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Definitions

• the Antisense Technology induces translation inhibition of the target mRNA as a consequence of the formation of a long RNA:RNA duplex molecule that, in principle, inhibits mRNA translation.
• This technology seemed initially very promising.
• its efficacy is negatively affected by the fact that long RNA:RNA duplex molecules are often recognized as virus replicative intermediates by the cell, which subsequently activates an antiviral response (known as the Interferon Response) leading to the damage and/or the death of the host cell.
• the authors present a novel gene expression downregulation technology aimed at minimizing or eliminating both the interferon response activation typical of the AS-based methods and the off-targeting limitation of the shRNA/siRNA/miRNAs gene silencing procedures.
• the present technology maintains the positive features of AS-based methods such as the target specificity associated with long AS RNA sequences and the gene expression downregulation efficiency of shRNA/siRNA/miRNAs.
• the authors described a set of noncoding snRNA-like transcription units with regulatory features. Two of them (called 21 A and 29A, respectively) were Alu/7SL-derived transcripts with an extragenic Pol III Type 3 promoter (4).
• the AIu similar secondary structure of 21 A/29 A transcripts exhibit the two canonical loops (called AIu module) of the 7SL-derived molecules known to bind srp9 and srpl4 molecules and responsible for a transient translational blockade in primates (6).
• AIu module the two canonical loops of the 7SL-derived molecules known to bind srp9 and srpl4 molecules and responsible for a transient translational blockade in primates (6).
• this very long (>100 base pairs) target determinant coupled to the stabilization of the ribonucleoparticle by a specific and well tolerated secondary structure complex formation that escapes the antiviral response is referred to as TaD/ AM (Target Determinant/ AIu Module) gene silencing configuration.
• TaD/ AM Target Determinant/ AIu Module
• AD Alzheimer's Disease
• Ib amyloid plaques in the brain
• the most important approaches for its therapy are based on the disaggregation of amyloid plaques by different reagents such as specific antibodies or other specific molecules.
• Other approaches attempt to block amyloid plaques formation (2b).
• the very detailed studies performed until now on Alzheimer's Disease allowed to identify a series of proteins (amyloyd Beta peptide and its precursor, the Presenilins 1 and 2, Alpha and Beta Secretase and others) whose function and/or misfunction is associated with Alzheimer phenotype generation.
• the authors propose in the present invemtion, a novel tool of analysis of AD together with an experimental plan for the identification of an efficacious therapeutic agent.
• the noncoding fraction of the human genome includes a larger than expected number of ncRNA genes controlled by distal sequence element (DSE) and proximal sequence element (PSE) Pol Ill-readable promoters, thus likely to be transcribed by Pol III. They referred to them as co-genes since they could specifically co-act with a protein-coding pol II gene (3b).
• DSE distal sequence element
• PSE proximal sequence element
• GPR51 is specifically expressed in the brain and it is part of the G protein-coupled receptor protein family whose misfunction has been associated to different neurodegenerative disorders (6b).
• 38A maps in antisense configuration in the intron I of KCNIP4 (GC04M020407, 1 Ip 15) in a chromosomal region where alternative splicing events take place.
• KCNIP4 interacts with one of the most studied components of AD pathways, the presenilin 2 protein.
• 45 A lies in AS configuration in the intron I (in a region alternatively spliced) of APBB2 gene (NP 775098.2, 4pl4); whose protein product is associated to late onset AD manifestations.
• the present model suggests that the expression modulation induced by 17A, 38A, 45 A and 51A might be specifically associated with AD and exerts a role in the onset of the pathological phenotype. Therefore 17A, 38A, 45A and 51A represent valuable targets for AD therapeutic agents. Results of specific experiments that demonstrate the action of 17 A, 38 A, 45 A and 51A in AD are reported and discussed below, together with their role as therapeutic molecular targets.
• TaD/ AM a novel gene expression regulatory tool with advantageous features. They refer to this novel molecular configuration as TaD/ AM being composed by a long Target Determinant aimed to specifically silence the target gene expression fused with an AIu Module that confers a high stability to the whole silencer and that participates to the silencing mechanism by increasing significantly its efficiency.
