AU2013202594B2 - MicroRNA molecules - Google Patents

Abstract

F:1IDPERffMPMe.PickCmSchalo407SnIV ,aadw 11700 In Caenorhabditis elegans, lin-4 and let-7 encode 22- and 21 -nucleotide RNAs, respectively, that function as key regulators of developmental timing. Because the appearance of these short RNAs is regulated during development, they are also referred to as "small temporal RNAs" (stRNAs). We show that many more 21- and 22-nt expressed RNAs, termed microRNAs, (miRNAs), exist in invertebrates and vertebrates, and that some of these novel RNAs, similar to let-7 stRNA, are also highly conserved. This suggests that sequence-specific post-transcriptional regulatory mechanisms mediated by small RNAs are more general than previously appreciated.

Description

MicroRNA molecules This application is a divisional of Australian Application No. 2011253686 filed 25 November 2011 (derived from Australian Patent Application No. 2008200680 filed 13 February 2008 which is a divisional of Australian Patent Application No. 2002347035 filed 27 September 2002), the entire contents of which are incorporated herein by reference. Description The -present invention relates to novel -small expressed (micro)RNA molecules associated with physiological regulatory mechanisms, particularly in developmental control. In Caenorhabditis elegans, Iin-4 and let-7 encode 22- and 21-nucleotide 15 RNAs, respectively (1, 2), that function as key regulators of developmental timing (3-5). Because the appearance of these short RNAs is regulated during development, they are also referred to as "microRNAs" (miRNAs) or small temporal RNAs (stRNAs) (6). lin-4 and let-21 are the only known 20 rniRNAs to date. Two distinct pathways exist in animals and plants in which 21- to 23 nucleotide RNAs function as post-transcriptional regulators of gene 25 expression. Small interfering RNAs (siRNAs) act as mediators of sequence specific mRNA degradation in RNA interference (RNAi) (7-11) whereas miRNAs regulate developmental timing by mediating sequence-specific repression of mRNA translation (3-5). siRNAs and miRNAs are excised from dbuble-stranded RNA (dsRNA) precursors by Dicer (12, 13, 29), a 30 multidomain RNase Ill protein, thus producing RNA species of similar size. However, siRNAs are believed to be double-stranded (8, 11, 12), while miRNAs are single-stranded (6). 35 We show that many more short, particularly 21- and 22-nt expressed RNAs, termed microRNAs (miRNAs), exist in invertebrates and vertebrates, and that some of these novel RNAs, similar to let-7 RNA (6), are also highly conserved. This suggests that sequence-specific post-transcriptional -2 regulatory mechanisms mediated by small RNAs are more general than previously appreciated. The present invention relates to an isolated nucleic acid molecule comprising: (a) a nucleotide sequence as shown in Table 1, Table 2, Table 3 or Table 4 (b) 'a nucleotide sequence which is the complement of (a), (c) a nucleotide sequence which has an 1dentity.of at least 80%, preferably of at least 90% and more preferably of at least 99%, to a sequence of (a) or (b) and/or (d) a nucleotide .sequence which hybridizes under stringent conditions to a sequence of (a), (b) and/or (c). In a preferred embodiment the invention relates to miRNA molecules and analogs thereof, to miRNA precursor molecules and to DNA molecules encoding miRNA or miRNA precursor molecules. Preferably the identity of sequence (c) to a sequence of (a) or (b) is at least 90%, more preferably at least 95%. The determination of identity (percent) may be carried out as follows: I= n :L wherein I is the identity in percent, n is the number of identical nucleotides between a given sequence and a comparative sequence as shown in Table 1, Table 2, Table 3 or Table 4 and L is the length of the comparative sequence. It should be noted that the nucleotides A, C, G and U as depicted in Tables 1, 2, 3 and 4 may denote ribonucleotides, -3 deoxyribonucleotides and/or other nucleotide analogs, e.g. synthetic non naturally occurring nucleotide analogs. Further nucleobases may be substituted by corresponding nucleobases capable of forming analogous H bonds to a complementary nucleic acid sequence, e.g. U may .be substituted by T. Further, the invention encompasses nucleotide sequences which hybridize under stringent conditions with the nucleotide sequence as shown in Table 1, Table'2, Table 3 or Table 4, a complementary sequence thereof or a highly identical sequence. Stringent hybridization conditions comprise washing for 1 h in 1 x SSC and 0.1% SOS -at 45"C, preferably at48"C and more preferably at 50"C, particularly for 1 h in 0.2 x SSC and 0.1% SDS. the isolated nucleic acid molecules of the invention preferably have a length of from 18 to 100 nucleotides, and more preferably from 18 to 80 nucleotides. It should be noted that mature miRNAs usually have a length of 19-24 nucleotides, particularly 21, 22 or 23 nucleotides. The miRNAs, however, may be also provided as a precursor which usually has a length of 50-90 nucleotides, particularly 60-80 nucleotides. It should be noted that the precursor may be produced by processing of a primary transcript which may have a length of >100 nucleotides. The nucleic acid molecules may be present in single-stranded or double stranded form. The miRNA as such is usually a single-stranded molecule, while the mi-precursor is usually an at least partially self-complementary molecule capable of forming double-stranded portions, e.g. stem-and loop structures. DNA molecules encoding the miRNA and miRNA precursor molecules. The nucleic acids may be selected from RNA, DNA or nucleic acid analog molecules, such as sugar- or backbone-modified ribonu cleotides or deoxyribonucleotides. It should be noted, however, that other nucleic analogs, such as peptide nucleic acids (PNA) or locked nucleic acids (LNA), are also suitable.

