that involved 5 'end as well as 3 'end diversity and because of this complexity could not be confirmed, and those that did not show a striking effect by Northern comparing controls to nbf' ⁇ mutant animals.
  • Table 5 (related to Figure 4).
  • Flies were grown in standard cornmeal molasses agar medium with dry yeast, at 25 °C unless otherwise specified.
  • General stock lines and GAL4 driver lines were obtained from the Drosophila Stock center at Bloomington.
  • nb 022S7 was obtained from the Exelixis collection (Harvard University). Fly transgenics were generated by standard procedures (Genetic Services, Inc).
  • Fly genomic DNA was prepared from whole flies with the Puregene DNA purification kit (Qiagen). Using genomic DNA as the template, a 286bp of miR-34 genomic sequence was amplified by PCR (primers: 5'-CCG TTA CAC ACG ACTA TTC TCA AT-375'-CCA TCT GAT ACA GGT CCT ACA TTT TCT AAA A-3'), and used to generate a miR-34 pUAST construct.
  • PCR amplification was conducted using single stranded cDNA as the template, with primer pairs of HA-Nbr (5'- GAA TTC ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT GCA CGC AAG AGC CAC ATG-375'- GGT ACC TCA CTT AAC ATG GGC ACC CCG). PCR products were then cloned into the pRmHa3 vector. mRNA Northern and small RNA Northerns
  • RNA probes were used that were made by in vitro transcription of cDNA templates using Maxiscript-T7 in vitro transcription kit (Ambion), supplemented with P 32 -labled UTP.
  • the cDNA templates were prepared from total RNA of DL1 cells by one-step RT-PCR (Superscript One-Step RT-PCR with Platinum Taq, Invitrogen, CA), with primers: ⁇ -nbr (5 ' -GAATTCATGGCACGCAAGAGCCACATG-3 '/ 5'-GAT AAT ACG ACT CAC TAT AGG GAG AGG CTT CAG AAT GAG CTC CAG-3') andl8S rRNA loading control (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-37 5'-AGG GAG CCT GAG AAA CGG CTA CCA CAT CTA AGG AAT CTC CCT ATA GTG AGT CGT ATT ATC -3').
  • RNA northerns 3-15ug of RNA was fractionated on a 15% Tris-UREA gel (NuPage) with 1XTBE buffer. The transfer was performed with 0.5X TBE buffer. Prior to hybridization, the RNA blots were first prehybridized with Oligohyb (Ambion), and then incubated with radioactive labeled RNA probes for -12 hr to overnight at 50°C. [00129] RNA probes were used, and made by in vitro transcription of oligo templates using Maxiscript-T7 in vitro transcription kit (Ambion), supplemented with P32-labled UTP.
  • Oligo DNA templates were prepared by annealing two single stranded DNA oligos into duplex (99°C 5min and cool down to room temperature). Oligos used were miR-2b-l (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TAT CAC AGC CAG CTT TGA GGA GCT CTC CCT ATA GTG AGT CGT ATT ATC-3'); miR-3 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TCA CTG GGC AAA GTG TGT CTC ATC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-7 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA ATG GAA GAC TAG TGA TTT TGT TGT TCT CCC TAT AGT GAG TCG TAT TAT C-3'); miR-10 (5' -GAT AAT ACG ACT
  • Drosophila DL1 cells were grown and maintained in Schneider's media supplemented with 10% FBS (JRH), penicillin/streptomycin and glutamine.
  • dsRNAs for RNAi were generated. Briefly, gene-specific primers containing T7 polymerase binding sites were used to amplify -500 nucleotide regions within genes of interest by PCR. PCR products were used as templates for in vitro transcription using MEGAscript T7 (Ambion), and dsRNA products were purified using RNeasy columns (Qiagen). For RNAi knockdowns, cells were bathed into serum free media containing dsRNA for 45min-lh. Complete media was then added and cells were incubated for three more days.
  • pMT-FLAG-Ran and pMT-FLAG- Ago 1 are as described.
  • 8xl0 6 cells were seeded into 10 cm plates and transfected the next day with 4 ⁇ g pMT-HA-Nbr and 4 ⁇ g of either pMT-Flag-Ran or pMT-Flag-Agol using Effectene (Qiagen). Plasmid expression was induced 24 hours later with 500 ⁇ CuS0 4 , and cells were collected 36 hours post- induction. Cells were processed.
  • cells were lysed into Buffer A+KOAc: 150mM KOAc, 30mM Hepes pH 7.4, 2mM MgOAc, 0.1% NP40, 5mM DTT, PMSF, and a complete protease inhibitor cocktail (Roche).
  • FLAG-tagged proteins were immunoprecipitated overnight at 4°C using anti-FLAG M2 agarose beads (Sigma). Beads were washed six times in Buffer A+KOAc, and bound proteins were separated by SDS-PAGE and immunoblotted with monoclonal anti-FLAG M2 antibody diluted 1:2,500 (Sigma #F3165) and HRP-conjugated anti-HA antibody diluted 1 :2,000 (Roche #12013819001).
  • RNA immunoprecipitation 1.2xl0 7 DL1 cells were seeded into 10 cm plates in serum- free media with 12 ⁇ g dsRNA. One hour later, complete media was added and cells were incubated for 5 days. Endogenous Agol was immunoprecipitated. Cells were lysed in lysis buffer: 20mM HEPES pH 7.0, 150mM NaCl, 2.5mM MgCl 2 , 0.3% Triton-X, 30% glycerol, PMSF, and a complete protease inhibitor cocktail (Roche).
  • Pre-cleared lysates were incubated with rabbit polyclonal AGOl antibody (1 :20; Abeam #ab5070) or control rabbit polyclonal GFP antibody (1 :20; Invitrogen #A-6455) overnight at 4°C.
  • AGOl and control antibodies were isolated using protein A/G beads (1 : 10; Pierce #20421) for 1 hour at 4°C. Beads were then washed 6 times, 10 minutes each in wash buffer: 30mM HEPES pH 7.4, 800mM NaCl, 2mM MgCl 2 , 0.1% NP-40, PMSF and a complete protease inhibitor cocktail (Roche). 1 mL Trizol (Invitrogen) was added to beads following the final wash. RNA was extracted and analyzed by small RNA northern blotting. Small RNA deep sequence analysis
  • RNA sequencing libraries total RNA was extracted using Trizol Reagent (Invitrogen) from -3d old nbr mutants and control 5905 flies (1 : 1 ratio between males and females). 40ug RNA was fractioned in a 15% TBE-Urea gel (Novex, Invitrogen), followed by gel-purification of small RNA ranging between 18nt and 30 nt. The library was then prepared following Small RNA vl.5 Sample Preparation Guide (Illumina) with some modifications.
  • adaptor sequences (5' adapter- 5'-GTT CAGA GTT CTA CAG TCC GAC GAT C-3' ; 3' adapter 5'-ATC TCG TAT GCC GTC TTC TGC TTG AA-3') were removed from the raw reads in the Illumina fastQ generated files using the FASTQ/A Clipper program in the fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Reads less than 16 bp or more than 30 bp were discarded.
  • ratio nbrlcontxoX ratio of the most frequent length to the sum of all other lengths in wild type was calculated, and compared to the ratio of that same most frequent form divided by the sum of all other lengths in nbr (ratio nbrlcontxoX), and ratios plotted (Fig. 4F).
  • the ratio equals [(the number in nbr of most common form in wild type/sum all forms in nbr) divided by (the most common form in wild type/sum of all forms in wild type)].
  • the ratio is excessively large or excessively low when the most common length in nbr is either much greater or much lower than the percentage of reads of that length isoform for the miRNA in wild type.
  • another deep sequencing dataset from Drosophila S2 cells GSM430030 was also used. Reads were mapped to the miRNA stemloop, delineated by read length and sequence, and analyzed.
  • the cDNA was labeled using the FL-OvationTM cDNA Biotin Module V2 (Ovation), hybridized to GeneChip Drosophila Genome 2.0 Arrays (Affymetrix) and scanned with an Axon Instruments 4000B Scanner using GenePix Pro 6.0 image acquisition software (Molecular Devices).
  • Affymetrix .eel probe intensity files were exported from GeneChip Operating Software (Affymetrix). The .eel files were imported to ArrayAssist Lite (Agilent) in which GCRMA probeset expression levels and Affymetrix absent/present/marginal flags were calculated. Statistical analysis for those genes passing the flag filter was performed using Partek Genomics Suite v6.6 (Partek). The signal values were log2 transformed and a 2-way ANOVA was performed. DataGraph 2.3.2 was used to generate the scatterplot (http://www.visualdatatools.com/DataGraph/).
  • RNA was prepared from DL-1 cells treated with dsRNAs (Renilla control or Nbr), and flies, control, nbr loqs po791 .
  • cDNA was synthesized by High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). The realtime-PCR reaction was performed by Power SYBR Green PCR Master Mix (Applied Biosystems) in 7500 Fast Realtime PCR System (Applied Biosystems). Each target gene was normalized to endogenous control (Rp49), followed by calculation of relative fold change compared to control. 500 Fast System SDS Software (Applied Biosystems) was used for data analyses (ddCt method).
  • Oligos used were CG9247/nbr (5'-GCT GGA ATC GAC GGC TGT AA-375'-AAA AAC TCC TCC GCC TTT GC-3'); mRpS25 (CG14413) (5'-CCAGGTGCTCACGCTGAA-375'-GAA GTA GCA GCG CAC AAA CG-3'); Git (5'-CGC AGG CTA CCT CCG AGT AC-375'-AAG CGG ACT TTG CTG ATT GTT T-3'); CG10232 (5'-GGC TGG GCG AGC ATG A-375'- GCA GCG CAG TTT CCT GTA AAG-3'); CG30359 (5' -ATC CAT CGG CCT GCA ACA- 375'-CGG ATC GCG GGA GTA CTG-3'); CG34051 (5'-TGG ATA CGA TTG GCA CAA ACA-375'-TGC AAC TGT CGC CTT TGG T-3');
  • microarray data and deep sequencing datasets can be found in the Gene Expression Omnibus (GEO) of NCBI through accession number GSE32564.
  • GEO Gene Expression Omnibus

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Abstract

In one embodiment, the invention relates to a method for enhancing the efficacy of microRNA (miRNA) mediated interference, the method comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the target specificity and/or efficacy of miRNAmediated interference. In another embodiment, the invention relates to a method for enhancing the efficacy of miRNA mediated interference or expression control, the method comprising: enhancing the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of one or more isoforms of miRNA, thereby enhancing the efficacy of miRNA mediated interference or expression control.

Description

METHODS FOR ENHANCING THE EFFICACY OF MICRORNA MEDIATED
INTERFERENCE
STATEMENT OF GOVERNMENT INTEREST
[0001] The work described herein was, in part, supported by National Institute of Health grants NS043578, AI074951, and AI057168. The United States government may have certain rights in this application.
FIELD OF THE INVENTION
[0002] The invention relates to methods for enhancing the efficacy of microRNA (miRNA) mediated interference. Specifically, the invention relates to inhibiting the expression or function of Nibbler (Nbr) to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA-mediated interference.
BACKGROUND OF THE INVENTION
[0003] miRNAs are endogenous non-coding small RNAs with important roles in many biological pathways, and as such, their generation and activity are under precise regulation.
[0004] miRNAs play critical roles in processes as diverse as normal development and cellular homeostasis. Moreover, recent evidence suggests that they can function as oncogenes or tumor suppressors.
[0005] In the Drosophila miRNA biogenesis pathway, long primary miRNA transcripts undergo sequential cleavage to release the embedded miRNAs. Mature miRNAs are then loaded into Argonaute 1 (Agol) within the RNA-induced silencing complex (RISC).
[0006] Emerging evidence suggests that miRNA pathways are precisely modulated with controls at the level of transcription, processing and stability, with miRNA deregulation linked with diseases and neurodegenerative disorders. It is likely that additional layers of regulation exist that modulate miRNA processing and activity.
[0007] Accordingly, there exists a need for identifying agents that modulate miRNA processing and activity.
SUMMARY OF THE INVENTION
[0008] In one embodiment, the invention relates to a method for enhancing the efficacy of microRNA (miRNA) mediated interference, the method comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the target specificity and/or efficacy of miRNA- mediated interference.
[0009] In another embodiment, the invention relates to a method for enhancing the efficacy of miRNA mediated interference or expression control, the method comprising: enhancing the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of one or more isoforms of miRNA, thereby enhancing the efficacy of miRNA mediated interference or expression control.
[0010] In another embodiment, the invention relates to a method for modulating the efficacy of miRNA mediated interference, the method comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby modulate the target specificity and/or efficacy of miRNA-mediated interference.
[0011] In another embodiment, the invention relates to a method for regulating the production of an miRNA in a cell, the method comprising: regulating the expression or function of Nbr, a variant, or a homolog thereof in said cell, thereby regulating the production of said miRNA in said cell.
[0012] In another embodiment, the invention relates to a method for enhancing silencing of a target RNA, the method comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA mediated interference of said target RNA, and thereby enhancing silencing of said target RNA.
[0013] In another embodiment, the invention relates to a method for identifying an RNAi modulatory compound, comprising, contacting a cell expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
[0014] In another embodiment, the invention relates to a method for identifying an RNAi modulatory compound, comprising, contacting a composition comprising Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified. [0015] In another embodiment, the invention relates to a method for identifying an RNAi modulatory compound, comprising, contacting an organism expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
[0016] In another embodiment, the invention relates to an assay for detecting modulation of RNA interference, comprising, contacting a reaction mixture comprising Nbr with a test compound, and evaluating the effect of the test compound on an indicator of RNA interference, such that modulation of RNA interference is detected.
[0017] In another embodiment, the invention relates to a method for identifying a molecule that regulates Nbr mediated processing of miRNA in a subject, the method comprising: screening a library of molecules; identifying a molecule that regulates the expression or function of Nbr, a variant, or a homolog thereof, thereby identifying said molecule that regulates Nbr mediated processing of miRNA in said subject.
[0018] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1. Fly miR-34 Shows Multiple Isoforms Whose Generation Appears Dependent on 3' End Trimming. (A) miR-34 has multiple forms in adult flies. Left, miR-34 precursor, mature 24-nt sequence in red. Right, Northern for miR-34. Isoforms of 24, 22 and 21nt, labeled a, b, c, respectively. (B) miR-34 isoforms from a deep sequencing fly S2 cell dataset [21]. In red, 24nt isoform a. In blue, isoforms b and c. These are 99.1% of the miR-34 reads. (C) Northern blot analysis of miR-34 isoform accumulation in vivo. Transient induction of pri-miR-34 by hs-GAL4 in adult flies leads to initial accumulation of isoform a, which is lost over time while the shorter isoforms accumulate. Arrowhead, pre-miR-34. (D) Quantification of miR-34 isoforms from pulse-chase in C. Values normalized to 2S rRNA. [0020] Figure 2. nbr is Required to Generate the Isoforms of miR-34. (A) Depletion of known factors in the small RNA biogenesis pathways has no effect on miR-34. (B) Depletion of candidate exoribonucleases shows that loss of CG9247/Nbr (red) leads to an accumulation of the 24nt isoform, with dramatic reduction of the shorter isoforms. (C) Cells depleted of Nbr are not altered in single isoform miRNAs or endogenous siRNA esi-2.1.