• TaD/AM shows several advantages: 1) This method allows the specific silencing of the desired target gene while minimizing the possibility of an undesired off-target phenomenon. This advantage is independent from the length of the Target Determinant of the sequence. 2) TaD/AM does not activate the antiviral response that is almost always triggered by the canonical antisense molecules.
• TaD/AM allows the use of a Target Determinant sequence of any length (virtually between 15 and hundreds of basepairs). This peculiar characteristic is most likely the consequence of an AIu Module-driven specific ribonucleoparticle assembly.
• TaD/AM configuration can be used both in vitro (by transfection/infection of in vitro cultured cells) and in vivo (by infecting mice with viral vectors harboring TaD/AM silencers or by the generation of TaD/AM-transgenic mice).
• TaD/AM silencing procedure does not require a specific equipment and is of easy use with common instruments.
• TaD/AM also comprises a pol III type 3 promoter selected from the group of:
• the model proposed implies that the regulation of the alternative splicing events has to be ascribed to the specific expression modulation of four correspondent Pol III small RNAs whose transcription regulation is under the control of Pol Ill-specific promoters.
• nucleic acid molecule comprising sequentially: a) a 7SL small-RNA derived sequence comprising at least the binding domain to srp9 and srpl4 proteins of the 7SL ribonucleocomplex b) a sequence identical or complementary to a target sequence; c) a pol III type 3 promoter.
• the 7SL small-RNA derived sequence is an Alu-derived sequence. More preferably, the Alu-derived sequence is a 29A derived sequence. Preferably, the Alu- derived sequence is a 21 A derived sequence.
• the sequence identical or complementary to a target sequence is of a length of at least 15 nucleotides. More preferably, it is of a length of at least 50 nucleotides.
• the pol III type 3 promoter has a sequence comprised in the group of SEQ ID No.l to SEQ ID No.34.
• an expression vector comprising the nucleic acid molecule as described above.
• a further object of the invention is a host cell transformed with the expression vector as described above.
• Another object of the invention is a non human transgenic animal bearing the nucleic acid molecule as described above.
• nucleic acid molecule as described above, or of the vector as described above or of the host cell as described above to modulate the expression of the target sequence in vivo or in vitro.
• the target sequence is involved in a pathological state.
• a nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for the diagnosis of an age-related pathology.
• the age-related pathology is a neurodegenerative disease. More preferably, the neurodegenerative disease is Alzheimer's disease.
• nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71 for medical use.
• Another object of the invention is a molecule able to be vehiculated into the CNS and to bind to the promoter activating region of a nucleic acid molecule comprised in at least one of the following sequence: Seq ID No. 68, Seq ID No. 69, Seq ID No. 70 and Seq ID No. 71.
• the use of the transcribed region of the nucleic acid molecule comprised in at least one of the following sequence: Seq ID No.
• the age-related pathology is a neurodegenerative disease. More preferably, the neurodegenerative disease is Alzheimer's disease.
• FIG.l Promoter activity transfection assay.
• a specific luciferase-silencing hairpin is transcribed by four PSE/DSE-dependent promoters (21A, U6, Hl, 29A).
• a view of the silencing constructs enclosing the hairpin sequence is enclosed.
• the promoter region encompasses the putative pol III Type 3 regulatory regions (PSE, DSE and TATA).
• pMock is a negative control being a luciferase hairpin construct lacking the entire PSE/DSE- dependent promoter region, thus resulting transcriptionally inactive.
• FIG. 2 TaD/AM Structure.
• FIG.3 GFP-specific Antisense TaD/AM transfection in HeLa cells.
• A Construct structures.
• B Representative fluorescence microscope fields showing the silencing effects.
• C Graphical quantitative determination of GFP silencing by different constructs.
• FIG. 4 GFP-specific Sense TaD/AM transfection in HeLa cells.
• FIG. 6 Luciferase gene expression silencing by TaD/AM in HeLA cells. Quantitative determination of luminescence emission after constructs transfection.
• FIG. 7 Nucleic acid sequence phylogenetic tree of 2 Kbp of the whole collection of promoters. The Alzheimer's disease associated promoters are indicated by the arrows.