-4 In an embodiment of the invention the nucleic acid molecule is an RNA- or DNA molecule, which contains at least one modified nucleotide analog, i.e. a naturally occurring ribonucleotide or deoxyribonucleotide is substituted by a non-naturally occurring nucleotide. The modified nucleotide analog may be located for example at the 5'-end and/or the 4'-end of the nucleic acid molecule. Preferred nucleotide analogs are selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase modified ribonucleotides, i.e.. ribonucleotides, containing a non-rttura.lly occurring nucleobase instead.of a naturally occurring nucleobase.suolh as uridines or cytidines modified at the 5-position, e.g. 5-(2-amino)propyl uridine, 5-bromo uridine; adenosines and guanosines modified at the 8 position, e.g. 8-bromo guanosine; deaza nucleotides, e.g. 7-deaza adenosine; 0- and N-alkylated nucleotides, e.g. N6-methyl adenosine are suitable.. In preferred sugar-modified ribonucleotides the 2-OH-group is replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is C 1

-C

8 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or . In preferred backbone-modified ribonucleotides the phosphoester group connecting to adjacent ribonucleotides is replaced by a modified group, e.g. of phosphothioate group. It should be noted that the above modifications may be combined. The nucleic acid molecules of the invention may be obtained by chemical synthesis methods or by recombinant methods, e.g. by enzymatic transcription from synthetic DNA-templates or from DNA-plasmids isolated from recombinant organisms. Typically phage RNA-polymerases are used for transcription, such as T7, T3 or SP6 RNA-polymerases. The invention also relates to a recombinant expression vector comprising a recombinant nucleic acid operatively linked to an expression control sequence, wherein expression, i.e. transcription and optionally further processing results in a miRNA-molecule or miRNA precursor molecule as described above. The vector is preferably a DNA-vector, e.g. a viral vector or a plasmid, particularly an expression vector suitable for nucleic acid expression in eukaryotic, more particularly mammalian cells. The recombinant nucleic acid contained in said vector may be a sequence which results in the transcription of the miRNA-molecule as- such, a precursor or a primary transcript thereof, which may be further processed to give the miRNA-molecule. Further, the invention relates to diagnostic- or therapeutic -applications of. the claimed nucleic acid molecules. For example, miRNAs may be detected: in biological samples, e.g. in tissue sections, in order to determine and classify certain cell types or tissue types or miRNA-associated pathogenic disorders which are characterized by differential expression of miRNA molecules or miRNA-molecule patterns. Further, the developmental stage of cells may be classified by determining temporarily expressed miRNA molecules. Further, the claimed nucleic acid molecules are suitable for therapeutic applications. For example, the nucleic acid molecules may be used as modulators or targets of developmental processes or disorders associated with developmental dysfunctions, such as cancer. For example, miR-15 and miR-16 probably function as tumor-suppressors and thus expression or delivery of these RNAs or analogs or precursors thereof to tumor cells may provide therapeutic efficacy, particularly against leukemias, such as B-cell chronic lymphocytic leukemia (B-CLL). Further, miR-10 is a possible regulator of the translation of Hox Genes, particularly Hox 3 and Hox 4 (or Scr and Dfd in Drosophila). In general, the claimed nucleic acid molecules may be used as a modulator of the expression of genes which are at least partially complementary to said nucleic acid. Further, miRNA molecules may act as target for therapeutic screening procedures, e.g. inhibition or activation of miRNA molecules might modulate a cellular differentiation process, e.g. apoptosis. Furthermore, existing miRNA molecules may be used as starting materials for the manufacture of sequence-modified miRNA molecules, in order to modify the target-specificity thereof, e.g. an oncogene, a multidrug resistance gene or another therapeutic target gene. The novel engineered miRNA molecules preferably have an identity of at least 80% to the starting miRNA, e.g. as depicted in Tables 1, 2,. 3 and 4. Further,. mIRNA molecules can be modified, in order that they are symetrically processed and then generated as double-stranded siRNAs which are again directed against therapeutically relevant targets. Furthermore, miRNA molecules may be used for tissue reprogramming procedures, e.g. a differentiated cell line might be transformed by expression of rniRNA molecules into a different cell type or a stem cell. For diagnostic or therapeutic applications, the claimed RNA molecules are preferably provided as a pharmaceutical composition. This pharmaceutical composition comprises as an active agent at least one nucleic acid molecule as described above and optionally a pharmaceutically acceptable carrier. The administration of the pharmaceutical composition may be carried out by known methods, wherein a nucleic acid is introduced into a desired target cell in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation and microinjection and viral methods [30, 31, 32, 33, 34]. A recent addition to this arsenal of techniques for the introduction of DNA into cells is the use of cationic liposomes [351.