[0021] Figure 3. Nbr Interacts with Agol-RISC. (A) Small RNA Northern blot analysis of mir-34 isoforms. Depletion of Agol phenocopies Nbr knockdown. (B) Agol and Nbr interact by co-IP. Cells were untreated or transfected with HA-Nbr and Flag- Agol or Flag-Ran (control). Following IP, interacting proteins were probed by immunoblot. Input, 10% of Flag- IP. (C) All miR-34 isoforms co-IP with Agol. Cells were treated with dsRNA to control (LacZ), Nbr or Agol, and IPs were performed anti-GFP (control) or Agol antibodies. Input and IP-ed RNA were analyzed by Northern blotting for miR-34.
[0022] Figure 4. nbr is Required in vivo to Process Select miRNAs and Silence Target mRNAs. (A) Genomic map of the nbr locus. Coding region in red, with transposon insertion highlighted. (B) Northern blot for nbr. The nbr ^02257 mutant shows complete mRNA loss. (C) Shorter isoforms of miR-34 are abolished in the nbr mutant. Arrow, isoform a. (D) Northern blot of single-isoform miR-277, which is not altered in nbr'7'. (E) Comparison of multiple- isoform miRNAs from control and nbr102257 flies. Some miRNAs require nbr (red arrowheads), while others are n&r-independent. (F) The ratio of the most frequent form of the miRNA in wild type, compared to the sum of all other forms, was generated for nbr and control. The ratios were compared {nbr ratio/control ratio), and plotted. The ratio was excessively large or low when isoform biogenesis is defective. Red boxes, miRNAs with extreme ratios that were further analyzed. Red symbols are confirmed Nbr-targets (Fig. 8). (G) Scatterplot of microarray data from cells treated with dsRNA against Nbr or Renilla control. Highlighted, all of the genes >1.5 fold changed in either direction. (H) Realtime PCR for mRNAs from nbr and logs mutant flies. (4-6 experiments; */?<0.05, **/?<0.01, ***/?<0.001).
[0023] Figure 5 (related to Figure 2). Reduction of nbr affects biogenesis of miR-34 shorter isoforms. (A) mRNA Northern shows that treating cells with two independent dsRNAs directed to different regions of the nbr gene depleted nbr mRNA levels. Loading control, 18S rRNA. (B) Upon reduction of nbr, biogenesis of miR-34 shorter isoforms was affected. Loading control, 2S rRNA. (C) Loss of nbr has minimal effects on the level or pattern of miR-
34. Northern analysis of miR-34* in wild type versus nbr mutant flies. (D) Neighbor joining homology tree showing the relatedness of the 3'→5' exoribonuclease domains of Nbr and other exoribonucleases, rooted to the E. coli RNase D exonuclease domain.
[0024] Figure 6 (related to Figure 3). The interaction between Nbr and Agol is not dependent on RNA. Agol and Nbr interact by co-immunoprecipitation in a manner that is RNA- independent. Cells were either left untreated or were transfected with HA-Nbr and either Flag- Ago 1 or Flag-Ran (control), in the presence or absence of added RNase. Flag-tagged protein was then immunoprecipitated, and interacting proteins were probed by Western immunoblot. Protein input is 10% of Flag-IP.
[0025] Figure 7 (related to Figure 4). New n&r-dependent candidate miRNAs. (A and B) Distribution of (A) overall reads and (B) reads mapping to the miRNA stemloops in control f02257
and nbr mutants. These show no overall difference in read lengths between control and mutant. (C and D) The left panels are the distribution plots of read lengths for these miRNAs, from which the ratio plot in Fig. 4F was generated. The right panels are small RNA Northerns from adult flies of control and nb mutants. In C are candidate miRNAs from the low ratio end of the plot in Fig. 4F where the most abundant isoform in wild type is trimmed. The mutant shift in the distribution is observed by Northern blot analysis. In D, are two miRNAs are from the high ratio side of the plot in Fig. 4F. For these miRNAs, only one major isoform is detectable by Northern in controls, but the level of this isoform becomes more abundant in nbfA (a 2.6-fold increase for miR-190, a 1.8-fold increase for miR-10). Deep sequencing analysis confirmed an impact on isoform distribution in the nbfA mutant (Table 3).
DETAILED DESCRIPTION OF THE INVENTION
[0026] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, it will be understood by those skilled in the art that the present invention may be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the invention.
[0027] The invention encompasses methods for enhancing or modulating the efficacy of microRNA (miRNA) mediated interference. Specifically, the invention encompasses modulating the expression or function of Nibbler (Nbr) to modulate the accumulation of one or more forms of miRNA, thereby enhancing or modulating the efficacy of miRNA-mediated interference. [0028] In one embodiment, provided herein is a method for enhancing the efficacy of miRNA-mediated interference, the method comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the target specificity and/or efficacy of miRNA-mediated interference. In another embodiment, provided herein is a method for enhancing the efficacy of miRNA mediated interference or expression control, the method comprising: enhancing the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of one or more isoforms of miRNA, thereby enhancing the efficacy of miRNA mediated interference or expression control.
[0029] In another embodiment, provided herein is a method for modulating the efficacy of miRNA mediated interference, the method comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby modulate the target specificity and/or efficacy of miRNA-mediated interference. In another embodiment, provided herein is a method for regulating the production of an miRNA in a cell, the method comprising: regulating the expression or function of Nbr, a variant, or a homolog thereof in said cell, thereby regulating the production of said miRNA in said cell.
[0030] In another embodiment, provided herein is a method for enhancing silencing of a target RNA, the method comprising: inhibiting the expression or function of Nbr, a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA mediated interference of said target RNA, and thereby enhancing silencing of said target RNA.
[0031] In another embodiment, provided herein is a method for identifying an RNAi modulatory compound, comprising, contacting a cell expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified. In another embodiment, provided herein is a method for identifying an RNAi modulatory compound, comprising, contacting a composition comprising Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified. In another embodiment, provided herein is a method for identifying an RNAi modulatory compound, comprising, contacting an organism expressing Nbr with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified. [0032] In another embodiment, provided herein is an assay for detecting modulation of RNA interference, comprising, contacting a reaction mixture comprising Nbr with a test compound, and evaluating the effect of the test compound on an indicator of RNA interference, such that modulation of RNA interference is detected.
[0033] In another embodiment, provided herein is a method for identifying a molecule that regulates Nbr mediated processing of miRNA in a subject, the method comprising: screening a library of molecules; identifying a molecule that regulates the expression or function of Nbr, a variant, or a homolog thereof, thereby identifying said molecule that regulates Nbr mediated processing of miRNA in said subject.
[0034] Surprisingly, the inventors of the instant application found that Drosophila miR-34 displays multiple isoforms that differ at the 3 'end, indicating a novel biogenesis mechanism involving 3 'end processing. To define the cellular factors responsible, the inventors of the instant application performed an RNAi screen and identified a putative 3'→5' exoribonuclease Nbr essential for the generation of the smaller isoforms of miR-34. These findings show that Nbr- mediated 3' end processing represents a critical step in miRNA maturation that impacts miRNA diversity.
[0035] As used herein, "Nbr," may refer to all isoforms, homologs, or variants of Nbr. The term "Nbr" is used interchangeably with the terms "Trimmer" or "Tmr."
[0036] In one embodiment, Nbr has the amino acid sequence as set forth in GenBank accession number NP_610094.1 (SEQ ID NO: 2).
[0037] In another embodiment, Nbr protein of the invention comprises the amino acid sequence:
MARKS HMYNA IPAGFESDEE NMENLMSNLK IKRLEDI TTG AGIDGCNFDA TLDAKAEEFF 60 KLFREKWNMY SKKKSPHLRQ EFGRALMGHQ DPLLLALKIF ANCPDSSNIK TKSLSHFVLD 120 TVCKLHKDFP HLGEGCDPNT SMIAFNFVKT SGLLALNNAV IHAYSLRQIR DLLLPKLREL 180 LDNGLYKEVT QWSISLQLTH EFDMLELAFP LIAIEKLPLA EEYLDHATQQ RLPFVKFLDS 240 LLHKEKSVLE LCEHLLDRYK NLKISHNVLS YRPMAK I VAR LAKKYGFDDA VTPNYKFTKT 300 CSYLHYLYRE YEKTRMNLAS FREWSVHAF NHELRTDFVK YLASAGAHSE AIYWYTEFNI 360 DPKDCPLE IE TQVSQNGAGK ASGWESPGKE RCPSSRCDMY LTMDLPDECL I IVNKADEFD 420 RMLYHLQQEC VIYLDSEWMQ SVCGDNQLCV LQIATGHNVY LIDCLARESL RSEHWRLLGA 480 NIFNNVNIRK VGFSMVSDLS VLQRSLPLQL RLQMPHHYLD LRNLWLELKK QRFGVELPFG 540 NVNRAGDALT DLSLACLGKK LNKSNQCSNW ANRPLRREQI LYAAIDARCL MLIYNTLIER 600 VSFIQAVIEK S IASNNFLRR GAHVK 625
(SEQ ID NO: 2).
[0038] In another embodiment, Nbr is a homologue, a variant, an isomer, or a functional fragment of SEQ ID NO: 2. In another embodiment, Nbr is a homologue, a variant, an isomer, or a functional fragment of SEQ ID NO: 2 having an exoribonuclease domain. [0039] In another embodiment, Nbr is homologous to human Exonuclease 3 '-5' domain- containing protein 3 (EXD3). Each possibility represents a separate embodiment of the present invention.
[0040] In some embodiments, Nbr includes polypeptides with amino acid sequences substantially similar to the amino acid sequence of SEQ ID NO.: 2. Substantially similar amino acid sequence may refer to a sequence with at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to a compared amino acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA §5:2444- 2448 (1988).
[0041] In another embodiment, Nbr is encoded by the nucleic acid sequence as set forth in GenBank accession number NM_136250.3 (SEQ ID NO: 1).
[0042] In another embodiment, Nbr protein of the invention is encoded by the nucleic acid sequence:
gtttgttcgc ctttaccgtc aattgcattt gcaattggca attgcacttg cttcgctctg 60 cagtggcacg ggaaacccgt gagaaatggc acgcaagagc cacatgtaca acgcaatacc 120 cgccggcttt gagtcggacg aggagaacat ggagaaccta atgagcaacc tgaagatcaa 180 gcgcttggag gacatcacga caggtgctgg aatcgacggc tgtaactttg acgccacctt 240 ggacgcaaag gcggaggagt tttttaagct gttccgcgag aagtggaaca tgtatagcaa 300 gaaaaagtcg ccacatctgc gccaggaatt tggaagggct ttaatgggcc atcaggatcc 360 tttgctcctg gccctgaaga tcttcgccaa ctgcccagac agcagcaaca tcaagacgaa 420 gagcttgtca cattttgtgc tggacacggt ttgcaagctc cacaaggatt tcccgcatct 480 tggcgagggc tgtgatccca acaccagcat gatagccttt aactttgtca agacctccgg 540 cttgctggcc ctcaacaatg ctgttatcca tgcctacagt ctacgtcaga tccgcgacct 600 actgcttccc aagttgcgcg aactcctgga caacgggctt tacaaagagg tcacccaatg 660 gtccatcagt ctgcagttga cgcacgagtt cgacatgctg gaactggctt tcccgctgat 720 tgcaattgag aaactaccac tggccgagga atatctggac catgccacac agcagaggtt 780 gccctttgtc aagttcttgg attcgttgct gcacaaggag aagtcggttt tagagctctg 840 cgagcatcta ctcgatcggt acaaaaatct caaaatttcc cataacgtgc tcagctatcg 900 gcctatggcc aagattgtgg cgcgactggc caagaagtat ggttttgacg acgccgtcac 960 gcccaactat aagttcacga agacgtgcag ctacttgcat tacctttacc gcgagtacga 1020 gaagacgcgc atgaacctgg ccagtttccg ggaggttgtg agcgtccatg cctttaacca 1080 cgagctgcgc acggacttcg taaagtattt ggcctctgct ggagctcatt ctgaagccat 1140 ctactggtac acggagttca atattgaccc gaaagattgt ccgttggaga tcgagacgca 1200 ggtatcgcaa aacggtgctg gaaaagcgtc aggctgggaa tcccccggca aggaacgctg 1260 tccttcgagt agatgtgata tgtacctgac catggacctg cccgacgagt gccttatcat 1320 tgtgaacaag gctgatgagt tcgatcgtat gttataccat ctgcagcagg agtgcgtcat 1380 atacctggat tctgaatgga tgcagagcgt gtgcggagac aatcagctgt gcgtgctaca 1440 gatcgccacc ggccacaatg tctacctgat cgattgcctg gcgcgggaga gcttacgctc 1500 ggaacactgg cgcttgctgg gcgcgaatat ctttaataac gtgaacatcc gtaaggtggg 1560 gttctctatg gtcagtgatc tcagtgtatt gcagcggtca ctgcccctgc aactgcgcct 1620 tcaaatgcct caccactact tggaccttcg caatctctgg ttggagttaa aaaagcagcg 1680 cttcggcgtt gaacttccct tcggcaatgt taaccgggca ggagacgccc ttacggatct 1740 ctcattggca tgccttggca agaaactgaa caaatcaaac cagtgctcaa actgggccaa 1800 tagaccgctg cgtcgcgagc agattcttta tgcagccatc gatgcgcggt gtttgatgct 1860 gatctacaac acgctcattg aacgcgtgtc cttcatacaa gcggttatcg aaaagagcat 1920 cgccagcaat aactttctca ggcggggtgc ccatgttaag tgattgagga gctggcctct 1980 gttataccca tgctcctttt gatggatagc gattatcacg aagacgacaa aggcgttcgc 2040 ccaacattta ttaagtgcaa atgttatatg tagttgcaaa gagtatttaa catatattta 2100 gtgactagcc aggcagctgg gttagtgccg agttaggcag tttgtacaca tgttacaaat 2160 cagaaggaaa ctatgtgact gcgagcagga tatgaaagaa tgaattttat tacgttatga 2220 tttcaagctc actgtatctc catactttaa atacttgcat tattccaatt cgtaaattcg 2280 cgtattattt gaattcacgc atgatcaaaa ctgaataaaa gcaatgaata ggattgaac 2339
(SEQ ID NO: 1). [0043] In another embodiment, Nbr protein of the invention is encoded by a homologue, a variant, an isomer, or a functional fragment of SEQ ID NO: 1. Each possibility represents a separate embodiment of the present invention.