• FIG. 8 KCNIP4 N-terminal fragment variants. In bold underlined is indicated the predicted signal peptide with IEA-GLED as cleavage site indicated by the arrow.
• VARIANT-I Ace. N°: NM025221 (gene 1-2361; CDS 121-873); VARIANT-II Ace.
• EF-hand calcium binding motif
• a diverse superfamily of calcium sensors and calcium signal modulators most examples in this alignment model have 2 active canonical EF hands.
• Ca2+ binding induces a conformational change in the EF-hand motif, leading to the activation or inactivation of target proteins.
• Epsl5 homology domain found in proteins implicated in endocytosis, vesicle transport, and signal transduction.
• the alignment contains a pair of EF-hand motifs, typically one of them is canonical and binds to Ca2+, while the other may not bind to Ca2+.
• FIG. 9 Promoter activity transfection assay.
• a specific luciferase-silencing hairpin is transcribed by three PSE/DSE-dependent promoter elements (pMock, pU6-ffl, p38A-ffl).
• the promoter region encompasses the putative pol III type 3 regulatory regions (TATA box, PSE, and DSE).
• pU6-ffl is the canonical pol III promoter used as positive control; pMock contains no promoter thus resulting transcriptionally inactive.
• FIG. 10 Graphical representation of 38A expression in AD patients and controls. mRNA product of the housekeeping gene GAPDH (Glyceraldehyde 3 Phosphate Dehydrogenase) was measured and subsequently all the 38A expression values were normalized to their GAPDH counterparts.
• FIG. 11 A) Dissociation curves. The merge of several analysis of the amplification product dissociation curves evidences that the two KCNIP4 splice variants [here referred to as Variant I (I) and Variant IV (IV)] are unambiguously distinguishable by two picks at different dissociation temperatures.
• the splice variant I is characterized by a pick at 83.3 + 0.5 0 C while the alternative splice variant IV is dissociated at 80.8 0 C in the same conditions.
• FIG. 12 In vitro transcription analysis of 17A and 38A.
• a In vitro transcription of 17A (lanes 2, 7) or empty vector (lanes 1, 4).
• Transcription products were either radiolabeled during synthesis, gel- fractionated and directly visualized (lanes 1, 2) or subjected to primer extension analysis (lanes 4-7; lane 5, no DNA during in vitro transcription; lane 6, no reverse transcriptase during primer extension). Radiolabeled RNA size markers were loaded on lane 3. The most abundant 17A-specif ⁇ c primer extension product is indicated by a red arrowhead. Shown in lanes 8-11 are the results of sequencing reactions primed with the same oligonucleotide utilized for primer extension, b: The in vitro transcription experiment was identical to the one reported in panel a, except that the specific template was the 38A transcription unit plus regulatory regions, and primer extension analysis was conducted using a 38 A- specific primer.
• RNA size markers run in parallel are indicated on the left, c:
• the in vitro transcription experiment was identical to the one reported in panel A, except that the specific templates were the human 7SK gene (lanes 1 and 10), promoter- less 7SK (lanes 3 and 9), the 7SK transcribed region fused with the 38A promoter region (lanes 2 and 8).
• panels a-c are the sequences of 17A, 38A and hybrid 38A-7SK transcription units, respectively.
• the PSE, TATA and terminator sequences are in red, the bases corresponding to the transcription start sites identified by primer extension are in blue, the transcribed regions are underlined.
• the 38A-derived upstream sequence is in italic character.
• a,b Real Time RT-PCR detection of KCNIP4 and GPR51 splice variants synthesis in SH-SY5Y (full bars) and SKNBE (striped bars) cell lines transiently transfected with either 17A or 38A expression plasmids.
• FIG. 14 ncRNA expression and alternative splicing in AD cases, a: 17A and 38A expression in AD cases (orange columns) and non-AD control individuals (green columns) as determined by Real-Time RT-PCR.
• AD cases 124 and 1024 have a familiar origin.
• the amplification product dissociation curves unambiguously distinguish the RNAs of interest by peaks at specific temperatures (insets). The averaged results are also reported.