7 Commercially available cationic lipid formulations are e.g. Tfx 50 (Promega) or Lipofectamin 2000 (Life Technologies). The composition may be in form of a solution, e.g. an injectable solution, a cream, ointment, tablet, suspension or the like. The composition may be administered in any suitable way, e.g. by injection, by oral, topical, nasal, rectal application etc- The carrier may be any suitable pharmaceutical carrier. Preferably, a carrier is used, which is capable of increasing the efficacy of the RNA molecules to enter the target-cells. Suitable examples 6f such carriers are liposorries, particularly cationic liposomes. Further, the invention relates to a method of identifying njvel microRNA molecules and precursors thereof, in eukaryotes, particularly in vertebrates and more particularly in mammals, such as humans or mice. This method comprises: ligating 5'- and 3'-adapter-molecules to the end of a size fractionated RNA-population, reverse transcribing said adapter-ligated RNA population, and characterizing said reverse transcribed RNA-molecules, e.g. by amplification, concatamerization, cloning and sequencing. A method as described above already has been described in (8), however, for the identification of siRNA molecules. Surprisingly, it was found now that the method is also suitable for identifying the miRNA molecules or precursors thereof as claimed in the present application. Further, it should be noted that as 3'-adaptor for derivatization of the 3' OH group not only 4-hydroxymethylbenzyl but other types of derivatization groups, such as alkyl, alkyl amino, ethylene glycol or 3'-deoxy groups are suitable. Further, the invention shall be explained in more detail by the following Figures and Examples: -8 Figure Legends Fig. 1A. Expression of D. melanogaster miRNAs. Northern blots of total RNA isolated from staged populations of D. melanogaster were probed for the Indicated miRNAs. The position of 76-nt val-tRNA is also indicated on the blots. 55 rRNA serves as loading control. E, embryo; L, larval stage; P, pupae; A, adult; S2, Schneider-2 cells. It should be pointed out, that S2 cells are polyclonal, derived from an unknown subset of embryonic tissues, and may have also lost soms features of their tissue of origin while maintained in culture. miR-3 to miR-6 RNAs were not detectable in S2 cells (data not shown). miR-14 was not.detected by-Northern- blotting and may be very weakly expressed, which is consistent with its cloning frequency. Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes. Fig. 1 B.' Expression of vertebrate miRNAs. Northern blots of total RNA isolated from HeLa cells, mouse kidneys, adult zebrafish, frog ovaries, and S2 cells were probed for the indicated miRNAs, The position of 76-nt val-tRNA is also indicated on the blots. 5S rRNA from the preparations of total RNA from the indicated species is also shown. The gels used for probing of miR-18, miR-1 9a, miR-30, and miR-31 were not run as far as the other gels (see tRNA marker position). miR-32 and miR-33 were not detected by Northern blotting, which is consistent with their low cloning frequency. Oligodeoxynucleotides used as Northern probes were: let-7a, 5' TACTATACAACCTACTACCTCAATTTGCC (SEQ ID NO:1); let-7d, 5' ACTATGCAACCTACTACCTCT (SEQ ID NO:2); let-7e, 5' ACTATACAACCTCCTACCTCA (SEQ ID NO:3); D. melanogasterval-tRNA, 5 ' TGGTGTTTCCGCCCGGGAA (SEQ ID NO:4); miR-1, 5' TGGAATGTAAAGAAGTATGGAG (SEQ ID NO:5); miR-2b, 5' GCTCCTCAAAGCTGGCTGTGATA (SEQ ID NO:6); miR-3, 5' TGAGACACACTTTGCCCAGTGA (SEQ ID NO:7); miR-4, 5' TCAATGGTTGTCTAGCTTTAT (SEQ ID NO:8); miR-5, 5' CATATCACAACGATCGTTCCTTT (SEQ ID NO:9); miR-6, 5' AAAAAGAACAGCCACTGTGATA (SEQ ID NO:10); miR-7, 5' TGGAAGACTAGTGATTTTGTTGT (SEQ ID NO:1 1); miR-8, 5' GACATCTACCTGACAGTATTA (SEQ ID NO:12); miR-9, 5' TCATACAGCTAGATAACCAAAGA (SEQ ID NO:13); miR-10, 5' ACAAATTCGGATCTACAGGGT (SEQ ID NO:14); miR-1 1, 5' GCAAGAACTCAGACTGTGATG (SEQ ID NO:15); miR-12, 5' ACCAGTACCTGATGTAATACTCA (SEQ ID NO:16); miR-13a, 5' ACTCGTCAAAATGGCTGTGATA (SEQ ID NO: 17); miR-1 4, 5' TAGGAGAGAGAAAAAGACTGA (SEQ ID NO: 18); miR-15, 5' TAGCAGCACATAATGGTTTGT (SEQ ID NO:19); miR-1 6, 5' GCCAATATTTACGTGCTGCTA (SEQ ID NO:20); miR-17, 5' TACAAGTGCCTTCACTGCAGTA (SEQ ID NO:2 1); miR-1 8, 5' TATCTGCACTAGATGCACCTTA (SEQ ID NO:22); miRI-1 9a, 5' TCAGTTTTGCATAGATTTGCACA (SEQ ID NO:23); miR-20,-5' TACCTGCACTATAAGCACTTTA (SEQ ID NO:24); miR-21, 5' TCAACATCAGTCTGATAAGCTA (SEQ ID NO:25); miR-22, 5' ACAGTTCTTCAACTGGCAGCTT (SEQ ID NO:26); miR-23, S' GGAAATCCCTGGCAATGTGAT (SEQ ID NO:27); miR-24, 5' CTGTTCCTGCTGAACTGAGCCA (SEQ ID NO:28); miR-25, 5' TCAGACCGAGACAAGTGCAATG (SEQ ID NO:29); miR-26a, 5' AGCCTATCCTGGATTACTTGAA (SEQ ID NO:30); miR-27; 5' AGCGGAACTTAGCCACTGTGAA (SEQ ID NO:3 1); miR-28, 5' CTCAATAGACTGTGAGCTCCTT (SEQ ID NO:32); miR-29, 5' AACCGATTTCAGATGGTGCTAG (SEQ ID NO:33); miR-30, 5' GCTGCAAACATCCGACTGAAAG (SEQ ID NO:34); miR-31, 5' CAGCTATGCCAGCATCTTGCCT (SEQ ID NO:35); miR-32, 5' GCAACTTAGTAATGTGCAATA (SEQ ID NO:36); miR-33, 5' TGCAATGCAACTACAATGCACC (SEQ ID NO:37). Fig. 2. Genomic organization of miRNA gene clusters. The precursor structure is indicated as box and the location of the miRNA within the -10 precursor is shown in gray; the chromosomal location is also indicated to the right. (A) D. melanogaster miRNA gene clusters. (B) Human mIRNA gene clusters. The cluster of let-7a-1 and let-7f-1 is separated by 26500 nt from a copy of let-7d on chromosome 9 and 17. A cluster of let-7a-3 and let-7b, separated by 938 nt on chromosome 22, is 'not illustrated. Fig. 3. Predicted precursor structures of D. melanogaster miRNAs. RNA secondary structure prediction was performed using fold version 3.1 (28] and manually refined to accommodate G/U wobble base pairs in the helical segments. The miRNA sequence is underlined.