[0044] In some embodiments, Nbr is encoded by nucleic acid sequences substantially similar to the nucleic acid sequence of SEQ ID NO.: 1. Substantially similar nucleic acid sequence may refer to a sequence with at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to a compared nucleic acid sequence, as determined by the FASTA search method in accordance with Pearson and Lipman, Proc. Natl. Acad. Sci. USA §5:2444-2448 (1988).
[0045] In some embodiments, the expression or function of Nbr, a variant, or a homolog thereof may be inhibited by any suitable Nbr antagonist, known to one of skilled in the art. In one embodiment, the expression of Nbr can be inhibited, for example, by antisense inhibition, by inactivation of an activator or agonist, by activation of an inhibitor or antagonist, by inhibition through adding inhibitory antibodies, by adding active compounds, or by introducing negative dominant mutants, etc.
[0046] In other embodiments, the step of regulating or modulating the expression or function may include enhancing the expression or function of Nbr, a variant, or a homolog thereof. The expression or function of Nbr, said variant, or said homolog thereof may be enhanced by any suitable Nbr agonist, known to one of skilled in the art
[0047] In another embodiment, provided herein is a method for identifying a molecule that modulates the efficacy of miRNA mediated interference in a subject, the method comprising: screening a library of molecules; identifying a molecule that regulates (e.g., upregulate or downregulate) the expression of Nbr, thereby identifying said molecule that modulates the efficacy of miRNA mediated interference in said subject. In one embodiment, a library of oligo nucleic acid molecules can be screened to identify a molecule that regulates the expression of Nbr. In another embodiment, a library of peptide or protein molecules can be screened to identify a molecule that regulates the expression of Nbr. In yet another embodiment, a library of small molecule compounds can be screened to identify a compound that regulates the expression of Nbr. In some embodiments, the screening can be accomplished by any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring decon volution; the "one-bead one- compound" library method; and synthetic library methods using affinity chromatography selection.
[0048] In another embodiment, the invention provides a method for screening or testing a library of molecules, the method comprising the steps of: contacting a plurality of test samples with said library of molecules; measuring the expression of Nbr; and identifying a molecule that is effective in regulating the expression of Nbr, thereby regulating miRNA production or processing.
[0049] Such molecules may include, but are not limited to, peptides made of D- and/or L- configuration amino acids (for example, the form of random peptide libraries; see e.g., Lam, K. S. et al., 1991, Nature 354:82-84), phosphopeptides (m, for example, the form of random or partially degenerate, directed phosphopeptide libraries; see, e.g., Songyang, Z. et al., 1993, Cell 72:767-778), antibodies, and small organic or inorganic molecules. Molecules identified may be useful, for example, in modulating the activity of target gene proteins, preferably mutant target gene proteins, may be useful in elaborating the biological function of the target gene protein, may be utilized in screens for identifying molecules.
[0050] In another embodiment, the invention relates to identifying a molecule that modulates the interaction of Nbr with its interactor or binding molecule. Such modulators may be useful in regulation of: processing of miRNA precursors; mediating mRNA cleavage; mediating assembly of RNA induced silencing complex (RISC); directing translation repression (e.g., via miRNAs); a ribonuclease activity (e.g., cleavage of dsRNA); and initiation of RNAi.
[0051] In one embodiment, a cell free assay, known to one of skilled in the art, can be used to identify a modulator. In a cell free assay, a test compound can contact with a composition comprising an assay reagent (e.g., Nbr), thereby the test compound's ability to modulate the interaction can be determined.
[0052] In some embodiments, the test compounds or polypeptides can be labeled with a radioisotope, for example, but are not limited to, 1251, 35S, 33P, 32P, 14C, or 3H, either directly or indirectly. The radioisotope can be detected by radioemmission counting or by scintillation counting. Alternatively, the test compounds or polypeptides can be enzymatically labeled with, for example, but are not limited to, horseradish peroxidase, alkaline phosphatase, or luciferase. The enzymatic label can be detected, for example, by determining the conversion of an appropriate protein to product. [0053] Binding of reagents can be determined using any suitable methods or techniques known to one of skilled in the art. In one example, a real-time Biomolecular Interaction Analysis (BIA) can be used. See Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705.
[0054] In one embodiment, Nbr can be used as bait proteins in a two-hybrid assay or three- hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268: 12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8: 1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with Nbr or Nbr interactor.
[0055] In another embodiment, an assay is a cell based assay. In a cell based assay, a cell expressing an Nbr is contacted with a test compound and the ability of the test compound to modulate the expression of Nbr is determined. The cell, for example, can be of mammalian origin, yeast, or others known to one skilled in the art.
[0056] In another embodiment, an assay is an organism based assay. In an organism based assay, an organism expressing an Nbr is contacted with a test compound and the ability of the test compound to modulate the expression of Nbr is determined.
[0057] In another embodiment, is an anti-Nbr antibody, or fragments thereof such as F(ab)2 fragments, Fv fragments, single chain antibodies and other forms of "antibodies" can be used as modulators. In a particular embodiment, the antibody is a monoclonal antibody. In another particular embodiment, the antibody is a humanized form of the murine monoclonal antibody. In another particular embodiment, the antibody is a human antibody.
[0058] In some embodiments, in the assays, the activity of Nbr can be determined by measuring the expression of its nucleic acids or amino acids. In other embodiments, the activity of Nbr can be determined by measuring a molecule in Nbr mediated pathway.
[0059] Nbr-mediated processing of regulated precursor microRNAs can also be measured in vitro or in vivo through the measurement of the levels of miRNA duplexes or mature miRNAs. The levels of mature longer miRNA are expected to be higher in cells with reduced or inhibited Nbr activity. The levels of the miRNA or various precursors can be measured using techniques known in the art, including, but not limited to, PCR (e.g., quantitative RT-PCR), Northern blots, and microRNA microarrays (see, for example,
Thomson et al. (2004) Nature Methods 1 :47-53). [0060] Alternatively, the level of Nbr or its activity can be indirectly measured by monitoring the expression of a target polynucleotide (either the target mRNA or polypeptide encoded thereby) that is known to be regulated by the mature miRNA using techniques known in the art. For example, PCR (e.g., quantitative RT-PCR), Northern blots, or microarrays can be used to measure the level of target mRNAs and Western blots or an ELISA, for example, can be used to measure the level of target polypeptides. In addition, assays that measure the activity of target polypeptides can be used as an indirect measure of miRNA production. It should be noted that the levels of miRNA and the expression of target polynucleotides can also be affected by various downstream mechanisms, including, but not limited to other post- transcriptional and translational regulatory mechanisms. In some embodiments, the levels of pre-miRNA can be measured in a cell.
[0061] The invention also relates, in part, to a method of using the nucleic acids of the invention to reduce expression of a target gene in a cell, tissue or organ. Expression of the target gene may be reduced by expressing a nucleic acid of the invention that comprises a sequence substantially complementary to one or more binding sites of the target mRNA. The nucleic acid may be a miRNA or a variant thereof. The nucleic acid may also be pri-miRNA, pre-miRNA, or a variant thereof, which may be processed to yield an miRNA. The expressed miRNA may hybridize to a substantially complementary binding site on the target mRNA, which may lead to translational repression or activation of RISC-mediated gene silencing.
[0062] The target of gene silencing may also be a gene or protein that causes the silencing of a second gene or protein. By repressing expression of the target gene, expression of the second protein may be increased. Examples for efficient suppression of miRNA expression are the studies by Esau et al 2004 JBC 275-52361; and Cheng et al 2005 Nucleic Acids Res. 33-1290, which are incorporated herein by reference.
[0063] The present invention also relates to a method of using the nucleic acids of the invention to increase expression of a target gene in a cell, tissue or organ. Expression of the target gene may be increased by expressing a nucleic acid of the invention that comprises a sequence substantially complementary to a pri-miRNA, pre-miRNA, miRNA or a variant thereof. The nucleic acid may be an anti-miRNA. The anti-miRNA may hybridize with a pri- miRNA, pre-miRNA or miRNA, thereby reducing its gene repression activity. Expression of the target gene may also be increased by expressing a nucleic acid of the invention that is substantially complementary to a portion of the binding site in the target gene, such that binding of the nucleic acid to the binding site may prevent miRNA binding. Alternatively, an miRNA may be used to increase gene expression. Recent evidence has shown that an miRNA may bind to a gene promoter and induce transcription (Place et al. PNAS. 2008 vol. 105 no. 5 1608-1613).
[0064] The nucleic acid sequences coding for the above-described molecules (e.g., Nbr) can be obtained using recombinant methods known in the art, such as, for example, by screening cDNA and genomic libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.
[0065] For example, a DNA of the present invention can be prepared from a cDNA library from cells which express a protein of the present invention by conducting hybridization using a partial sequence of a DNA of the present invention as a probe. A cDNA library can be prepared, for example, by the method described by Sambrook J. et al. (Sambrook J. et al. (1989) Molecular Cloning, Cold Spring Harbor Laboratory Press), or by using commercially available cDNA libraries. A cDNA library can be also prepared by extracting RNA from cells expressing a protein of the present invention, synthesizing cDNA using reverse transcriptase, synthesizing an oligo DNA base on the sequence of the DNA of the present invention, conducting PCR using these as primers, and amplifying cDNA encoding the protein of the present invention.
[0066] In addition, by sequencing the nucleotides of the obtained cDNA, a translation region encoded by the cDNA can be determined, and the amino acid sequence of a protein of the present invention can be obtained. Moreover, by screening the genomic DNA library using the obtained cDNA as a probe genomic DNA can be isolated.
[0067] More specifically, mRNAs may first be prepared from a cell, tissue, or organ (for example, testis, brain, heart, liver, and kidney) in which a protein of the invention is expressed. Known methods can be used to isolate mRNAs. For instance, total RNA is prepared by the guanidine ultracentrifugation method (Chirgwin J M. et al. (1979) Biochemistry 18, 5294-5299) or by the AGPC method (Chomczynski P. and Sacchi N. (1987) Anal. Biochem. 162, 156-159), and mRNA is purified from total RNA using mRNA Purification Kit (Pharmacia). Alternatively, mRNA may be directly purified by the QuickPrep mRNA Purification Kit (Pharmacia). [0068] The obtained mRNA is used to synthesize cDNA using reverse transcriptase. A cDNA may be synthesized using kits, such as the AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo). Alternatively, a cDNA may be synthesized and amplified according to the 5'-RACE method (Frohman M A. et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8998-9002; Belyavsky A. et al. (1989) Nucleic Acids Res. 17:2919-2932) wherein primers, described herein, the 5'-Ampli FINDER RACE Kit (Clontech), and polymerase chain reaction (PCR) are used.
[0069] A desired DNA fragment is prepared from the PCR products and ligated with a vector DNA. The recombinant vectors are used to transform E. coli and a desired recombinant vector is prepared from a selected colony. The nucleotide sequence of the desired DNA can be verified by conventional methods, such as, the dideoxynucleotide chain termination method.
[0070] A DNA of the invention may be also designed to have a sequence that is expressed more efficiently by taking into account the frequency of codon usage in the host to be used for expression (Grantham R. et al. (1981) Nucleic Acids Res. 9, 43-74). A DNA of the present invention may be altered by a commercially available kit or a conventional method. For instance, a DNA may be altered by digestion with restriction enzymes, insertion of synthetic oligonucleotides or appropriate DNA fragments, addition of linkers, or insertion of the initiation codon (ATG) and/or the stop codon (TAA, TGA, or TAG).
[0071] The DNAs of this invention include a DNA that (a) hybridizes with a DNA consisting of the nucleotide sequence of Nbr; and (b) encodes a protein that is functionally equivalent to a protein of this invention mentioned above. Conditions for hybridization can be selected appropriately by those skilled in the art, and those conditions specifically mentioned above may be used. Under these conditions, DNA having higher homology is obtained as the temperature is raised. The above-mentioned DNA to be hybridized is preferably a naturally occurring DNA, for example, a cDNA or chromosomal DNA.
[0072] The present invention also provides vectors into which a DNA of the present invention is inserted. The vectors of the present invention are useful to retain a DNA of the present invention in host cell, or to express a protein of the present invention
[0073] A particular embodiment of the invention encompasses a polypeptide comprising all or a portion of the amino acid sequence of Nbr. Where a portion of the Nbr is used, the portion most preferably retains function so as to regulate miRNA processing. In addition, Nbr protein may contain one or more mutations or deletions, so long as the product functions to regulate miRNA processing.
[0074] The polypeptides of the invention can be prepared by methods known in the art. For example, chemical synthesis, such as the solid phase procedure described by Houghton et al., 1985, Proc. Natl. Acad. Sci. U.S.A., 82: 5131-5, can be used. A preferred method involves the recombinant production of protein in host cells transfected within a vector containing polynucleotide sequence(s) encoding Nbr, described above.
[0075] In another aspect, the invention includes an expression vector comprising a nucleic acid sequence containing an open reading frame (ORF) that encodes the Nbr, described herein. The vector further includes regulatory sequences effective to express the ORF in a host cell; such sequences may be endogenous or heterologous (such as a secretory signals recognized in yeast, mammalian cells, insect cells, tissue culture or bacterial expression systems). In the expression vector, regulatory sequences may also include, 5' to the nucleic acid sequence, a promoter region and an ATG start codon in-frame with the hybrid fusion polypeptide coding sequence (chimeric nucleic acid molecule), and 3' to the coding sequence, a translation termination signal followed by a transcription termination signal. Further, the invention includes a method of recombinantly producing a transcriptional regulating hybrid fusion polypeptide using an expression vector.
[0076] Expression vectors are usually plasmids, but the invention includes other vector forms, such as viral vectors, including recombinant viral vectors known and used by those skilled in the art, as well as vectors that serve equivalent functions and become known in the art subsequently hereto. The polynucleotide sequences encoding Nbr proteins can be stably integrated into the chromosome of an appropriate host cell using direct DNA introduction methods as practiced in the art. Suitable expression vectors include, but are not limited to, mammalian cell expression vectors, such as pcDNA3 (available from Invitrogen), bacterial cell expression vectors, such as pET-30 (available from Novagen or Promega) or yeast expression vectors. Preferred are mammalian cell expression vectors.
[0077] Expression vectors typically contain regulatory elements capable of affecting expression of the Nbr protein. Typically, a vector contains an origin of replication, a promoter, and a transcription termination sequence. The vector may also include other regulatory sequences, including mRNA stability sequences, which provide for stability of the expression product; secretory leader sequences, which provide for secretion of the expression product; environmental feedback sequences, which allow expression of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium); marking sequences, which are capable of providing phenotypic selection in transformed host cells; restriction sites, which provide sites for cleavage by restriction endonucleases; and sequences which allow expression in various types of host cells, including prokaryotic cells, yeast, fungi, algae, plant cells, insect cells, mammalian cells, including human cells and non-human animal cells, cells of non-human primates, and cells of higher eukaryotes.