• Asterisks indicate the concomitant highest expression of 17A and 38A in the same individuals keeping in line with a common deregulation of the members of the AP-cluster.
• FIG. 15 Expression analysis of 38A a sequence variation in three different cell lines.
• FIG. 16 ncRNA-induced perturbation of A ⁇ secretion a: Increased amyloid ⁇ secretion and perturbation of A ⁇ 42/A ⁇ 40 ratio in 17A/38A-overexpressing SHSY5Y cells.
• X axis transfected plasmids.
• Y axis quantitative determination of A ⁇ (pg/ml) secreted in the medium 48 hours after transfection as determined by sandwich ELISA (results were normalized to the pMock-transfected cell line).
• FIG. 17 Schematic view of GPR51 (G protein-coupled receptor 51) locus.
• Variant I is referred to as Variant a in AceView,http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly and Variant II represents Variant b as referred to Aceview).
• the alternatively spliced Variant II sequence is homologous to Pan troglodytes GPR51 (XP 520146.2) except for the additional 20 residues long predicted sequence stretch (underlined) most likely constituting a misread protein product. Both the variants encompass a predicted ANF receptor domain (receptor family ligand binding domain).
• Variant I contains a 7tm 3 (7 transmembrane receptor, metabotropic glutamate domain) missed in Variant II.
• GPR51 was reminescent of a possible involvement in AD generation due to the fact that: i) Alterations in signal transduction pathway of G-protein-coupled receptors (GPCRs) were found in the cerebrocortex and in the peripheral cultured tissues of AD patients (Leosco D. et AL, 2007) and ii) the membrane G protein-coupled receptors Kinases were significantly reduced in brain cortices of an early-onset AD transgenic model (Suo Z. et al., 2004).
• FIG. 18 Schematic view of KCNIP4 locus.
• Variant IV variant b in AceView
• Variant I Variant a in Aceview
• 38A maps in intron I of KCNIP4 gene (GC04M020407, I lpl5.31) in a region where alternative splicing events give rise to the distinct I and IV protein variants.
• KCNIP4 regulates the electrophysiological properties of Kv Kb channel with a specific role of the splice variant IV in determining an altered kinetics of potassium channels (Holmqvist, M. H et al., 2002).
• KCNIP4 is a Presenilin 1 and 2 protein interactor (Morohashi H. et al., 2002).
• FIG. 19 Cerebral cortex samples.
• FIG. 20 Speculative model of the AP cluster contribution to Alzheimer's disease.
• EXAMPLE 1 TARGET DETERMINANT/ALU MODULE (TAD/AM) GENE EXPRESSION REGULATORY CONFIGURATION Materials and Methods
• HeIa cells grown in DMEM supplemented with 10% FCS
• the expression constructs containing the regions of interest cloned in the pTopo vectors were introduced into the cells using the Fugene 6 transfection reagent (Roche) according to the manufacturer's instructions.
• a plasmid Expressing Luciferase (pGL3) and one expressing Renilla (pRL) were used as control of transfection efficiency (to which all the results were normalized).
• RNAi-silencing assay 24, 48 and 72 hours after transfection cells were harvested and both firefly luciferase and Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to manufacturer's protocol. RNAi-silencing assay
• the hairpin sequence [targeting a firefly luciferase mRNA from a co-transfected expression plasmid (Promega)] is:5'GGAUUCCAUUCAGCGGAGCCACCUGAUGAAGCUUGAUCGGGUCUCGCUG AGUUGGAAUCCAUU-3' (SEQ ID No. 35).
• Oligos used to subclone the novel Pol III Type 3 promoters within Not I/HinD III restriction sites were the following: HlFprom Not I: 5 '-ATGCGCGGCCGCATTTGCATGTCGCTATGTG-S ' (SEQ ID NO. 36)
• Firefly (ffl) Luciferase as target to be silenced and with a pRL plasmid (Promega) expressing a Renilla Luciferase to which all the determinations were normalized. 24, 48 and 72 hours after transfection cells were harvested and firefly/Renilla luciferase activities were measured by Dual-Luciferase reporter assay system (Promega) according to the manufacturer's protocol.