-The actual size of the stem loop structure is not known experimentally and may be slightly shorter or longer than represented. Multicopy miRNAs and their corresponding precursor structures are also shown. Fig. 4. Predicted precursor structures of human miRNAs. For legend, see Fig. 3. Fig. 5. Expression of novel mouse miRNAs. Northern blot analysis of novel mouse miRNAs. Total RNA from different mouse tissues was blotted and probed with a 5 '-radiolabeled oligodeoxynucleotide complementary to the indicated miRNA. Equal loading of total RNA on the gel was verified by ethidium bromide staining prior to transfer; the band representing tRNAs is shown. The fold-back precursors are indicated with capital L. Mouse brains were dissected into midbrain, mb, cortex, ox, cerebellum, cb. The rest of the brain, rb, was also used. Other tissues were heart, ht, lung, Ig, liver, lv, colon, co, small intestine, si, pancreas, pc, spleen, sp, kidney, kd, skeletal muscle, sm, stomach, st, H, human Hela SS3 cells. Oligodeoxynucleotides used as Northern probes were: miR-1 a, CTCCATACTTCTTTACATTCCA (SEQ ID NO:38); miR-30b, GCTGAGTGTAGGATGTTTACA (SEQ ID NO:39); miR--30a-s, GCTTCCAGTCGAGGATGTTTACA (SEQ ID NO:40); miR-99b, CGCAAGGTCGGTTCTACGGGTG (SEQ ID NO:41); - 11 miR-1 01, TCAGTTATCACAGTACTGTA (SEQ ID NO:42); miR-1 22a, ACAAACACCATTGTCACACTCCA (SEQ ID NO:43); miR-124a, TGGCATTCACCGCGTGCCTTA (SEQ ID NO:44); miR-125a, CACAGGTTAAAGGGTCTCAGGGA (SEQ ID NO:45); miR-1 25b, TCACAAGTTAGGGTCTCAGGGA (SEQ ID NO:46); miR-127, AGCCAAGCTCAGACGGATCCGA (SEQ ID NO:47); miR-1 28, AAAAGAGACCGGTTCACTCTGA (SEQ ID NO:48); mR-1 29, GCAAGCCCAGACCGAAAAAAG (SEQ ID NO:49); rniR-1 30, GCCCTTTTAACATTGCACTC (SEQiD NO-50);iniR-1 31, ACTTTCGGTTATCTAGCTTTA (SEQ ID NO:51); miR-132, ACGACCATGGCTGTAGACTGTTA (SEQ ID NO:52)-; miR-1 43, TGAGCTACAGTGCTTCATCTCA (SEQ ID NO:53). Fig.6. Potential orthologs of lin-4 stRNA. (A) Sequence alignment of C. elegans 'lin-4 stRNA with mouse miR-125a and miR-125b and the D. rnelanogaster miR-125. Differences are highlighted by gray boxes. (B) Northern blot of total RNA isolated from staged populations of D. melanogaster, probed for miR-1 25. E, embryo; L, larval stage; P, pupae; A, adult; S2, Schneider-2 cells. Fig. 7. Predicted precursor structures of miRNAs, sequence accession numbers and homology information. RNA secondary structure prediction was performed using fold version 3.1 and manually refined to accommodate G/U wobble base pairs in the helical segments. Dashes were inserted into the secondary structure presentation when asymmetrically bulged nucleotides had to be accommodated. The excised miRNA sequence is underlined. The actual size of the stem-loop structure is not known experimentally and may be slightly shorter or longer than represented. Multicopy miRNAs and their corresponding precursor structures are also shown. In cases where no mouse precursors were yet deposited in the database, the human orthologs are indicated. miRNAs -12 which correspond to D. melenogaster or human sequences are included. Published C. elegans miRNAs [36, 37] are also included in the table. A recent set of new HeLa cell miRNAs is also indicated [46]. If several ESTs were retrieved for one organism in the database, only those with different precursor sequences are listed. rniRNA homologs-found in-other species are indicated. Chromosomal location and sequence accession -numbers, and clusters of miRNA genes are indicated; Sequences from cloned miRNAs were searched against mouse and human in GenBank (including trace data), and against Fugu rubripes and Dania rerio at www.jgi.doe-.gov and www.sanger.ac.uk, respectively. EXAMPLE 1: MicroRNAs from D. melanogaster and human. We previously developed a directional cloning procedure to isolate siRNAs after processing of long dsRNAs in Drosophila melanogaster embryo lysate (8). Briefly, 5' and 3' adapter molecules were ligated to the ends- of a size-fractionated RNA population, followed by reverse transcription,.PCR amplification, concatamerization, cloning and sequencing. This method, originally intended to isolate siRNAs, led to the simultaneous identification of 14 novel 20- to 23-nt short RNAs which are encoded in the D. melanogaster genome and which are expressed in 0 to 2 h embryos (Table 1). The method was adapted to clone RNAs in a similar size range from HeLa cell total RNA (14), which led to the identification of 19 novel human stRNAs (Table 2), thus providing further evidence for the existence of a large class of small RNAs with potential regulatory roles. According to their small size, we refer to these novel RNAs as microRNAs or miRNAs. The miRNAs are abbreviated as miR-1 to miR-33, and the genes encoding miRNAs are named mir-1 to mir-33. Highly homologous miRNAs are classified by adding a lowercase letter, followed by a dash and a number for designating multiple genomic copies of a mir gene.