[0078] As will be appreciated by the skilled practitioner, expression vectors comprise a nucleic acid sequence encoding Nbr, or their combinations, operably linked to at least one regulatory sequence or element. Operably linked is intended to mean that the nucleic acid sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence. Regulatory sequences are known in the art and are selected to direct expression of the desired protein in an appropriate host cell. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements (see, D. V. Goeddel, 1990, Methods Enzymol., 185:3-7). It will be appreciated by the skilled practitioner that the design of the expression vector can depend on such factors as the choice of the host cell to be transfected and/or the type of protein to be expressed.
[0079] Vectors can contain one or more replication and inheritance systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and/or one or more expression cassettes. The inserted coding sequences can be synthesized by standard methods, isolated from natural sources, or prepared as hybrids. Ligation of the coding sequences to transcriptional regulatory elements (e.g., promoters, enhancers, and/or insulators) and/or to other amino acid encoding sequences can be carried out using established methods.
[0080] Host cells containing an expression vector that comprises a nucleic acid sequence encoding the Nbr fusion proteins of the present invention can be cultured under conditions suitable for the expression and recovery of the expressed protein, e.g., from cell membranes or cell lysates, using methods known and practiced by those in the art. In particular, the host cells can contain an expression vector which comprises all or a portion of the DNA sequence encoding Nbr.
[0081] Suitable host cells include both prokaryotic cells (e.g., without limitation, E. coli strains HB 101, DH5a, XL1 Blue, Y1090 and JM101), plant cells, fungal cells, and eukaryotic cells. Eukaryotic recombinant host cells are preferred. Examples of eukaryotic host cells include, but are not limited to, yeast, e.g., S. cerevisiae cells, cell lines of human, bovine, porcine, monkey, and rodent origin, as well as insect cells, including but not limited to, Spodoptera frugiperda insect cells and Drosophila-derived insect cells. Mammalian species- derived cell lines suitable for use and commercially available include, but are not limited to, L cells, CV-1 cells, CHO cells, (CHO-K1, ATCC CCL 61), COS-1 cells (ATCC CRL 1650), COS-7 cells (ATCC CRL 1651), HEK 293 cells, human skin fibroblasts, 3T3 cells (ATCC CCL 92), HeLa cells (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
[0082] According to another embodiment, the expression construct of the invention is delivered to a cell. A wide variety of methods can be used to deliver the expression constructs to cells. Such methods include, for example, but are not limited to, DEAE dextran-mediated transfection, calcium phosphate precipitation, polylysine- or polyomithine-mediated transfection, electroporation, sonoporation, protoplast fusion, liposomes, peptoid delivery, or microinjection.
[0083] In some embodiments, the expression constructs are packaged in liposomes prior to delivery to the cells. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. Liposomal preparations for use with the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, known in the art.
[0084] Depending on the expression system and host selected, the molecules are produced by growing host cells transformed by an expression vector described above whereby the protein is expressed. The expressed protein is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the product can be purified directly from the media. If it is not secreted, it can be isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. For example, once expressed, the product may be isolated and purified by any number of techniques, well known in the art. A protein of the present invention obtained as above may be isolated from the interior or exterior (e.g., medium) of the cells or hosts, and purified as a substantially pure homogeneous protein. The method for protein isolation and purification is not limited to any specific method. In fact, any standard method may be used. For instance, column chromatography, filtration, ultrafiltration, salt precipitation, solvent precipitation, solvent extraction, distillation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, isoelectric point electrophoresis, dialysis, and recrystallization may be appropriately selected and combined to isolate and purify the protein.
[0085] For chromatography, for example, affinity chromatography, ion-exchange chromatography, hydrophobic chromatography, gel filtration, reverse phase chromatography, adsorption chromatography, and such may be used (ed. Daniel R. Marshak et al. (1996) Strategies for Protein Purification and Characterization: A Laboratory Course Manual., Cold Spring Harbor Laboratory Press). These chromatographies may be performed by liquid chromatography, such as, HPLC and FPLC.
[0086] The expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy, using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties. In another embodiment, the invention provides a gene therepy vector comprising a nucleic acid sequence encoding Nbr protein.
[0087] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art.
[0088] In one embodiment, the expression constructs of the present invention are delivered without a viral vector. For example, the construct can be delivered directly, or packaged in liposomes prior to delivery to the subject or to cells derived therefrom, as described above. In another embodiment, the expression constructs are encapsulated, adsorbed to, or associated with, particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG.
[0089] In some embodiments, biolistic delivery systems employing particulate carriers, such as gold and tungsten, are used for delivering the expression constructs of the present invention. The particles are coated with the construct to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a gene gun. For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744. In another embodiment, the invention provides a transgenic stem cell having up-regulated or down- regulated Nbr. In some embodiments, the invention provides transgenic animals having up- regulated or down-regulated Nbr. Transgenic animals can be farm animals (pigs, goats, sheep, cows, horses, rabbits, and the like), rodents (such as rats, guinea pigs, and mice), non-human primates (for example, baboons, monkeys, and chimpanzees), and domestic animals (for example, dogs and cats). Invertebrates such as Caenorhabditis elegans or Drosophila can be used as well as non-mammalian vertebrates such as fish (e.g., zebrafish) or birds (e.g., chickens).
[0090] Pharmaceutically useful compositions comprising agonists or antagonists of Nbr can be formulated as compositions, preferably physiologically acceptable compositions, according to known methods, such as by admixture with a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions may also include compounds which activate cellular interferon stimulated genes (ISGs). The compositions can comprise Nbr agonists or antagonists.
[0091] The pharmaceutical compositions comprising the same can be, in another embodiment, administered to a subject by any method known to a person skilled in the art, such as parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intra-dermally, subcutaneously, intra-peritonealy, intra- ventricularly, intra- cranially, intra- vaginally or intra-tumorally.
[0092] In another embodiment of methods and compositions of the present invention, the pharmaceutical compositions are administered orally, and are thus formulated in a form suitable for oral administration, i.e. as a solid or a liquid preparation. Suitable solid oral formulations include tablets, capsules, pills, granules, pellets and the like. Suitable liquid oral formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment of the present invention, the active ingredient is formulated in a capsule. In accordance with this embodiment, the compositions of the present invention comprise, in addition to the active compound (e.g. the mimetic compound, peptide or nucleotide molecule) and the inert carrier or diluent, a hard gelating capsule.
[0093] In another embodiment, the pharmaceutical compositions are administered by intravenous, intra- arterial, or intra-muscular injection of a liquid preparation. Suitable liquid formulations include solutions, suspensions, dispersions, emulsions, oils and the like. In another embodiment, the pharmaceutical compositions are administered intravenously and are thus formulated in a form suitable for intravenous administration. In another embodiment, the pharmaceutical compositions are administered intra- arterially and are thus formulated in a form suitable for intra- arterial administration. In another embodiment, the pharmaceutical compositions are administered intra-muscularly and are thus formulated in a form suitable for intra-muscular administration.
[0094] In another embodiment, the pharmaceutical compositions are administered topically to body surfaces and are thus formulated in a form suitable for topical administration. Topical formulations include, in another embodiment, gels, ointments, creams, lotions, drops and the like.
[0095] In another embodiment, the pharmaceutical composition is administered as a suppository, for example a rectal suppository or a urethral suppository. In another embodiment, the pharmaceutical composition is administered by subcutaneous implantation of a pellet. In another embodiment, the pellet provides for controlled release of active agent over a period of time.
[0096] In another embodiment, the active compound is delivered in a vesicle, e.g. a liposome.
[0097] In other embodiments, carriers or diluents used in methods of the present invention include, but are not limited to, a gum, a starch (e.g. corn starch, pregeletanized starch), a sugar (e.g., lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g. microcrystalline cellulose), an acrylate (e.g. polymethylacrylate), calcium carbonate, magnesium oxide, talc, or mixtures thereof.
[0098] In other embodiments, pharmaceutically acceptable carriers for liquid formulations are aqueous or non-aqueous solutions, suspensions, emulsions or oils. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
[0099] In another embodiment, parenteral vehicles (for subcutaneous, intravenous, intraarterial, or intramuscular injection) include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Examples of oils are those of animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, olive oil, sunflower oil, fish-liver oil, another marine oil, or a lipid from milk or eggs.
[00100] In other embodiments, the compositions further comprise binders (e.g. acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone), disintegrating agents (e.g. cornstarch, potato starch, alginic acid, silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodium starch glycolate), buffers (e.g., Tris-HCL, acetate, phosphate) of various pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g. sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g. hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosity increasing agents(e.g. carbomer, colloidal silicon dioxide, ethyl cellulose, guar gum), sweeteners (e.g. aspartame, citric acid), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants (e.g. stearic acid, magnesium stearate, polyethylene glycol, sodium lauryl sulfate), flow-aids (e.g. colloidal silicon dioxide), plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers (e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymer coatings (e.g., poloxamers or poloxamines), coating and film forming agents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/or adjuvants. Each of the above excipients represents a separate embodiment of the present invention.
[00101] In another embodiment, the pharmaceutical compositions provided herein are controlled-release compositions, i.e. compositions in which the active compound is released over a period of time after administration. Controlled- or sustained-release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). In another embodiment, the composition is an immediate-release composition, i.e. a composition in which of the active compound is released immediately after administration. [00102] In another embodiment, the pharmaceutical composition is delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials are used; e.g. in microspheres in or an implant. In yet another embodiment, a controlled release system is placed in proximity to the target cell, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984); and Langer R, Science 249: 1527-1533 (1990).
[00103] The compositions also include, in another embodiment, incorporation of the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts.) Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.
[00104] Also included in the present invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.
[00105] Also comprehended by the invention are compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline. The modified compounds are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981 ; Newmark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound. [00106] Each of the above additives, excipients, formulations and methods of administration represents a separate embodiment of the present invention.
[00107] In one embodiment, the methods of the present invention comprise administering an active compound as the sole active ingredient. However, also encompassed within the scope of the present invention are methods for treating diseases and disorders that comprise administering the active compound in combination with one or more therapeutic agents.
[00108] The methods described herein comprise in one embodiment, using a combination therapy. In another embodiment, the term "combination" is used in its broadest sense and means that a subject is treated with at least two therapeutic regimens.
[00109] The term "subject," as used herein, refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term "subject" does not exclude an individual that is normal in all respects.
[00110] In one embodiment, the term "treatment," as used herein, refers to any process, action, application, therapy, or the like, wherein a subject, including a human being, is subjected to medical aid with the object of improving the subject's condition, directly or indirectly. In another embodiment, the term "treating" refers to reducing incidence, or alleviating symptoms, eliminating recurrence, preventing recurrence, preventing incidence, improving symptoms, improving prognosis or combination thereof in other embodiments. "Treating" embraces in another embodiment, the amelioration of an existing condition. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. Treatment also embraces palliative effects: that is, those that reduce the likelihood of a subsequent medical condition. The alleviation of a condition that results in a more serious condition is encompassed by this term
[00111] The terms "polypeptide" and "protein," as used herein, refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full- length proteins and fragments thereof are encompassed by the definition. The terms also include postexpression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like. Furthermore, for purposes of the present invention, a "polypeptide" refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site- directed mutagenesis, or may be serendipitous, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
[00112] The term "nucleic acid," as used herein, can include both double- and single-stranded sequences and refers to, but not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral (e.g. DNA viruses and retroviruses) or procaryotic DNA, and especially synthetic DNA sequences. The term also captures sequences that include any of the known base analogs of DNA and RNA.
[00113] The term "isolated," as used herein with respect to a polynucleotide, refers to a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
[00114] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
EXAMPLES EXAMPLE 1
The exoribonuclease Nibbler controls 3' end processin2 of microRNAs in Drosophila
[00115] Surprisingly, we found that Drosophila miR-34 displays multiple isoforms that differ at the 3 'end, suggesting a novel biogenesis mechanism involving 3 'end processing. To define the cellular factors responsible, we performed an RNAi screen and identified a putative 3'→5' exoribonuclease CG9247 /nibbler essential for the generation of the smaller isoforms of miR- 34. Nibbler (Nbr) interacts with Agol and processes miR-34 within RISC. Deep sequencing analysis revealed a larger set of multi-isoform miRNAs that are controlled by nibbler. These findings shows that Nbr-mediated 3' end processing represents a critical step in miRNA maturation that impacts miRNA diversity.
[00116] While miRNAs are typically annotated and observed as a single species, we found that miR-34 showed a pattern of three major isoforms of 24, 22 and 21 nt in Northern blots from adult Drosophila (Fig. 1A). Deep sequencing analysis also showed that miR-34 is present in multiple forms that all bear the same 5' terminus but differ at their 3' ends, presenting a nested series (Fig. IB). To assess the relationship among these, we designed a pulse-chase experiment to follow miR-34 biogenesis. Heat shock driven primary miR-34 was tightly induced for 30 min, then monitored over time in adult flies. The longest isoform, isoform a (24nt), was predominant initially, while the accumulation of the shorter isoforms was delayed, but then increased over time (Fig. 1C). Moreover, as the 21 nt isoform accumulated, the 24 nt form was lost in a seemingly reciprocal manner, suggesting that the 24-mer may be converted into the 21-mer.
[00117] To define the mechanism, we treated cells with dsRNA targeting specific genes within the small RNA biogenesis pathways, and assessed the miR-34 pattern by Northern blot. Imprecise cleavage of the precursor transcript could result in the production of the multiple forms. However, reduction of either Drosha or Dcr-1, or their binding partners Pasha and Loquacious, or Dicer-2 (Dcr-2), responsible for siRNA generation, did not alter the pattern (Fig. 2A). Therefore, we reasoned that the smaller isoforms may instead be generated by an exonuclease that sequentially processes the longest isoform into the nested series observed. To test this hypothesis, we performed an RNAi screen against the predicted 3'→5'exonucleases in Drosophila, including components of the RNA exosome (Table 1). This identified one gene, CG9247 (which we named nibbler/nbr), with a striking effect: depletion of nbr led to a dramatic accumulation of the miR-34 large isoform with a concomitant loss of the shorter isoforms (Fig. 2B; Fig. 5). In contrast, loss of nbr did not appear to alter the sizes or levels of miRNAs that normally show a single isoform by Northern, such as miR-14 and miR-277 (Fig. 2C). We also examined whether nbr knockdown had an effect on endogenous siRNAs, but saw no impact on esi-2.1 (Fig. 2C). These data indicated that the novel putative exoribonuclease Nbr is required to generate the shorter isoforms of the multi-isoform miRNA miR-34, but is not required for general small RNA biogenesis.