• the original plasmid constructs p21A and p29A were generated amplifying from a genomic DNA preparation the regions of interest; the PCR products were then subcloned into the pNEB193 vector.
• the oligos used to generate p21A and p29A PCR fragments were the fo Ho wing :
• the TaD/ AM constructs were generated by ligating together in the pSHAG vector the three elements of their construction (the 29A and or U6 promoter, the GFP sense Target Determinant, the GFP antisense Target Determinant, The Luciferase sense Target
• the authors fused the two promoters of the Alu/7SL-derived transcription units (2 IA and 29A) with an hairpin able to down-regulate the expression of a co-transfected Luciferase plasmid (pGL3) by a mechanism of RNA interference.
• pGL3 co-transfected Luciferase plasmid
• luciferase hairpin construct lacking the entire promoter region was prepared (here referred to as pMock).
• pMock As positive controls two similar constructs were prepared substituting the 21 A and/or 29 A promoters with that of two well known Pol III Type 3 transcription unit, Hl and U6 respectively.
• the luciferase emission was 0.5 + 0.1 for the 21A-driven construct, 0.1 + 0.005 for the U6-driven sample, 0.2 + 0.01 for the Hl-driven construct and 0.1 + 0.02 for the 29A-driven construct (Fig. IA).
• a AIu module expected to bind srp9 and srp 14 proteins (Fig. 2). All the constructs were then sequenced in order to assess the correct cloning procedures.
• the authors analyzed the samples by a fluorescence microscope and counted several microscope fields for GFP positive cells evidencing that: a) the promoter without TaD/ AM shows a rate of positive cells (1.1 + 0.09) comparable to the unsilenced sample transfected with the GFP 47 plasmid.
• results showed that: a) As previously demonstrated, the promoter alone does not induce variation in the number of GFP positive cells (1.1 + 0.2) when compared to the unsilenced sample; b) The construct enclosing the Target Determinant in Sense configuration butr lacking the AIu module inhibits with low efficiency the GFP expression (0.86 + 0.2); c) Strikingly, the GFP-sense TaD/ AM construct is the most efficient configuration for gene expression silencing (0.25 + 0.03). This could be explained by a conjugating and unexpected silencer effect of the sense configuration with the stability conferred by the AIu Module (Fig. 4). The constructs did not induce any other changes that could be associated with a possible antiviral response.
• the authors prepared a Luciferase-specif ⁇ c Sense TAD/AM construct.
• a 270 bp long luciferase fragment was subcloned in the GFP TaD/AM construct substituting the GFP sense sequence moiety.
• the construct was co-transfected with a plasmid expressing luciferase (pGL3) and a plasmid expressing Renilla luminescent protein (pRL) in HeLa cell.
• pRL Renilla luminescent protein
• HeIa cells grown in DMEM supplemented with 10% FCS were grown in multiwell Petri dishes 16 hours before transfection.
• a plasmid Expressing Luciferase (pGL3) and one expressing Renilla (pRL) were used as control of transfection efficiency (to which all the results were normalized).
• the hairpin sequence [targeting a firefly luciferase mRNA from a co-transfected expression plasmid (Promega)] is: 5 ' GGAUUCCAUUCAGCGGAGCCACCUGAUGAAGCUUGAUCGGGUCUCGCUGA GUUGGAAUCCAUU-3' (SEQ ID No. 58).
• Oligos used to subclone the novel Pol III Type III promoters within Not I/HinD III restriction sites were the following: 38AFprom Not I: 5 '-ATGCGCGGCCGCTTCACTAAGATCCAGTGC-S ' (SEQ ID NO. 59) 38ARprom HinDIII:5 '-GATCAAGCTTCATCAGGTGGCTCCCGCTGAATTGGAAT CCGATTCATGAAC ACAGAAT ATT-3' (SEQ ID No. 60).
• the original plasmid construct p38A was generated amplifying from a genomic DNA preparation the regions of interest; the PCR products were then subcloned into the pNEB193 vector.