-13 The expression and size of the cloned, endogenous short RNAs was also examined by Northern blotting (Fig. 1, Table 1 and 2). Total RNA isolation was performed by acid guanidinium thiocyanate-phenol-chloroform extraction [45]. Northern analysis was performed as described [1], except that the total RNA was resolved on a 15% denaturing polyacrylamide-gel,. transferred onto Hybond-N +membrane -(Amersham Pharmacia Biotech), and the hybridization and -wash steps were performed at 50"C. Oligodeoxynucleotides. used as- Northern probes were 5'-32P phosphorylated, complementary. to the miRNA sequence and 20 to 25 nt-in length. 5S rRNA was detected by ethidium staining of polyacrylamide gels prior to transfer. Blots were stripped by boiling in 0.1% aqueous sodium dodecylsulfate/0. 1x SSC (15 mM sodium chloride, 1.5. mM sodium citrate, pH 7.0) for 10 min, and were re-probed up to 4 times until the 21-nt signals became too weak for detection. Finally, blots were probed for val-tRNA as size marker. For analysis of D. melanogaster RNAs, total RNA was prepared from different developmental stages, as well as cultured Schneider-2 (S2) cells, which originally derive from 20-24 h D. melanogaster embryos [15] (Fig. 1, Table 1). miR-3 to miR-7 are expressed only during embryogenesis and not at later developmental stages. The temporal expression of miR-1, miR-2 and miR-8 to miR-13 was less restricted. These miRNAs were observed at all developmental stages though significant variations in the expression levels were sometimes observed. Interestingly, m!R-1, miR-3 to mi!R-6, and miR-8 to miR-1 1 were completely absent from cultured Schneider-2 (S2) cells, which were originally derived from 20-24 h D. melanogaster embryos [15], while miR-2, miR-7, miR-12, and miR-13 were present in S2 cells, therefore indicating cell type-specific miRNA expression. miR-1, miR-8, and miR-12 expression patterns are similar to those of lin-4 stRNA in C. elegans, as their expression is strongly upregulated in larvae and sustained -14 to adulthood [161. miR-9 and miR-1 1 are present at all stages but are strongly reduced in the adult which may reflect a maternal contribution from germ cells or expression in one sex only. 5 The mir-3 to mir-6 genes are clustered (Fig.. 2A),- and mir-6 is present as triple repeat with slight variations in the mir-6 precursor sequence but not in the miRNA sequence itself: The expression profiles of miR-3 to miR-6 are highly similar (Table 1), which -suggests that a single embryo-specific precursor transcript may give rise to the -different miRNAs, or- that the same enhancer regulates miRNA-specific.promoters. .Several other fly miRNAs are also found in gene clusters (Fig. 2A-). . The expression of HeLa cell miR-1 5 to miR-33 was examined by Northern blotting using HeLa cell total RNA, in addition to total RNA prepared from mouse kidneys, adult zebrafish, Xenopus laevis ovary, and D. melanogaster S2 cells. (Fig. 1B, Table 2). miR-15 and miR-16 are encoded in a gene cluster (Fig. 2B) and are detected in mouse kidney, fish, and very weakly in frog ovary, which may result from miRNA expression in somatic ovary tissue rather than oocytes. mir-17 to mir-20 are also clustered (Fig. 25), and are expressed in HeLa cells and fish, but undetectable in mouse kidney and frog ovary (Fig. 1, Table 2), and therefore represent a likely case of tissue-specific miRNA expression. The majority of vertebrate and invertebrate mIRNAs identified in this study are not related by sequence, but a few exceptions, similar to the highly conserved let-7 RNA [6], do exist. Sequence analysis of the D. melanogaster miRNAs revealed four such examples of sequence conservation between invertebrates and vertebrates. miR-1 homologs are encoded in the genomes of C. elegans, C. briggsae, and humans, and are found in cDNAs from zebrafish, mouse, cow and human. The expression of mir-1 was detected by Northern blotting in total RNA from adult zebrafish and C. elegans, but not in total RNA from HeLa cells or mouse kidney -15 (Table 2 and data not shown). Interestingly, while mir-1 and let-7 are expressed both in adult flies (Fig. IA) [6] and are both undetected in S2 cells, miR-1 is, in contrast to let-7, undetectable in HeLa cells. This represents another case of tissue-specific expression of a miRNA, and a indicates that mIRNAs may not only play a regulatory role in developmental timing, but also in tissue specification. miR-7 homologs were found by database searches in mouse and human genomic and expressed sequence tag sequences (ESTs). Two mammalian miR-7 variants are predicted by sequence analysis in mouse and human, and were detected by Northern. blotting in HeLa cells and fish, but not in mouse kidney (Table 2). Sirrilarly, we identified mouse and human miR-9 and miR-l0 homologs by database searches but only detected mir-10 expression in mouse kidney. The identification of evolutionary related miRNAs, which have already acquired multiple sequence mutations, was not possible by standard bioinformatic searches. Direct comparison of the D. melanogaster mIRNAs with the human miRNAs identified an 11-nt segment shared between D. melanogaster miR-6 and HeLa miR-27, but no further relationships were detected. One may speculate that most miRNAs only act on a single target and therefore allow for rapid evolution by covariation, and that highly conserved miRNAs act on more than one target sequence, and therefore have a reduced probability for evolutionary drift by cbvariation [6]. An alternative interpretation is that the sets of miRNAs from D. melanogaster and humans are fairly incomplete and that many more miRNAs remain to be discovered, which will provide the missing evolutionary links. lin-4 and let-7 stRNAs were predicted to be excised from longer transcripts that contain approximately 30 base-pair stem-loop structures [1, 61. Database searches for newly identified miRNAs revealed that all miRNAs are flanked by sequences that have the potential to form stable stem-loop structures (Fig. 3 and 4). In many cases, we were able to detect the predicted, approximately 70-nt precursors by Northern blotting (Fig. 1).