[00118] The Nbr exoribonuclease domain shows closest sequence homology to human EXD3, falling within the E. coli RNAse D protein family; this includes the Werner exoribonuclease and C. elegans Mut-7 involved in transposon silencing (Fig. 5). Nbr, however, showed no predicted RNA binding domain, suggesting that it may function with a partner with RNA binding capacity, to bring Nbr activity to RNA substrates. To define these, we then performed a second RNAi screen genes known to bind RNA or associate with small RNA silencing pathways, including the two somatic RISC-associated Argonautes (Table 1). Strikingly, loss of Agol phenocopied nbr depletion: accumulation of the 24 nt isoform occurred, with reduction of the shorter isoforms (Fig. 3A). Controls indicated that knockdown of Agol had no effect on nbr expression, and nbr knockdown had no effect on Agol expression. These data suggested that Agol is also required for trimming, and that Nbr and Agol may act in a complex. Co-immunoprecipitation (IP) studies indicated that HA-tagged Nbr associates with Flag-tagged Agol, but not with a control protein (Flag-Ran) (Fig. 3B). RNase treatment indicated the association was not RNA-dependent (Fig. 6). Proteomic studies have identified both Agol and Nbr as small RNA associated proteins, underscoring the specificity of the interaction. Since Nbr associates with Agol, we hypothesized that miR-34 3 'end processing may occur in the context of RISC. Indeed, IP of Agol revealed that all miR-34 isoforms were bound (Fig. 3C). Furthermore, when Nbr was depleted, the longest miR-34 isoform remained bound to Agol (Fig. 3C). Altogether, these data show that the 24 nt miR-34 isoform is first generated by Dcr-1 then loaded into RISC. Next, Nbr, in association with Agol, processes the long 24 nt isoform into shorter isoforms that remain loaded in RISC.
[00119] To assess the in vivo role of Nbr, we analyzed the expression and function of nbr in flies. Northerns revealed that nbr is expressed during development and in the adult, with peaks during the late larval/early pupal stage, and in adults. Analysis of nbr mRNA levels in animals with a transposon insertion in the coding region (nbr102257) showed that homozygous mutants {nbr'1') lacked nbr expression (Fig. 4A, B). nbr'1' flies were semi-lethal, and sterile, indicating that nbr function is critical. Given the homology to Mut-7, we examined levels of transposons, but found no evidence linking nbr to transposon silencing. Assessment of miR-34 expression in nbr'1' flies phenocopied cells treated with dsRNA: the shorter isoforms were abolished, while the 24-nt form accumulated (Fig. 4C). As in cells, there was no striking effect on single-form miRNAs like miR-277 (Fig. 4D). Furthermore, miR-34* levels and isoform distribution appeared unaffected (Fig. 5). These data indicated that nbr modulates the isoform abundance of miR-34.
[00120] To assess the broader impact of Nbr function, we screened 65 miRNAs by Northern blot of RNA from cultured cells and flies. We identified 9 additional miRNAs with multiple isoforms: mir-2, miR-3, miR-12, miR-79, miR-263a/b, miR-274, miR-279, miR-281-1/2 and miR-305. The expression patterns of five of these were altered in nbr'1' mutants, exhibiting accumulation of the longest isoforms with concomitant loss of the shorter isoforms (Fig. 4E; miR-2 family not studied further due to cross hybridization between members). Analysis of small RNA profiling data from cells confirmed that two of these {miR-263a, miR-305) had significant levels of multiple forms that differed at the 3'end (Table 2); miR-3, miR-12, miR- 281, and miR-274 were too low for analysis. Three multiple-isoform miRNAs {miR-79, miR- 274, and miR-279) did not show an altered pattern in nbr'1' flies (Fig. 4E). The deep sequencing dataset revealed that miR-279 displays a series of isoforms that do differ at the 3 'end; since miR-279 processing is nZ?r-independent, nbr may be one member of a larger set of genes or mechanisms responsible for 3 'end diversity. miR-79 isoforms differed at the 5 'end, suggesting that mechanisms also exist for 5' end diversity of miRNAs.
[00121] We further investigated the extent to which trimming is involved in miRNA processing by deep sequencing the small RNAs from flies, comparing nbr mutants to controls. There was no major impact on the size distribution of small RNAs as a whole or miRNAs in particular (Fig. 7A-B). To more carefully assess isoforms, reads were mapped to the miRNA stem-loop sequences and analyzed for length. For each miRNA, we calculated a ratio of the most frequent length in wild type to the sum of all other lengths, and compared this ratio between nbr and control. The distribution of the length ratios highlighted a cohort of miRNAs with extreme differences between nbf and control. At the two ends of the plot were miRNAs where the most common length isoform of the miRNA was present at a much higher or much lower level in nbr' than in wild type, reflecting an altered pattern of isoform distribution or relative abundance for these miRNAs in the absence of nbr. These included miRNAs we had defined as trimmed and modulated by nbr (miR-34, miR-263a, miR-263b), with additional candidates (Fig. 4F, red boxes). Northern blots were performed on the top and bottom 8 miRNAs that we had not tested; we confirmed 7 new n/?r-dependent miRNAs (miR- 7, miR-10, miR-11, miR-31b, miR-100, miR-190, miR-317; Fig. 7, Table 3, 4). Northern revealed some miRNAs that were trimmed that were not detected as so by deep sequencing and the reverse: for any given miRNA, the extent of trimming had to be greater than -10% in isoform level to detect a consistent change by Northern blot, while deep sequence analysis suggested that not all isoforms were cloned with equal efficiency.
[00122] Trimming exerts a profound and diverse impact on miRNA sequence profiles: nbr promotes the diversity of some miRNAs (miR-34, miR-7, miR-317), and alters the relative abundance of the most prominent isoform of others (miR-190 and miR-10; Fig. 7C-F, Table 3). To identify potential Nbr-dependent miRNA targets, we performed transcriptional profiling of cells. This would allow identification of mRNA targets whose stability was altered by miRNA trimming, but not targets primarily controlled by translational repression [24]. This identified 12 genes whose levels were affected by nbr depletion by >1.5-fold (Fig.
4G, Table 5); of these, one was reduced (nbr), the others were upregulated. Assessing the levels of 8 of these by realtime PCR confirmed increased expression of 6/8 mRNAs (75%) in n/?r-depleted cells (Table 5). Next, we assessed expression of 9 of these genes in nb '~ flies, compared to wild type and loquacious mutant flies, loq 00791 mutants are viable and show deficiency in miRNA maturation and function, thus allow assessment of miRNA function in adults. We reasoned that genes regulated by miRNAs that are impacted by n&r-processing would also show dependence on loqs. We validated 5/9 genes (55%) as upregulated in both nbf'~ and loqs100791 (two additional genes were upregulated, although did not reach statistical significance in nbr1') (Fig. 4H, Table 5). Sequence analysis of these mRNA targets revealed that 4/7 genes (57%) have potential sites for the miRNAs that showed n&r-dependent processing (Table 5). It is unclear, however, if existing algorithms for miRNA targeting efficiently predict binding sites for miRNAs with 3 'end diversity; targets for trimmed miRNAs may use non-canonical recognition motifs that are more dependent on 3 'end pairing than seed complementarity.
[00123] These data provide evidence for a novel step in miRNA biogenesis: miRNA 3' end terminal trimming mediated by the 3'→5' exoribonuclease Nbr. Notably, small RNA deep sequencing has unveiled a rich pattern of miRNA sequence isoforms, although miRNAs have routinely been annotated as a single mature form. Our findings show that miRNA processing by Nbr alters the repertoire of at least a subset of miRNAs in cells and whole animals, contributing to the diversity of the small RNA profile and potentially impacting post- transcriptional gene regulation in Drosophila. Mechanistically, our data indicate that, upon nbr knockdown, miR-34 is still associated with RISC, thus trimming is not a pre-requisite to miR-34 loading, and likely occurs after loading.
[00124] Nbr may impact strand selection within RISC because strand selection is influenced by the extent of 3' overhang and degree of pairing for any miRNA-miRNA duplex. Nbr may impact miRNA stability, as previous studies have demonstrated that tailing and trimming of mature Drosophila miRNAs influences their turnover. Trimming may also impact mRNA silencing by favoring alterative miRNA sites within mRNA targets. Although canonical miRNA-target specificity is thought to be driven largely by complementarity within the seed, non-canonical interactions can depend more heavily on 3' compensatory sites. Therefore, differences in the length of the 3' end of miRNAs may influence both target selection, as well as silencing efficiency of targets that require extensive 3 'end pairing.
[00125] The modification of mature miRNAs and their precursors is an emerging facet of miRNA-mediated gene regulation. Our data demonstrates that Nbr represents a central player in a larger spectrum of factors that shape miRNA repertoire and function, through the generation of multi-isoform miRNAs.
Table 1 : dsRNA amplicons used in this study
Figure imgf000030_0001
I Core Components of small RNA silencing pathways
Dcr-1 miRNA biogenesis TAATACGACTCACTATAGGCGCAACACGGTGACAATATC TAATACGACTCACTATAGGCGGAACACGATTATT
TGCCT
Dcr-2 siRNA biogenesis TAATACGACTCACTATAGGAAGAGCAAGTGCTCACGGTT TAATACGACTCACTATAGGTCTTGCAGTTTTTGC
ACCAG
Agol miRNA biogenesis TAATACGACTCACTATAGGCGCATGAAATTGCGCTAC TAATACGACTCACTATAGGCTGCTGGG
TGAAATTACGC
Ago2 siRNA biogenesis TAATACGACTCACTATAGGAGGATGGAGCAACTCAGGT TAATACGACTCACTATAGGATTCTAAACTGAGGA
ATAATCACA
Drosha miRNA biogenesis TAATACGACTCACTATAGGGAAACAGTCTCATGCTCCTT TAATACGACTCACTATAGGTCGAAGCCCTCTTTC
AAGGA
Pasha miRNA biogenesis TAATACGACTCACTATAGGACCGATACGGTGGCGGAGTA TAATACGACTCACTATAGGGAAGCCATGGACGT
GGAGGG
loq miRNA biogenesis TAATACGACTCACTATAGGCTTGGCCTCCTTCTTCGATC TAATACGACTCACTATAGGTCCTGTCCAGACGGG
ATTTG
Bgal Negative Control TAATACGACTCACTATAGGATGACGGAACAGGTATTCGC TAATACGACTCACTATAGGCACCCGAGTGTGATC
ATCTG
Predicted exonuclease-domain containing proteins
CG6744 3'→5 exonuclease activity TAATACGACTCACTATAGGTTATGGCGGTGG TAATACGACTCACTATAGGTACGCATCCCGGTCA
CCATTTA CC
CG6744 3'→5 exonuclease activity TAATACGACTCACTATAGGAGGCCGAAATGG TAATACGACTCACTATAGGAAGATCGGCTTTATC
AAAAGACT GATGC
CG9247/tmr 3'→5 exonuclease activity TAATACGACTCACTATAGGCAAAAATCTCAA TAATACGACTCACTATAGGCCTGCTGCAGATGGT
AATTTCCCATAA ATAAC
CG9247/tmr 3'-»5 exonuclease activity TAATACGACTCACTATAGGAGTATGGTTTTG TAATACGACTCACTATAGGAATGATAAGGCACTC
ACGACGCC GTCGG
egl 3'→5 exonuclease activity TAATACGACTCACTATAGGGGACAGAGCCA TAATACGACTCACTATAGGGGTGTTCTGCAGCAC
GGGGTT CTT
egl 3'→5 exonuclease activity TAATACGACTCACTATAGGCCGAATAGCTGA TAATACGACTCACTATAGGACCCTGATTGAGATT
TCGTGGAT GGCAC
mus205 3'→5 exonuclease activity TAATACGACTCACTATAGGTGGTGGCCTGGC TAATACGACTCACTATAGGACTGGCGCAGAAAG
AATGT GAAC
mus205 3'→S exonuclease activity TAATACGACTCACTATAGGGATCCCCTAATC TAATACGACTCACTATAGGGGCGGTGTAGCCAT
GTGCTTGA AGGTAA
CG10214 3'→S exonuclease activity TAATACGACTCACTATAGGTGGATGGACTTG TAATACGACTCACTATAGGAATACGCAAGCTCTT
GAGATGAC TGATG