• the oligos used to generate p38A PCR fragments were the following: 38A Forward: 5 '-AGCAATAGCAATCAGACCAG-S ' (SEQ ID NO. 61); 38A Reverse: 5 '-GTCCTGTTGGTACCCTTGT-S ' (SEQ ID NO. 62).
• the insert obtained was then subcloned in pTOPO vector (Invitrogen) following manufacturer's instructions. Prior to transfection all the plasmids were sequenced by DNA Sequencing Kit (Applied Biosystems) following manufacturer's instructions.
• RNA preparations of 8 post-mortem brain biopsis derived from Alzheimer's disease patients and 8 derived from healthy donors (no correlation with AD) were subjected to reverse transcription by Superscript II First Strand Synthesis Kit (Invitrogen) following manufacturer's instructions.
• the cDNA obtained was measured by real-time quantitative RT-PCR using SYBR GREEN gene expression quantization, on PE ABI PRISM@ 7700 Sequence Detection System (Perkin Elmer).
• the sequences of forward and reverse primers as designed by the Primer Express 1.5 software were KCNIP4 Sybr For (varl) 5'-ATGAAGCTCTTGCCCTGCTC-S', (SEQ ID NO.
• Threshold cycle CT, which correlates inversely with the target mRNA levels, was measured as the cycle number at which the reporter fluorescent emission increases above a threshold level.
• Glyceraldehyde 3 phosphate dehydrogenase (GAPDH) gene was examined by quantitative RT-PCR as described above.
• the sequences for human GAPDH primers were 5'-GAAGGTGAAGGTCGGAGTC-S' (SEQ ID NO. 66), 5'- GAAGATGGTGATGGGATTTC-3' (SEQ ID No. 67).
• Relative transcript levels were determined from the relative standard curve constructed from stock cDNA dilutions, and divided by the target quantity of the calibrator following manufacturer's instructions. Results Co-evolution of the four AD-associated Pol III promoter elements.
• Table 1 Sequences of 2000 bp of 17A, 38A, 45A, 51A nucleic acid promoter sequences; the PSE elements are in bold, the TATA boxes are underlined. Promoters are followed by the transcript sequences indicated in italics. Transcript sequences are followed by 600 bp of downstream regions. 17A promoter (2000bp) Seq ID No. 68
• a splice variant of KCNIP 4 gene contains a potential signal peptide region
• 38A maps in the first intron of its putative gene target (KCNIP4, Uniprot/SWISSPROT Acc:Q6PIL6; from human gene ENSGOOOOO 185774, mapping at 4pl5.31) in antisense configuration and being, once expressed, most likely able to modulate its splicing mechanism
• KCNIP4 putative gene target
• KNIP4 does not contain a KDEL sequence motif in its C-terminal moiety (that would redirect the protein into the endoplasmic reticulum)
• the authors fused its promoter to a luciferase-specific silencer hairpin; 48 hours after co-transfection of sk-NBE neuron-like cells with this construct together with a plasmid expressing luciferase (pGL3), the authors detected a significant decrease in luciferase activity (0.27 fold of the negative control emission, a DNA construct harboring the same hairpin without promoter, here referred to as pMock) thus demonstrating an efficient hairpin transcription driven by 38A Pol III promoter.
• pMock plasmid expressing luciferase
• KCNIP4 Variant IV is specifically expressed in Alzheimer's Disease patients.
• EXAMPLE 3 RNA POLYMERASE III TRANSCRIPTS CONTRIBUTE TO ALZHEIMER'S DISEASE Material and methods
• Transcription reactions were carried out in a final volume of 25 ⁇ l in the presence of 2 ⁇ g of template DNA and HeLa cell nuclear extract (100 ⁇ g) supplemented with 50 ng of recombinant human TBP.
• the standard transcription mix contained: 5 mM creatine phosphate, 70 mM KCl , 5 mM MgCl 2 , 20 mM Tris/HCl pH 8, 1 mM DTT, 2 ⁇ g/ml ⁇ - amanitin, 0.5 mM CTP,ATP,GTP, 25 ⁇ M/ 10 ⁇ Ci UTP /[ ⁇ - 32 P]UTP, SUPERase IN (Ambion, 10 U), glycerol 10 % (v/v).