-16 Some mIRNA precursor sequences were also identified in mammalian cDNA (EST) databases [27], indicating that primary transcripts longer than 70-nt stem-loop precursors do also exist. We never cloned a 22-nt RNA complementary to any of the newly identified miRNAs, and it is as yet e unknown how the'cellular processing machinery distinguishes between the mIRNA and its complementary strand. Comparative analysis. of the precursor stem-loop structures indicates that the loops adjacent to the base-paired miRNA segment can be located.on either side of the mIRNA sequence (Fig. 3 and 4), suggesting that the 5' or..3' location-of the stem closing loop is not the determinant-of miRNA excision. It is also unlikely that the structure, length or stability of the precursor stem is the critical determinant as the base-paired structures are frequently imperfect and interspersed by less stable, non-Watson-Crick base pairs such as G/A, U/U, C/U, A/A, and G/U wobbles. Therefore, a sequence-specific recognition process is a likely determinant for miRNA excision, perhaps mediated by members of the Argonaute (rde-1/agol/piwi) protein family. Two members of this family, alg-1 and alg-2, have recently been shown to be critical for stRNA processing in C. elegans [13]. Members of the Argonaute protein family are also involved in RNAi and PTGS. In D. melanogaster, these include argonaute2, a component of the siRNA-endonuclease complex (RISC) [173, and its relative aubergine, which is important for silencing of repeat genes [181. In other species, these include rde-1, argonautel, and qde-2, in C. elegans [19], Arabidopsis thaliana [20], and Neurospora crassa [21], respectively. The Argonaute protein family therefore represents, besides the RNase III Dicer [12, 13], another evolutionary link between RNAi and miRNA maturation. Despite advanced genome projects, computer-assisted detection of genes encoding functional RNAs remains problematic [22]. Cloning of expressed, short functional RNAs, similar to EST approaches (RNomics), is a powerful alternative and probably the most efficient method for identification of such novel gene products [23-26]. The number of functional RNAs has been - 17 widely underestimated and is expected to grow rapidly because of the development of new functional RNA cloning methodologies. The challenge for the future is to define the function and the potential targets of these novel miRNAs by-using bloinformatics as-well as genetics, and to establish a complete catalogue of time- and tissue-specific distribution of the already identified and yet to be uncovered mlRNAs. lin-4 and let-7 stRNAs negatively regulate the expression of proteins encoded by mRNAs whose 3' untranslated regions contain sites of complementarity to the stRNA [3-51. Thus, a series of 33 novel genes, coding for 19- to 23-nucleotide microRNAs (miRNAs), has been cloned from fly embryos and human cells. Some of these mIRNAs are highly conserved between vertebrates and invertebrates and are developmentally or tissue-specifically expressed. Two of the characterized human miRNAs may function as tumor suppressors in B-cell chronic lymphocytic leukemia. miRNAs are related to a small class of previously described 21- and 22-nt RNAs (lin-4 and let-7 RNAs), so-called small temporal RNAs (stRNAs), and regulate developmental timing in C. elegans and other species. Similar to stRNAs, miRNAs are presumed to regulate translation of specific target mRNAs by binding to partially complementary sites, which are present in their 3'-untranslated regions. Deregulation of mIRNA expression may be a cause of human disease, and detection of expression of miRNAs may become useful as a diagnostic. Regulated expression of miRNAs in cells or tissue devoid of particular miRNAs may be useful for tissue engineering, and delivery or transgenic expression of miRNAs may be useful for therapeutic intervention. miRNAs may also represent valuable drug targets itself. Finally, miRNAs and their precursor sequences may be engineered to recognize therapeutic valuable targets.