CG10214 3'→5 exonuclease activity TAATACGACTCACTATAGGCTGTGGCTTAGA TAATACGACTCACTATAGGGCTCTTTGATGCTTT
CACGGACA CTCGG
CsL4 3'→S exonuclease activity TAATACGACTCACTATAGGGAACCGAGGACA TAATACGACTCACTATAGGTCCTTGATCGTGGTC
GCATAGT TGG
CsL4 3'→S exonuclease activity TAATACGACTCACTATAGGCAGGTGGTTAGT TAATACGACTCACTATAGGTTTTAGTTCCACAAA
GTGCATAA TGAATTTAAG
mRPL28 3'→S exonuclease activity TAATACGACTCACTATAGGCAAGCCGGTGCA TAATACGACTCACTATAGGATGCGGCACCACCTT
GAACA TTC
mRPL28 3'→S exonuclease activity TAATACGACTCACTATAGGAGTGGGAACGA TAATACGACTCACTATAGGAGATAGCGGCGGTA
GATGAGGTG TTCCTT
MTR3 3'→5 exonuclease activity TAATACGACTCACTATAGGCCCGCCAAAGGA TAATACGACTCACTATAGGAGGTGGCTGGACTG
GCTAAT AGATA
MTR3 3'→S exonuclease activity TAATACGACTCACTATAGGCTATGTGAATTTT TAATACGACTCACTATAGGCAGAGCAGCTCCTCA
GCGGCCT ACCTT
rrp42 3'→S exonuclease activity TAATACGACTCACTATAGGTGCCACGCCGGA TAATACGACTCACTATAGGACCCGAAAGGAATG
GTTT CCTG
rrp42 3'→S exonuclease activity TAATACGACTCACTATAGGCTGTTCAACACA TAATACGACTCACTATAGGTCCTACTATTTGCGG
AAGCTGCC TCGCT
ski6 3'→S exonuclease activity TAATACGACTCACTATAGGTACATGGAGCAG TAATACGACTCACTATAGGCAAGTGATCGATGT
GGAAACAC GGAACC
ski6 3'→5 exonuclease activity TAATACGACTCACTATAGGAAACTGGGAGTC TAATACGACTCACTATAGGCTCGATGACGGTTTC
TTCGAGCA CAAGT
Mrell exonuclease activity TAATACGACTCACTATAGGTTCCATCTGTTAG TAATACGACTCACTATAGGCCAAACCGAATCGCA
TGGTACATC TTAAA
Mrell exonuclease activity TAATACGACTCACTATAGGTTCCATCTGTTAG TAATACGACTCACTATAGGATTAAACATGCAGG
TGGTACATC ACTCATCT DIS3 3'— >5' exonuclease activity TAATACGACTCACTATAGGATCATCGTAACG TAATACGACTCACTATAGGCTTCATTGTCCACTTC
ATTGACACA CCAC WRNexo 3'— >5' exonuclease activity TAATACGACTCACTATAGGGAAAGCTGGCAC TAATACGACTCACTATAGGGATGGCGGCGTACA
GTGATTT TTAG RRP6 3'— >5' exonuclease activity TAATACGACTCACTATAGGTTATCGTTGTATA TAATACGACTCACTATAGGGCATCTCCCTTGGAA
TCGTCAACAT GACT
Rrp45 3'— >5' exonuclease activity TAA TAC GAC TCA CTA TAG GCG AAT ACC TAA TAC GAC TCA CTA TAG GTC GAG CAG GGA
CAG GAT GTT CCA GTT CAG TTT CG 16940 3'— >5' exonuclease activity TAA TAC GAC TCA CTA TAG GCG AAT ACC TAA TAC GAC TCA CTA TAG GTC GAG CAG GGA
CAG GAT GTT CCA GTT CAG TTT
CG12877 exonuclease activity TAA TAC GAC TCA CTA TAG GAC CAG CAG TAA TAC GAC TCA CTA TAG GAT CGC ACT GGT
AAC CTA TGG ATG GGG GTC
CG6833 exonuclease activity TAA TAC GAC TCA CTA TAG GGT GCA ACA TAA TAC GAC TCA CTA TAG GTG CAC AGC GGC
GGT GAA CAC AAG TTA TAA TG
Rrpl 3'— >5' exonuclease activity TAA TAC GAC TCA CTA TAG GAG AGC CGC TAA TAC GAC TCA CTA TAG GGG CTT GGT TTC
CGA AAC AAC AGT AGA GG
Rrp40 3'— >5' exonuclease activity TAA TAC GAC TCA CTA TAG GAC ATT AAA TAA TAC GAC TCA CTA TAG GAC GAT ATG GAG
ATG AGC GCG ACC GCT GTG TCC
Rrp46 3'— >5' exonuclease activity TAA TAC GAC TCA CTA TAG GTG CTA CAG TAA TAC GAC TCA CTA TAG GCT GAA AGA TCT
GTC TCA TAG GC CGG CAC TG CG11337 3'— >5' exonuclease activity TAA TAC GAC TCA CTA TAG GCG GCT GAG TAA TAC GAC TCA CTA TAG GGT GGG AAG
AGA GAT CTT C CAA CGA TCT CT
Snp 3'— >5' exonuclease activity TAA TAC GAC TCA CTA TAG GGC ACC ATC TAA TAC GAC TCA CTA TAG GTC CCA GCA GTG
TTG CAC ATG AG CTT GTT AA
RNA interacting proteins screened
CG1434 double-stranded RNA binding TAA TAC GAC TCA CTA TAG GTG TGC TCC TAA TAC GAC TCA CTA TAG GCC AGC TCC CAG
GGC TTG GAC ATC TCG
CG5641 double-stranded RNA binding TAA TAC GAC TCA CTA TAG GCT GAC GGG TAA TAC GAC TCA CTA TAG GAG CCC GGC GGA
CAA CAA TGT AG GAG T
CG8273 double-stranded RNA binding TAA TAC GAC TCA CTA TAG GGA ATC CAA TAA TAC GAC TCA CTA TAG GGG TAG GGA
CAC CAG GAG AAA C GGG CAA CGG
Adar double-stranded RNA binding TAA TAC GAC TCA CTA TAG GCA AAA AGA TAA TAC GAC TCA CTA TAG GAT TGC ACT GAA
CGG CGA AAA ATG GGT CCA GC
mle double-stranded RNA binding TAA TAC GAC TCA CTA TAG GAT TTC CCG TAA TAC GAC TCA CTA TAG GAT CAA TTA CGA
TGC AGC AAT TTT AAA CAA TGT CG
Msp-300 double-stranded RNA binding TAA TAC GAC TCA CTA TAG GAA ACA ACG TAA TAC GAC TCA CTA TAG GAG GCG GTG
AGG TGC AGA AAC AAG GTA GAG A
DIP1 double-stranded RNA binding TAA TAC GAC TCA CTA TAG GCA CTC GGG TAA TAC GAC TCA CTA TAG GGC ACT GGC GGC
ACA AGA AGT TG TTC TTT
stau double-stranded RNA binding TAA TAC GAC TCA CTA TAG GGA CCG TTT TAA TAC GAC TCA CTA TAG GTT GGT GGG CGT
CCT CCG AAG TT AAG GGG
Tudor-SN RNA-induced silencing complex TAA TAC GAC TCA CTA TAG GCC GCC GCG TAA TAC GAC TCA CTA TAG GCG CTG GCG ACA
TGA CCA A AAC TCT
Fmrl RNA-induced silencing complex TAA TAC GAC TCA CTA TAG GCG TGC CCG TAA TAC GAC TCA CTA TAG GAT TGT GCG CTG
AGA GTA TGA AAT AAA CTC CTT
bel RNA-induced silencing complex: TAA TAC GAC TCA CTA TAG GCA ACA GCC TAA TAC GAC TCA CTA TAG GGT TCT TTT CGT
(41 TAG AAG GGA TCG TCC TCG CAC
Rbp4 single-stranded RNA binding TAA TAC GAC TCA CTA TAG GCA CTG GAT TAA TAC GAC TCA CTA TAG GCA CGG GCG CCG
TCT TCA GAC ATT G AAA TC
Ssrp single-stranded RNA binding TAA TAC GAC TCA CTA TAG GTC CCG ATG TAA TAC GAC TCA CTA TAG GTT GTC CGA ATC
AGG AGA CCA C ACC AAA GTC
gw gene silencing by miRNA TAA TAC GAC TCA CTA TAG GAA TCC AAG TAA TAC GAC TCA CTA TAG GAT TGC TTG CTT
TAA TCC TAT AAG CAG TGC TTA ATG A
AG03 RNA binding TAA TAC GAC TCA CTA TAG GAT CTT TAT TAA TAC GAC TCA CTA TAG GCA TTT GAC GCC
CAG CAA AAT GTA GAG CAC TTA TCT
snRNP-Ul-C mRNA binding ; 14. TAA TAC GAC TCA CTA TAG GCA AAG TAC TAA TAC GAC TCA CTA TAG GCT TGG GTC CGT
TAT TGC GAC TAC T TCA TGA TTC
mRNA binding ; (4. TAA TAC GAC TCA CTA TAG GTT CGA GCA TAA TAC GAC TCA CTA TAG GTT GCT CGC AAC
ATT TGT ACG CAG ACA AAG TTC
Psi mRNA binding : 14. TAA TAC GAC TCA CTA TAG GGC TGA ATG TAA TAC GAC TCA CTA TAG GTT GCT GCT CGA
TCA TTT CTC GCA TCA TTT CAG
nito mRNA binding : 14 TAA TAC GAC TCA CTA TAG GAC ACT GTT TAA TAC GAC TCA CTA TAG GGG CTT TGA GCT
TGC AGG AAA TCT CTT GAA AG
Nelf-E mRNA binding : 14. TAA TAC GAC TCA CTA TAG GGT GAA GTG TAA TAC GAC TCA CTA TAG GGT TGA TGC CAT
GCC AGG AAA CT TCA CAT TCT T
Hrb98DE mRNA binding ; 14. TAA TAC GAC TCA CTA TAG GTT CCT CAG TAA TAC GAC TCA CTA TAG GCG TTT CTT GCC
GAC TCC ATC ACC AGT CTC CTT
Glo mRNA binding : 14. TAA TAC GAC TCA CTA TAG GCA ACA ACA TAA TAC GAC TCA CTA TAG GCG AAG TTC GAG
TGC TGG GCT TC CCG ACA
Spen mRNA binding : 14. TAA TAC GAC TCA CTA TAG GCT TCC ACA TAA TAC GAC TCA CTA TAG GCT CGG GAA ACT
GCG GAT ACT TC GTG ATT CTT Table 2: miRNA reads from deep sequencing data miR-263a Read count Percentage
AAUGGCACUGGAAGAAUUCACGGG 24 552 56 .3%
AAUGGCACUGGAAGAAUUCACGG 23 170 17 .3%
AAUGGCACUGGAAGAAUUCACG 22 97 9 .9%
AAU GGC ACU GGAAG AAUUC AC 21 112 11 .4%
AAUGGCACUGGAAGAAUUCA 20 36 3 .7%
AAUGGCACUGGAAGAAUUC 19 14 1 .4%
99. 6% of reads
miR-279
UGACUAGAUCCACACUCAUUAAU 23 5 0 .1%
UGACUAGAUCCACACUCAUUAA 22 785 18 .8%
UGACUAGAUCCACACUCAUUA 21 757 18 .2%
UGACUAGAUCCACACUCAUU 20 1858 44 .6%
UGACUAGAUCCACACUCAU 19 758 18 .2%
UGACUAGAUCCACACUCA 18 6 0 .1%
99. 1% of reads
miR-305
AUUGUACTJUCAUCAGGUGCUCUGGU 25 8 0 .4%
AUUGUACUUCAUCAGGUGCUCUGG 24 225 10 .9%
AUUGUACUUCAUCAGGUGCUCUG 23 63 3 .0%
AUUGUACUUCAUCAGGUGCUCU 22 295 14 .2%
AUUGUACUUCAUCAGGUGCUC 21 1110 53 .6%
AUUGUACUUCAUCAGGUGCU 20 357 17 .2%
AUUGUACUUCAUCAGGUGC 19 12 0 .6%
AUUGUACUUCAUCAGGUG 18 2 0 .1%
99. 6% of reads
miR-79
AUAAAGCU GAUU C CAAAGC AU 23 885 17 .7%
UAAAGC UAGAUUAC C AAAGC AU 22 4044 81 .0%
AAAGC UAGAUUAC C AAAGCAU 21 66 1 .3%
98.1% of reads
Reads from a Drosophila S2 cell library were mapped to the miRNA stem loop sequences, and miRNAs that showed multiple forms by small RNA northern were analyzed. Four of these miRNAs had significant reads in the library. Analysis showed evidence for 3 'end diversity of miR-263a, miR-279 and miR-305, and 5 'end diversity of miR-79. By small RNA northern, processing of miR-263a and miR-305 are affected by nbr mutation; miR-279
(showing diversity at the 3 'end) is not affected by nbr f02257 , indicating additional mechanisms modulate 3 'end diversity. miR-79 (unaffected by nbr102257) indicates mechanisms for 5 'end diversity also exist.
Table 3 (related to Figure 4). Sequence counts of miRNAs from ends of the ratio plot (Fig.
4F) in control (WT) and Nbr mutants.
Norm, normalized counts. Specific % of reads, specific percentage of read counts that started at
that 5' position. Red are those miRNAs that were previously known or validated by Northern.
The nine miRNAs from end of the ratio plot with smallest ratios
WT specific Tmr Tmr specific
miR~7 length WT norm of reads norm % of reads
UGGAAGACUAGTJGAUUUUGUUGUUU 25 4 0 7 1
UGGAAGACUAGUGAUUUUGUUGUU 24 321 16 513 49
UGGAAGACUAGUGAUUUUGUUGU 23 432 21 339 32
UGGAAGACUAGUGAUUUUGUUG 22 902 45 151 14
UGGAAGACUAGUGAXJUUUGUU 21 255 13 25 2
UGGAAGACUAGUGAUUUUGU 20 94 5 12 I
UGGAAGACUAGUGAUUDUG 19 13 1 3 0
80.9% of reads 75% of reads
WT specific Tmr Tmr specific
length WT norm of reads norm % of reads
CAUCACAGUCUGAGUUCUUGCU 22 406 19 255 52
CAUCACAGUCUGAGUUCDUGC 21 1526 72 190 38
CAIJCACAGUCUGAGUUCDUG 20 153 7 41 8
CAUCACAGUCUGAGUUCUU 19 15 1 3 1
CAUCACAGUCUGAGUUCD 18 9 0 3 1
97% of reads 95% of reads
WT specific Tmr Tmr specific
!tiiR..317 length WT rjorm of reads norm % of reads
UGAACACAGCUGGUGGUAUCCAGU 24 64 2 123 17
UGAACACAGCUGGUGGUAUCCAG 23 104 3 161 22
UGAACACAGCUGGUGGUAUCCA 22 162 5 120 17
UGAACACAGCUGGUGGUAUCC 21 1564 49 179 25
UGAACACAGCUGGDGGUAUC 20 989 31 93 13
UGAACACAGCUGGUGGUAU 19 153 5 18 2
UGAACACAGCUGGUGGUA IS 103 3 20 3
UGAACACAGCUGGUGGU 17 33 1 5 1
97% of reads 92% of reads
WT specific Tmr Tmr specific
miS-283 length WT norm of reads norm % of reads
AAAUAUCAGCUGGUAAUUCUGGG 23 171 23 170 39
AAAUAUCAGCUGGUAAUUCUGG 22 S3 11 74 17
AAAUAUCAGCUGGUAAUUCUG 21 466 63 182 42
AAAUAUCAGCUGGUAAUUCU 20 12 2 5 1
AAAUAUCAGCUGGUAAUUC 19 4 0 0 0
96% of reads 97% of reads
WT specific Tmr Tmr specific
miR-210 length WT norm of reads norm % of reads
CUUGUGCGUGUGACAGCGGCUAUU 24 15 5 18 1 )
CUUGUGCGUGUGACAGCGGCUAU 23 86 29 86 52
CUUGUGCGUGUGACAGCGGCUA 22 26 9 20 12
CTJUGUGCGUGUGACAGCGGCO 21 121 40 29 17
CUUGUGCGUGUGACAGCGGC 20 47 16 8 5
CUUGUGCGUGUGACAGCGG 19 5 2 4 2
56% of reads 56% of reads
UTJGUGCGUGUGACAGCGGCUAUD 23 17 7 12 9
UUGUGCGUGUGACAGCGGCUAU 22 99 42 67 53
UUGUGCGUGUGACAGCGGCUA 21 30 13 17 13
UUGUGCGUGUGACAGCGGCU 20 72 31 21 16
UUGUGCGUGUGACAGCGGC 19 13 6 9 7
UDGUGCGUGUGACAGCGG 18 2 1 0 1
44% of reads 43% of reads
WT specific Tmr Tmr specific
mUt-307 length WT norm of reads norm % of reads
DCACAACCUCCUUGAGUGAGCGA 23 2 3 6 9
UCACAACCUCCUUGAGUGAGCG 22 6 11 14 21
UCACAACCDCCUUGAGUGAGC 21 47 81 43 65
UCACAACCUCCUUGAGUGAG 20 2 4 2 4
UCACAACCUCCUUGAGUGA 19 0 1 0 1
90% of reads 92% of reads WT specific % Tmr Tmr specific length T norm of reads norm % of reads
CUUGGCACUGGGAGAAUUCACAGU 24 3 0 4 0
CUUGGCACUGGGAGAAUUCACAG 23 233 8 151 15
CUUGGCACUGGGAGAAUUCACA 22 492 17 337 33
CUUGGCACUGGGAGAAUUCAC 21 1557 54 402 39
CUUGGCACUGGGAGAAUUCA 20 487 17 106 10
CUUGGCACUGGGAGAAUUC 19 93 3 22 2
CUUGGCACUGGGAGAAUU 18 6 0 1 0
CUUGGCACUGGGAGAAU 17 2 0 0 0
99% of re ds 9S% of reads
WT specific % Tmr Tmr specific miR-315 length WT norm of reads norm % of reads
UUUUGAUUGUUGCUCAGAAAGCC 23 71 27 75 40
UUTJUGAUUGUUGCUCAGAAAGC 22 187 70 104 56
UUUUGAUUGUUGCUCAGAAAG 21 6 2 4 2
UUUUGAUUGUUGCUCAGAAA 20 3 1 1 1
99% of reads 98% of reads
The nine miRNAs from end of ratio plof with largest ratws
WT specific % Tmr Tmr specific imR-!M) length WT norm of reads norm of reads
AGAUAUGUUUGAUAUUCUUGGUUGUU 26 0 0 0 J
AGAUAUGUUUGAUAUUCUUGGUUGU 25 9 5 13 9
AGAUAUGUUUGAUAUUCUUGGUUG 24 49 26 78 55
AGAUAUGUUUGAUAUUCUUGGUU 23 42 23 29 20
AGAUAUGUUUGAUAUUCUUGGU 22 31 17 12 8
AGAUAUGUUUGAUAUUCUUGG 21 38 20 7 5
AGAUAUGUUUGAUAUUCUUG 20 14 8 3 2
AGAUAUGUUUGAUAUUCUU 19 2 1 0
97% of reads 99% of reads
WT specific Tmr Tmr specific tmti-34 length WT norm of reads norm % of reads
UGGCAGUGUGGUUAGCUGGUUGUGUA 26 0 0 2 0
UGGCAGUGUGGUUAGCUGGUUGUGU 25 13 1 102 8