• a double scale transcription reaction was performed as described, without including radiolabeled UTP.
• the purified transcripts were resuspended in a final volume of 12 ⁇ l in the presence of 0.5 mM dNTPs and 1 pmole of specific 5 '-end-radio labeled probe.
• the mixture was heated at at 65 0 C for 5 min and a mixture providing 50 mM Tris/HCl pH 8, 75 mM KCl, 3 mM MgCl 2 ,5 mM DTT, SUPERase IN (Ambion, 10 U) and 200 U Superscript III reverse transcriptase (Invitrogen) was added to a final volume of 20 ⁇ l.
• the reactions were incubated for Ih at 60 0 C and subsequently for 15 min at 70 0 C to inactivate the enzyme.
• the products were precipitated with ammonium acetate and gel- fractionated.
• sequences of 17A forward and reverse primers were 5'-CCACCCTGCAACTGACACAT-S' (Seq ID No. 76) and 5'- GCAAAGGTGCTAATCTTGACTCTTG-3' (Seq ID No. 77).
• sequences of 38A forward and reverse primers were 5'-CTATCAAAATTTCAAGGATATGCATCA-S' (Seq ID No. 78) and 5'-GATGCCTCAAGCTTTGTTTTGC-S' (Seq ID No. 79).
• sequences of GPR51 (Vl) forward and reverse primers were 5'-TCCGTCACATCCATCATTGC-S' (Seq ID No.
• KCNIP4 (V4) forward and reverse primers were 5'-TGGAACAGTTTGGGCTGATTG-S' (Seq ID No. 86) and 5'-CGGTGGCCATCTCCAGTT-S' (Seq ID No. 87).
• G3PDH Glyceraldehyde 3 phosphate dehydrogenase
• human G3PDH primers were 5'- GAAGGTGAAGGTCGGAGTC-3' (Seq ID No. 88) and 5'- GAAGATGGTGATGGGATTTC-3'(Seq ID No. 89) .
• the sequences for human 5s rRNA primers were 5'-TACGGCCATACCACCCTGAA-S' (Seq ID No. 90) and 5'- GCGGTCTCCCATCCAAGTAC-3' (Seq ID No. 91).
• the sequences for human 7SK RNA primers were 5'-AGGACCGGTCTTCGGTCAA-S' (Seq ID No. 92) and 5'- TCATTTGGATGTGTCTGCAGTCT-3' (Seq ID No. 93).
• the sequences for human c- Myc primers were 5'-CGTCTCCACACATCAGCATAA-S' (Seq ID No.
• AD Alzheimer's Disease
• a ⁇ beta- amyloid
• RNAs originate from transcription units mapping in the intronic regions of protein-coding genes of brain- specific proteins: Potassium Channel Interacting Protein, KCNIP4 5c 7c ; G Protein Coupled Receptor 51, GPR51 8c l lc ; Sortilin-Related Receptor 1, SORLl 12c l4c and Amyloid Peptide Beta Binding 2, APBB2 15c ' 16c .
• KCNIP4 5c 7c G Protein Coupled Receptor 51, GPR51 8c l lc
• Sortilin-Related Receptor 1 SORLl 12c l4c
• Amyloid Peptide Beta Binding 2 APBB2 15c ' 16c .
• the authors here show that two of these Pol III transcripts used as experimental models drive AD-specific alternative splicing events leading to the synthesis of distinct protein isoforms and influence A ⁇ secretion.
• RNA Polymerase RNA Polymerase Ill-dependent non-coding RNAs
• ncRNAs RNA Polymerase Ill-dependent non-coding RNAs
• 17A SEQ. ID No. 68
• 38A SEQ. ID No. 69
• 45A SEQ. ID No. 70
• 51A SEQ. ID No. 71
• Pol III transcript-driven alternative splicing was also evidenced at protein level by immunofluorescence microscopy.