- 18 EXAMPLE 2: miRNAs from mouse. To gain more detailed insights into the distribution and function of miRNAs in mammals, we investigated the tissue-specific distribution of mIRNAs in adult mouse. Cloning of mJRNAs from specific tissues was preferred over whole organism-based. cloning because low-abundance miRNAs that normally go undetected by Northern blot analysis are identified clonally. Also, in situ hybridization techniques for detecting 21-nt RNAs have not yet been developed. Therefore, 19- to 25-nucleotide RNAs were cloned and sequenced from total -RNA, which was isolated.from 18.5-weeks old BL6 mice. Cloning of miRNAs was-performed as follows: 0.2 to .1 mg, of total RNA was separated on a 15% denaturing polyacrylamide gel and RNA of 19- to 25-nt size was recovered. A 5'-phosphorylated 3'-adapter oligonucleotide (5'-pUUUaaccgcgaattccagx: uppercase, RNA; lowercase, DNA; p, phosphate; x, 3'-Amino-Modifier C-7, ChemGenes, Ashland, Ma, USA, Cat. No. NSS-1004; SEQ ID NO:54) and a 5 '-adapter oligonucleotide (5'-acggaattcctcactAAA: uppercase, RNA; lowercase, DNA; SEQ ID NO:55) were ligated to the short RNAs. RT/PCR was performed with 3' primer (5'-GACTAGCTGGAATTCGCGGTTAAA; SEQ ID NO:56) and 5' primer (5'-CAGCCAACGGAATTCCTCACTAAA; SEQ ID NO:57). In order to introduce Ban I restriction sites, a second PCR w~s performed using the primer pair 5'-CAGCCAACAGGCACCGAATTCCTCACTAAA (SEQ ID NO:57) and 5'-GACTAGCTTGGTGCCGAATTCGCGGTTAAA (SEQ ID NO:56), followed by concatamerization after Ban I digestion and T4 DNA ligation. Concatamers of 400 to 600 basepairs were cut out from 1.5% agarose gels and recovered by Biotrap (Schleicher & Schuell) electroelution (1 x TAE buffer) and by ethanol precipitation. Subsequently, the 3' ends of the concatamers were filled in by incubating for 15 min at 720C with Taq polymerase in standard PCR reaction mixture. This solution was diluted 3 fold with water and directly used for ligation into pCR2.1 TOPO vectors. Clones were screened for inserts by PCR and 30 to 50 samples were subjected to sequencing. Because RNA was prepared from combining - 19 tissues of several mice, minor sequence variations that were detected multiple times in multiple clones may reflect polymorphisms rather then RT/PCR mutations. Public database searching was used to Identify the genomic sequences encoding the.approx. 21-nt RNAs. The occurrence of s a 20 to 30 basepair fold-back structure involving the immediate upstream or downstream flanking sequences was used to assign miRNAs [36-381. We examined 9 different mouse tissues and identified 34 novel miRNAs, some of which are highly tissue-specifically expressed (Table 3 and Figure 5). Furthermore, we identified 33 new miRNAs from different mouse tissues and also from human-Soas-2 osteosarcoma-cells'(Table 4). miR-1 was previously shown by Northern analysis to be strongly expressed in adult heart, but not in brain, liver, kidney, lung or colon [37]. Here we show that miR-1 accounts for 45% of all mouse miRNAs found in heart, yet miR-1 was still expressed at a low level in liver and midbrain even though it remained undetectable by Northern analysis. Three copies or polymorphic alleles of miR-1 were found in mice. The conservation of tissue-specific miR-1 expression between mouse and human provides additional evidence for a conserved regulatory role of this miRNA. In liver, variants of miR-1 22 account for 72% of all cloned miRNAs and miR-1 22 was undetected in all other tissues analyzed. In spleen, miR-143 appeared to be most abundant at a frequency of approx. 30%. In colon, miR-1 42-as, was cloned several times and also appeared at a frequency of 30%. In small intestine, too few miRNA sequences were obtained to permit statistical analysis. This was due to strong RNase activity in this tissue, which caused significant breakdown of abundant non-coding RNAs, e.g. rRNA, so that the fraction of miRNA in the cloned sequences was very low. For the same reason, no miRNA sequences were obtained from pancreas. To gain insights in neural tissue miRNA distribution, we analyzed cortex, cerebellum and mid brain. Similar to heart, liver and small intestine, variants -20 of a particular miRNA, miR-124, dominated and accounted for 25 to 48% of all brain miRNAs. miR-101, -127, -128, -131, and -132, also cloned from brain tissues, were further analyzed by Northern blotting and shown to be predominantly brain-specific. Northern blot analysis was performed as described in Example 1.-tRNAs and 5S rRNA were detected by ethidium staining of polyacrylamide gels prior to transfer to verify equal loading. Blots were stripped by boiling in demonized water for 5 min, and reprobed up to 4 times until the 21-nt signals became too weak for detection. miR-125a and miR-125b are very similar to the sequence of C. elegans lin-4 stRNA and may represent its orthologs (Fig. 6A). This is of great interest because, unlike let-7 that was readily detected in other species, lin.-4 has acquired a few mutations in the central region and thus escaped bloinformatic database searches. Using the mouse sequence miR- 1 25b, we could readily identify its ortholog in the D. melanogaster genome. miR-1 25a and miR-1 25b differ only by a central diuridine insertion and a U to C change. miR-125b is very similar to lin-4 stRNA with the differences located only in the central region, which is presumed to be bulged out during target mRNA recognition [411. miR-1 25a and miR-1 25b were cloned from brain tissue, but expression was also detected by Northern analysis in other tissues, consistent with the role for lin-4 in regulating neuronal remodeling by controlling lin-14 expression [43]. Unfortunately, orthologs to C. elegans lin-14 have not been described and miR-125 targets remain to be identified in D. melanogaster or mammals. Finally, miR-1 25b expression is also developmentally regulated and only detectable in pupae and adult but not in embryo or larvae of D. melanogaster (Fig. 66). Sequence comparison of mouse miRNAs with previously described miRNA reveals that miR-99b and miR-99a are similar to D. melanogaster, mouse and human miR-10 as well as C. e/egans miR-51 [36], miR-141 is similar to D. melanogaster miR-8 , miR-29b is similar to C. elegans miR-83 , and miR-131 and miR-142-s are similar to D. melanogaster miR-4 and C.