UGGCAGUGUGGUUAGCUGGUUGUG 24 807 52 1004 76
UGGCAGUGUGGLrUAGCUGGUUGU 23 79 5 111 8
UGGCAGUGUGGUUAGCUGGUUG 22 157 10 60 5
UGGCAGUGUGGUUAGCUGGUU 21 304 20 11 1
UGGCAGUGUGGUUAGCUGGU 20 93 6 14 1
UGGCAGUGUGGUUAGCUGG 19 82 5 11 1
UGGCAGUGUGGUUAGCUG 18 12 1 3 0
UGGCAGUGUGGirUAGCU 17 1 0 0 0
UGGCAGUGUGGUUAGC 16 3 0 2 0
91% of reads 93% of reads
WT specific % Tmr Tmr specific miK-10 length WT norm of reads norm % of reads
CAAAUUCOGUUCUAGAGAGGUUUG 24 0 0 0 0
CAAAUUCGGUUCUAGAGAGGUUU 23 873 46 479 64
CAAAUUCGGUUCUAGAGAGGUU 22 714 38 190 26
CAAAUUCGGUUCUAGAGAGGU 21 181 10 44 6
CAAAUUCGGUUCUAGAGAGG 20 87 5 19 3
CAAAUUCGGUUCUAGAGAG 19 7 0 3 0
CAAAUUCGGUUCUAGAGA 18 8 0 4 1
CAAAUUCGGUUCUAGAG 17 1 0 0 0
CAAAUUCGGUUCUAGA 16 8 0 4 0
9 % of reads 99% of reads
WT specific Tmr Tmr specific length WT norm of reads norm % of reads
AACCCGUAAAUCCGAACUUGUG 22 110 86 133 93
AACCCGUAAAUCCGAACUUGU 21 14 11 7 5
AACCCGUAAAUCCGAACUUG 20 3 3 2 2
97% of reads 97% of reads
Tim- Tmr specific
miR-1 10 length WT norm norm % of reads
UUUCACCUAUCGUUCCAUUUGCAG 24 38 76 22 86
UUUCACCUAUCGUUCCAUUUGCA 23 2 4 2 7
UUUCACCUAUCGUUCCAUUUGC 22 9 18 1 5
UUUCACCUAUCGUUCCAUUUG 21 0 1 0 2
88% of reads 87% of reads
WT specific Tmr Tmr specific
miR-263it length WT norm of reads norm % of reads
AAUGGCACUGGAAGAAUUCACGGGG 25 1 0 4 0
AAUGGCACUGGAAGAAUUCACGGG 24 20494 55 18394 66
AAUGGCACUGGAAGAAUUCACGG 23 10116 27 8037 29
AAUGGCACUGGAAGAAUUCACG 22 389S 11 1225 4
AAUGGCACUGGAAGAAUUCAC 21 2044 6 237 1
AAUGGCACUGGAAGAAUUCA 20 340 1 35 0
AAUGGCACUGGAAGAAUUC 19 129 0 14 0
AAUGGCACUGGAAGAAUU 18 27 0 5 0
AAUGGCACUGGAAGAAU 17 9 0 3 0
AAUGGCACUGGAAGAA 16 4 0 1 0
100% of reads 100% of reads
WT specific Tmr Tmr specific
miR-989 length WT norm of reads norm % of reads
UGUGAUGUGACGUAGUGGAACAU 23 27 0 19 0
UGUGAUGUGACGUAGUGGAACA 22 4256 32 3196 22
UGUGAUGUGACGUAGUGGAAC 21 8600 64 1027S 72
UGUGAUGUGACGUAGUGGAA 20 294 2 406 3
UGUGAUGUGACGUAGUGGA 19 263 2 408 3
UGUGAUGUGACGUAGUGG 18 8 0 14 0
UGUGAUGUGACGUAGUG 17 9 0 6 0
UGUGAUGUGACGUAGU 16 1 0 2 0
99% oj reads 99% of reads
WT specific Tmr Tmr specific
length WT norm of reads norm % of reads
UGGCAAGAUGUCGGAAUAGCUGA 23 555 35 347 30
UGGCAAGAUGUCGGAAUAGCUG 22 784 49 674 58
UGGCAAGAUGUCGGAAUAGCU 21 182 11 99 8
UGGCAAGAUGUCGGAAUAGC 20 62 4 38 3
UGGCAAGAUGUCGGAAUAG 19 13 1 7 1
97% of reads 98% of reads
Table 4 (related to Figure 4). Summary of results on miRNAs from the two ends of the length ratio plot.
Figure imgf000035_0001
miRNAs that could not be confirmed included those that were not detectable by small RNA northerns, those with more complex isoforms that involved 5 'end as well as 3 'end diversity and because of this complexity could not be confirmed, and those that did not show a striking effect by Northern comparing controls to nbf'~ mutant animals. Table 5 (related to Figure 4). Genes identified by microarray that changed in cells upon Nbr knockdown
List of all of the genes with a change of 1.5-fold or greater in either direction with a 20% false discovery rate or less (see Fig. 4G). One probe (1629669_x_at) does not correspond to a unique gene and was not pursued.
Not done (specific realtime primers could not be designed). For CG34051, amplification in cells showed multiple products, but amplication was specific in flies.
1, miRNA sites predicted by TargetScan, PicTar, or miRanda
*, significant effects indicated.
Figure imgf000036_0001
Additional Experimental Details:
Fly stocks and culture
[00126] Flies were grown in standard cornmeal molasses agar medium with dry yeast, at 25 °C unless otherwise specified. General stock lines and GAL4 driver lines were obtained from the Drosophila Stock center at Bloomington. nb 022S7 was obtained from the Exelixis collection (Harvard University). Fly transgenics were generated by standard procedures (Genetic Services, Inc).
Constructs
[00127] Fly genomic DNA was prepared from whole flies with the Puregene DNA purification kit (Qiagen). Using genomic DNA as the template, a 286bp of miR-34 genomic sequence was amplified by PCR (primers: 5'-CCG TTA CAC ACG ACTA TTC TCA AT-375'-CCA TCT GAT ACA GGT CCT ACA TTT TCT AAA A-3'), and used to generate a miR-34 pUAST construct. To generate Nbr constructs, PCR amplification was conducted using single stranded cDNA as the template, with primer pairs of HA-Nbr (5'- GAA TTC ATG TAC CCA TAC GAT GTT CCA GAT TAC GCT GCA CGC AAG AGC CAC ATG-375'- GGT ACC TCA CTT AAC ATG GGC ACC CCG). PCR products were then cloned into the pRmHa3 vector. mRNA Northern and small RNA Northerns
[00128] Total RNA was isolated from cells or flies using Trizol reagent (Invitrogen) according to manufacturer's protocol. For mRNA northern, 5μg RNA was run on a 1% MOPS/formaldehyde gel, and transferred onto nylon plus (Northernmax, Ambion). The RNA blots were then hybridized following standard procedures at 68 °C, with prehybridization (~ 1 hr), hybridization (~ 12 hr or overnight) with P32 labeled probe, washed and exposed to Phosphoimager (Amersham). RNA probes were used that were made by in vitro transcription of cDNA templates using Maxiscript-T7 in vitro transcription kit (Ambion), supplemented with P32-labled UTP. The cDNA templates were prepared from total RNA of DL1 cells by one-step RT-PCR (Superscript One-Step RT-PCR with Platinum Taq, Invitrogen, CA), with primers: ΊΊ-nbr (5 ' -GAATTCATGGCACGCAAGAGCCACATG-3 '/ 5'-GAT AAT ACG ACT CAC TAT AGG GAG AGG CTT CAG AAT GAG CTC CAG-3') andl8S rRNA loading control (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-37 5'-AGG GAG CCT GAG AAA CGG CTA CCA CAT CTA AGG AAT CTC CCT ATA GTG AGT CGT ATT ATC -3'). For small RNA northerns, 3-15ug of RNA was fractionated on a 15% Tris-UREA gel (NuPage) with 1XTBE buffer. The transfer was performed with 0.5X TBE buffer. Prior to hybridization, the RNA blots were first prehybridized with Oligohyb (Ambion), and then incubated with radioactive labeled RNA probes for -12 hr to overnight at 50°C. [00129] RNA probes were used, and made by in vitro transcription of oligo templates using Maxiscript-T7 in vitro transcription kit (Ambion), supplemented with P32-labled UTP. Oligo DNA templates were prepared by annealing two single stranded DNA oligos into duplex (99°C 5min and cool down to room temperature). Oligos used were miR-2b-l (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TAT CAC AGC CAG CTT TGA GGA GCT CTC CCT ATA GTG AGT CGT ATT ATC-3'); miR-3 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TCA CTG GGC AAA GTG TGT CTC ATC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-7 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA ATG GAA GAC TAG TGA TTT TGT TGT TCT CCC TAT AGT GAG TCG TAT TAT C-3'); miR-10 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA ACC CTG TAG ATC CGA ATT TGT TTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-11 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA ACA TCA CAG TCT GAG TTC TTG CTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-12 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TGA GTA TTA CAT CAG GTA CTG GTT CTC CCT ATA GTG AGT CGT ATT ATC); miR-3 lb (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TGG CAA GAT GTC GGA ATA GCT GTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-34 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375 '-AAA AAA TGG CAG TGT GGT TAG CTG GTT GTG TCT CCC TAT AGT GAG TCG TAT TAT C-3'); miR-34* (GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA CAG CCA CTA TCT TCA CTG CCG CCT CTC CCT ATA GTG AGT CGT ATT ATC-3'); miR-100 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'-AAA AAA AAC CCG TAA ATC CGA ACT TGT GTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-190 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA AGA TAT GTT TGA TAT TCT TGG TTG TCT CCC TAT AGT GAG TCG TAT TAT C-3'); miR-210 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'-AAA AAA ATT GTG CGT GTG ACA GCG GCT ATC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-263 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'-GTT AAT GGC ACT GGA AGA ATT CAC TCT CCC TAT AGT GAG TCG TAT TAT C-3 ' ) ; miR-277 (5 ' - GAT AAT ACG ACT CAC TAT AGG GAG A-375' -TAA ATG CAC TAT CTG GTA CGA CAT AAA TGC ACTATCTGGTACGACA TCT CCC TAT AGT GAG TCG TAT TAT C- 3'); miR-274 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TTT TGT GAC CGA CAC TAA CGG GTA ATT CTC CCT ATA GTG AGT CGT ATT ATC-3'); miR-281rev (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'-TGT CAT GGA ATT GCT CTC TTT GTT GTC ATG GAA TTG CTC TCT TTG TTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-283 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375' AAA AAA TAA ATA TCA GCT GGT AAT TCT TCT CCC TAT AGT GAG TCG TAT TAT C- 3'); miR-305 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'-AAA AAA ATT GTA CTT CAT CAG GTG CTC TGT CTC CCT ATA GTG AGT CGT ATT ATC-3'); miR-307 (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'-AAA AAA TCA CAA CCT CCT TGA GTG AGT CTC CCT ATA GTG AGT CGT ATT ATC-3'); miR-307rev (5'-GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TCA CAC CCA GGT TGA GTG AGT CTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-315 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TTT TGA TTG TTG CTC AGA AAG CTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-317 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA ATG AAC AC A GCT GGT GGT ATC CAG TTC TCC CTA TAG TGA GTC GTA TTA TC-3'); miR-986 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA ATC TCG AAT AGC GTT GTG ACT GAT CTC CCT ATA GTG AGT CGT ATT ATC-3'); miR-1010 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'- AAA AAA TTT CAC CTA TCG TTC CAT TTG CAG TCT CCC TAT AGT GAG TCG TAT TAT C-3'); esi2.1 (5' -GAT AAT ACG ACT CAC TAT AGG GAG A-375'-TTG ACT CCA ACA AGT TCG CTC CTC TCC CTA TAG TGA GTC GTA TTA TC-3') and 2S rRNA (5'- GAT AAT ACG ACT CAC TAT AGG GAG A-375'-TGC TTG GAC TAC ATA TGG TTG AGG GTT GTA TCT CCC TAT AGT GAG TCG TAT TAT C-3').
Cell culture, dsRNA synthesis and RNAi
[00130] Drosophila DL1 cells were grown and maintained in Schneider's media supplemented with 10% FBS (JRH), penicillin/streptomycin and glutamine. dsRNAs for RNAi were generated. Briefly, gene-specific primers containing T7 polymerase binding sites were used to amplify -500 nucleotide regions within genes of interest by PCR. PCR products were used as templates for in vitro transcription using MEGAscript T7 (Ambion), and dsRNA products were purified using RNeasy columns (Qiagen). For RNAi knockdowns, cells were bathed into serum free media containing dsRNA for 45min-lh. Complete media was then added and cells were incubated for three more days.
Protein and RNA Immunoprecipitations
[00131] pMT-FLAG-Ran and pMT-FLAG- Ago 1 are as described. For protein immunoprecipitations, 8xl06 cells were seeded into 10 cm plates and transfected the next day with 4 μg pMT-HA-Nbr and 4 μg of either pMT-Flag-Ran or pMT-Flag-Agol using Effectene (Qiagen). Plasmid expression was induced 24 hours later with 500 μΜ CuS04, and cells were collected 36 hours post- induction. Cells were processed. Briefly, cells were lysed into Buffer A+KOAc: 150mM KOAc, 30mM Hepes pH 7.4, 2mM MgOAc, 0.1% NP40, 5mM DTT, PMSF, and a complete protease inhibitor cocktail (Roche). FLAG-tagged proteins were immunoprecipitated overnight at 4°C using anti-FLAG M2 agarose beads (Sigma). Beads were washed six times in Buffer A+KOAc, and bound proteins were separated by SDS-PAGE and immunoblotted with monoclonal anti-FLAG M2 antibody diluted 1:2,500 (Sigma #F3165) and HRP-conjugated anti-HA antibody diluted 1 :2,000 (Roche #12013819001).