• a polyclonal antibody raised against GPR51 N-terminal epitopes the authors observed a well-marked immuno fluorescent signal in pMock-transfected SHSY5Y cells (Fig. 13c); in contrast, a strongly decreased signal was detected in cells overexpressing 17A where the alternatively spliced GPR51 protein form (lacking the long N-terminal portion) is synthesized and not recognized by the same IgGs (FIG. 13d).
• the same experiment was performed challenging 38A-transfected cells with a KCNIP4-specific antibody raised against the N-terminus of the protein.
• AD Alzheimer's disease
• the authors postulated a possible active role of the ncRNA cluster in this neurodegenerative disorder.
• the authors measured by quantitative Real Time RT-PCR 17A and 38A transcript amounts in post-mortem cerebral cortex samples obtained from 18 AD patients and from 10 non-Alzheimer control individuals (Fig. 19).
• AD cases exhibit a significantly increased expression level of 17A and 38A (13.3-fold and 10.5-fold on average, respectively) with respect to the non-AD control individuals suggesting the existence of a correlation between the expression of these ncRNAs and the AD phenotype (Fig. 14a).
• the authors investigated the alternatively spliced forms of their corresponding mRNAs.
• the authors measured in cortical extracts from AD and non-AD control individuals the relative amount of: 1) GPR51 splice variants 1 and 2 (the latter lacking a long N-terminal portion) 210 and 2) KCNIP4 splice variants I and IV (the latter harboring a predicted N-terminal signal peptide) 220 .
• Table II 38A AD-associated genetic variations, 38A promoter sequence analysis of AD cases.
• the three 38A novel alleles are indicated with ⁇ , ⁇ and ⁇ , and compared to the wild type promoter sequence.
• the functional promoter elements (DSE, PSE and TATA box) are indicated in italics.
• the base substitutions are indicated in red and bold whereas the point deletions are indicated by a star.
• the underscored sequence corresponds to the transcribed region.
• AAAATTATCTCT,4 TTTGCA GATGATATAATCCCTTT - ⁇ * G * G GAAAACCCTAAAGATTCCACAA AAAACTGACAGAATGAATTAATTCAGTAAACTTGCAGGATACAAAATCAACATACAAAAATCA GTAGCATTTTTATACACTAATAACAACATATCTGAAAAAGACGCTTTAAAATCCCATTTATGAA AGCATAAAAATAGTTAGAAATAAATTZ ⁇ CCA TAAA GGTGAAA ZATTTGTATACCGATAACZ4Z4 A4CCTTTGATAAAAAAAA ⁇ GTTGAAGAAGACACATATAAATAGAATAATATTCTGTGTTCATGAAT CAAAAAATTTAACAATGTTAAAATGTCTGTATTAACCAAAGCAATATACAAATTCAATGCAATT TCTATCAAAATTTCAAGGATATGCTTCACAGAAATAGAAAAAAAATTCTTGAAATTCATATGGA ACCACAGACACATAAAAACAGAAfAGGCAAAGGAACAATGAAAAAACAATGGAACCACAGACA
• the authors fused the 38 Aa promoter to a luciferase silencer hairpin; 48 hours after co-transfection of this construct with a plasmid expressing luciferase in HeLa cells they detected a very strong decrease of luciferase activity in the samples promoted by the ⁇ genetic variant and a less pronounced luciferase inhibition in the sample transfected with a hairpin construct which expression was promoted by the wild type allele, thus, demonstrating an increased hairpin transcription driven by the novel altered 38A ⁇ Pol III promoters.
• the experiment was repeated in parallele in three different cell lines providing the same result (Fig. 15).
• Pol Ill-specific inhibition was confirmed by a decreased synthesis of Pol Ill-transcribed RNAs (5S rRNA, 7SK RNA, 17A RNA, 38A RNA) and a concomitantly unaffected expression of Pol II-dependent transcripts (cMyc, Glyceraldehyde Phosphate Dehydrogenase) (Fig. 16).
• ncRNAs significantly up-regulates the synthesis of alternatively spliced transcript variants specifically in the brain of AD subjects; ii) causes a perturbation of the subcellular localization and/or abundance of the canonical splice variants; iii) increase the A ⁇ secretion and generate an unbalanced A ⁇ ratio, favoring the production of A ⁇ x-42 species.

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