[00132] For RNA immunoprecipitation, 1.2xl07 DL1 cells were seeded into 10 cm plates in serum- free media with 12 μg dsRNA. One hour later, complete media was added and cells were incubated for 5 days. Endogenous Agol was immunoprecipitated. Cells were lysed in lysis buffer: 20mM HEPES pH 7.0, 150mM NaCl, 2.5mM MgCl2, 0.3% Triton-X, 30% glycerol, PMSF, and a complete protease inhibitor cocktail (Roche). Pre-cleared lysates were incubated with rabbit polyclonal AGOl antibody (1 :20; Abeam #ab5070) or control rabbit polyclonal GFP antibody (1 :20; Invitrogen #A-6455) overnight at 4°C. AGOl and control antibodies were isolated using protein A/G beads (1 : 10; Pierce #20421) for 1 hour at 4°C. Beads were then washed 6 times, 10 minutes each in wash buffer: 30mM HEPES pH 7.4, 800mM NaCl, 2mM MgCl2, 0.1% NP-40, PMSF and a complete protease inhibitor cocktail (Roche). 1 mL Trizol (Invitrogen) was added to beads following the final wash. RNA was extracted and analyzed by small RNA northern blotting. Small RNA deep sequence analysis
[00133] To make small RNA sequencing libraries, total RNA was extracted using Trizol Reagent (Invitrogen) from -3d old nbr mutants and control 5905 flies (1 : 1 ratio between males and females). 40ug RNA was fractioned in a 15% TBE-Urea gel (Novex, Invitrogen), followed by gel-purification of small RNA ranging between 18nt and 30 nt. The library was then prepared following Small RNA vl.5 Sample Preparation Guide (Illumina) with some modifications. To perform sequence analysis, adaptor sequences (5' adapter- 5'-GTT CAGA GTT CTA CAG TCC GAC GAT C-3' ; 3' adapter 5'-ATC TCG TAT GCC GTC TTC TGC TTG AA-3') were removed from the raw reads in the Illumina fastQ generated files using the FASTQ/A Clipper program in the fastx-toolkit (http://hannonlab.cshl.edu/fastx_toolkit/). Reads less than 16 bp or more than 30 bp were discarded. Remaining reads were then mapped to the Drosophila genome (Flybase v5.34), and to the microRNA stemloop sequences (BDGP5.0, http://www.mirbase.org/) using Bowtie. Mapped files generated from Bowtie were formatted and analyzed by customized Perl scripts. To enrich miRNAs affected by nbr function, the length distribution for all reads corresponding to individual miRNAs was analyzed. To be included in the analysis the miRNA had to have more than 70 reads. The ratio of the most frequent length to the sum of all other lengths in wild type was calculated, and compared to the ratio of that same most frequent form divided by the sum of all other lengths in nbr (ratio nbrlcontxoX), and ratios plotted (Fig. 4F). miRNAs whose trimming is impacted by nbr presented at either end of the ratio graph. At one end of the graph were miRNAs with exceptionally high ratios of {nbrlcontxoX) and at the other end of the graph were miRNAs with exceptionally low ratios of nbrlcontxoX. The ratio equals [(the number in nbr of most common form in wild type/sum all forms in nbr) divided by (the most common form in wild type/sum of all forms in wild type)]. Thus, the ratio is excessively large or excessively low when the most common length in nbr is either much greater or much lower than the percentage of reads of that length isoform for the miRNA in wild type. In addition, another deep sequencing dataset from Drosophila S2 cells GSM430030 was also used. Reads were mapped to the miRNA stemloop, delineated by read length and sequence, and analyzed.
Transcriptional profiling
[00134] For microarray analysis, DL1 cells were treated with dsRNAs (Renilla control or nbr). Total RNA was extracted from 2.5 million DL1 cells per replicate with Trizol Reagent (Invitrogen). Microarray hybridization and reading was performed at the Penn Microarray Core Facility. For mRNA microarrays, total RNA was reverse-transcribed to ss-cDNA, followed by two PCR cycles using the Ovation RNA amplification system V2 (Ovation). Quality control on both RNA and ss-cDNA was performed using 2100 Agilent Bioanalyzer (Quantum Analytics). The cDNA was labeled using the FL-Ovation™ cDNA Biotin Module V2 (Ovation), hybridized to GeneChip Drosophila Genome 2.0 Arrays (Affymetrix) and scanned with an Axon Instruments 4000B Scanner using GenePix Pro 6.0 image acquisition software (Molecular Devices).
[00135] Five biological replicates of each set of cells, and each genotype of flies were used. Affymetrix .eel (probe intensity) files were exported from GeneChip Operating Software (Affymetrix). The .eel files were imported to ArrayAssist Lite (Agilent) in which GCRMA probeset expression levels and Affymetrix absent/present/marginal flags were calculated. Statistical analysis for those genes passing the flag filter was performed using Partek Genomics Suite v6.6 (Partek). The signal values were log2 transformed and a 2-way ANOVA was performed. DataGraph 2.3.2 was used to generate the scatterplot (http://www.visualdatatools.com/DataGraph/).
Realtime RT-PCR analysis
[00136] Total RNA was prepared from DL-1 cells treated with dsRNAs (Renilla control or Nbr), and flies, control, nbr loqspo791. cDNA was synthesized by High-Capacity cDNA Reverse Transcription kit (Applied Biosystems). The realtime-PCR reaction was performed by Power SYBR Green PCR Master Mix (Applied Biosystems) in 7500 Fast Realtime PCR System (Applied Biosystems). Each target gene was normalized to endogenous control (Rp49), followed by calculation of relative fold change compared to control. 500 Fast System SDS Software (Applied Biosystems) was used for data analyses (ddCt method). Oligos used were CG9247/nbr (5'-GCT GGA ATC GAC GGC TGT AA-375'-AAA AAC TCC TCC GCC TTT GC-3'); mRpS25 (CG14413) (5'-CCAGGTGCTCACGCTGAA-375'-GAA GTA GCA GCG CAC AAA CG-3'); Git (5'-CGC AGG CTA CCT CCG AGT AC-375'-AAG CGG ACT TTG CTG ATT GTT T-3'); CG10232 (5'-GGC TGG GCG AGC ATG A-375'- GCA GCG CAG TTT CCT GTA AAG-3'); CG30359 (5' -ATC CAT CGG CCT GCA ACA- 375'-CGG ATC GCG GGA GTA CTG-3'); CG34051 (5'-TGG ATA CGA TTG GCA CAA ACA-375'-TGC AAC TGT CGC CTT TGG T-3'); GluRIIA (5'-ATT TTC GGA CCA AGT TCA AAG G-375'-CGC GTT CGC GAT TTG C-3'); MESK4 (5'-GAG GAC AAC GGA CGC ATT TC-375'-CGC CGC GGA ATT GTC TT-3'); nub (5'-TCG CCG GAG GAA ACC A-375'-TGC TTG AAG GTC TTG GCA AA); Fas3 (5'-CGC ACC GAG CTG CTC TGT- 375'-GGA ATC TCA ATG CGG CAG TAG-3'); CG3328 (5'- GGTGCTGCCCAATGGAAAC-375'-CCATCAGGATGCGGTCCTT-3'); d (CG42840) (5'- CAT TCA TCC CGC AGT CGA A-375'-CCA AAG GGC AGA GTC TTC ACA-3'); Rp49 (5' -CAA CAT CGG TTA CGG ATC GA-3 5'-AAT CCG GTG GGC AGC AT-3').
Accession numbers :
[00137] The microarray data and deep sequencing datasets can be found in the Gene Expression Omnibus (GEO) of NCBI through accession number GSE32564.
[00138] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method for enhancing the efficacy of microRNA (miRNA) mediated interference, the method comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA-mediated interference.
2. The method of claim 1, wherein said Nbr is encoded by the nucleic acid sequence that comprises exoribonuclease domain.
3. The method of claim 1, wherein said Nbr is encoded by the nucleic acid sequence homologous to the sequence of human Exonuclease 3'-5' domain-containing protein 3 (EXD3).
4. The method of claim 1, wherein said Nbr is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1.
5. The method of claim 1, wherein said Nbr is encoded by a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.
6. The method of claim 1, wherein said Nbr is encoded by a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.
7. The method of claim 1, wherein said Nbr is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.
8. The method of claim 1, wherein said Nbr is encoded by a nucleic acid sequence that is complementary to the nucleic acid sequence set forth in SEQ ID NO: 1.
9. The method of claim 1, wherein said Nbr is encoded by a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence set forth in SEQ ID NO: 1.
10. The method of claim 1, wherein said Nbr is a protein that comprises the amino acid sequence set forth in SEQ ID NO: 2.
11. The method of claim 1, wherein said Nbr is a protein that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 2.
12. The method of claim 1, wherein said Nbr is a protein that comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 2.
13. The method of claim 1, wherein said Nbr is a protein that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2.
14. The method of claim 1, wherein the expression or function of Nbr, said variant, or said homolog thereof is inhibited by contacting said cell with an Nbr antagonist.
15. The method of claim 1, wherein the expression or function of Nbr, said variant, or said homolog thereof is inhibited by antisense inhibition.
16. The method of claim 1, wherein the expression or function of Nbr, said variant, or said homolog thereof is inhibited by knocking out of Nbr.
17. The method of claim 1, wherein the expression or function of Nbr, said variant, or said homolog thereof is inhibited by introducing dominant mutants.
18. The method of claim 1, wherein the step of inhibiting the expression or function of Nbr, said variant, or said homolog thereof comprising enabling Nbr, said variant, or said homolog thereof to interact with Agol.
19. A method for enhancing the efficacy of microRNA (miRNA) mediated interference or expression control, the method comprising: enhancing the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of one or more isoforms of miRNA, thereby enhancing the efficacy of miRNA mediated interference or expression control.
20. The method of claim 19, wherein said Nbr is encoded by the nucleic acid sequence that comprises exoribonuclease domain.
21. The method of claim 19, wherein said Nbr is encoded by the nucleic acid sequence homologous to human Exonuclease 3'-5' domain-containing protein 3 (EXD3).
22. The method of claim 19 wherein said Nbr is encoded by the nucleic acid sequence set forth in SEQ ID NO: 1.
23. The method of claim 19, wherein said Nbr is encoded by a nucleic acid sequence that is at least 70% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.
24. The method of claim 19, wherein said Nbr is encoded by a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.
25. The method of claim 19, wherein said Nbr is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.
26. The method of claim 19, wherein said Nbr is encoded by a nucleic acid sequence that is complementary to the nucleic acid sequence set forth in SEQ ID NO: 1.
27. The method of claim 19, wherein said Nbr is encoded by a nucleic acid sequence that hybridizes under stringent conditions with the nucleic acid sequence set forth in SEQ ID NO: 1.
28. The method of claim 19, wherein said Nbr is a protein that comprises the amino acid sequence set forth in SEQ ID NO: 2.
29. The method of claim 19, wherein said Nbr is a protein that comprises an amino acid sequence that is at least 70% identical to the amino acid sequence set forth in SEQ ID NO: 2.
30. The method of claim 19, wherein said Nbr is a protein that comprises an amino acid sequence that is at least 80% identical to the amino acid sequence set forth in SEQ ID NO: 2.
31. The method of claim 19, wherein said Nbr is a protein that comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 2.
32. The method of claim 19, wherein the expression or function of Nbr, said variant, or said homolog thereof is enhanced by contacting said cell with an Nbr agonist.
33. A method for regulating the production of a microRNA (miRNA) in a cell, the method comprising: regulating the expression or function of Nibbler (Nbr), a variant, or a homolog thereof in said cell, thereby regulating the production of said microRNA in said cell.
34. The method of claim 33, wherein said Nbr is encoded by the nucleic acid sequence that comprises exoribonuclease domain.
35. The method of claim 33, wherein said Nbr is encoded by the nucleic acid sequence homologous to human Exonuclease 3'-5' domain-containing protein 3 (EXD3).
36. The method of claim 33, wherein the step of regulating the expression or function comprising inhibiting the expression or function of Nbr, said variant, or said homolog thereof in said cell.
37. The method of claim 36, wherein the expression or function of Nbr, said variant, or said homolog thereof is inhibited by contacting said cell with an Nbr antagonist.
38. The method of claim 33, wherein the step of regulating the expression or function comprising enhancing the expression or function of Nbr, said variant, or said homolog thereof in said cell.
39. The method of claim 36, wherein the expression or function of Nbr, said variant, or said homolog thereof is enhanced by contacting said cell with an Nbr agonist.
40. A method for enhancing silencing of a target RNA, the method comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby enhancing the efficacy of miRNA mediated interference of said target RNA, and thereby enhancing silencing of said target RNA.
41. A method for identifying an RNAi modulatory compound, comprising, contacting a cell expressing a Nibbler (Nbr) with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
42. A method for identifying an RNAi modulatory compound, comprising, contacting a composition comprising a Nibbler (Nbr) with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
43. A method for identifying an RNAi modulatory compound, comprising, contacting an organism expressing a Nibbler (Nbr) with a test compound, and determining the ability of the test compound to modulate an Nbr activity, such that the RNAi modulatory compound is identified.
44. The method of any of claims 41-43, wherein determining the ability of the test compound to modulate an Nbr activity comprises at least one of the following: detecting Nbr activity, detecting RISC assembly, detecting RISC activation, and miRNA processing for incorporation into a RISC.
45. The method of claim 41, wherein the cell expressing an Nbr is selected from the group consisting of embryonic cells, ovarian cells, Drosophila melanogaster cells, Drosophila melanogaster cell lines, mammalian cells, and mammalian cell lines.
46. The method of claim 42, wherein the composition comprising an Nbr is selected from the group consisting of embryonic cell lysates, ovarian cell lysates, Drosophila melanogaster cell lysates, Drosophila melanogaster cell line lysates, mammalian cell lysates and mammalian cell line lysates.
47. An assay for detecting modulation of RNA interference, comprising, contacting a reaction mixture comprising Nbr with a test compound, and evaluating the effect of the test compound on an indicator of RNA interference, such that modulation of RNA interference is detected.
48. A method for identifying a molecule that regulates Nibbler (Nbr) mediated processing of microRNA in a subject, the method comprising: screening a library of molecules; identifying a molecule that regulates the expression or function of Nbr, a variant, or a homolog thereof, thereby identifying said molecule that regulates Nbr mediated processing of microRNA in said subject.
49. A method for modulating the efficacy of microRNA (miRNA) mediated interference, the method comprising: inhibiting the expression or function of Nibbler (Nbr), a variant, or a homolog thereof to enhance the accumulation of a longer form of miRNA, thereby modulating the efficacy of miRNA-mediated interference.
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