- 21 elegans miR-79 [36]. miR-124a is conserved between invertebrates and vertebrates. In this respect it should be noted that for almost every miRNA cloned from mouse was also encoded in the human genrome, and frequently detected in other vertebrates, such as the pufferfish, Fuga 5 rubrlpes, and the zebrafish, Danio rerlo. Sequence conservation may point to conservation in function of these miRNAs. Comprehensive information about orthologous sequences is listed in Fig. 7. In two cases both -strands of mIRN-A precursors were cloned (Table 3), which was previously observed once for a C elegans rniRNA [36]. It is thought that the most frequently cloned strand of a mIRNA precursor represents the functional miRNA, which is miR-30c-s and mIR-142-as, s and as indicating the 5' or 3' side of the fold-back structure, respectively. The mir-142 gene is located on chromosome 17, but was also found at the breakpoint junction of a t(8;17) translocation, which causes an aggressive B-cell leukemia due to strong up-regulation of a translocated MYC gene [441. The translocated MYC gene, which was also truncated at the first exon, was located only 4-nt downstream of the 3'-end of the miR-1 42 precursor. This suggests that translocated MYC was under the control of the upstream miR-1 42 promoter. Alignment of mouse and human miR-1 42 containing EST sequences indicate an approximately 20 nt conserved sequence element downstream of the mir-1 42 hairpin. This element was lost in the translocation. It is conceivable that the absence of the conserved downstream sequence element in the putative miR-1 42/mRNA fusion prevented the recognition of the transcript as a miRNA precursor and therefore may have caused accumulation of fusion transcripts and overexpression of MYC. miR-155, which was cloned from colon, is excised from the known noncoding BIC RNA [471. BIC was originally identified as a gene transcriptionally activated by promoter insertion at a common retroviral -22 integration site in B cell lymphomas induced by avian leukosis virus. Comparison of BIC cDNAs from human, mouse and chicken revealed 78% identity over 138 nucleotides [47]. The identity region covers the miR-1 55 fold-back precursor and a few conserved boxes downstream of the fold-back sequence. The relatively high level of expression of -BIC in lymphoid organs and cells in human, mouse and chicken implies an evolutionary conserved function, but BIC RNA has also been detected -at low levels in non-hematopoletic tissues [471. Another interesting observation was that segments of perfect complementarity to miRNAs are not observed in mRNA sequences or in genomic sequences outside the miRNA inverted repeat. Although this could be fortuitous, based on the link between RNAI and miRNA processing [11, 13, 43] it may be speculated that miRNAs retain the potential to cleave perfectly complementary target RNAs. Because translational control without target degradation could provide more flexibility it may be preferred over mRNA degradation. In summary, 63 novel miRNAs were identified from mouse and 4 novel miRNAs were identified from human Soas-2 ostaosarcoma cells (Table 3 and Table 4), which are conserved in human and often also in other non-mammalian vertebrates. A few of these miRNAs appear to be extremely tissue-specific, suggesting a critical role for some miRNAs in tissue-specification and cell lineage decisions. We may have also identified the fruitfly and mammalian ortholog of C. elegans lin-4 stRNA. The establishment of a comprehensive list of miRNA sequences will be instrumental for bioinformatic approaches that make use of completed genomes and the power of phylogenetic comparison in order to identify miRNA-regulated target mRNAs.

-23 References and Notes 1. R. C. Lee, R. L. Feinbaum, V. Ambros, Cell 75, 843 (1993). 2. B. J. Reinhart et al., Nature 403, 901 (2000). 3. V. Ambros, Curr. Opin. Gartet. Dev. 10, 428 (2000). 4. E. G. Moss, Curr. Biol. 10, R436 (2000). 5. F. Slack, G. Ruvkun, Annu. Rev. Genet. 31, 611 (1997). 6. A. E. Pasquinelli et al., Nature 408, 86 (2000). 7. S. M. Elbashir St al., Nature 411, 494 (2001). 8. S. M. Elbashir, W. Lendeckel, T. Tuschl, Genes & Dev. 15,. 188 (2001). 9. A. J. Hamilton, D. C. Baulcombe, Science 286, 950 (1999). 10. S. M. Hammond, E. Bernstein, D. Beach, G. J. Hannon, Nature 404, 293 (2000). 11. P. D. Zamore, T. Tuschi, P. A. Sharp, D. P. Bartel, Cell 101, 25 (2000). 12. G. Hutvhgner, J. McLachlan, t. Bdlint, T. Tuschl, P. D. Zamore, Science 93, 834 (2001). 13. A. Grishok et al., Cell 106, 23 (2001). 14. Cloning of 19- to 24-nt RNAs from D. melanagaster 0-2 h embryo lysate was performed as described (8). For cloning of HeLa mIRNAs, 1 mg of HeLa total RNA was separated on a 15% denaturing polyacrylamide gel and RNA of 19- to 25-nt size was recovered. A 5' phosphorylated 3' adapter oligonucleotide (5' pUUU aaccgogaattccagx: uppercase, RNA; lowercase, DNA; p, phosphate; x, 4-hydroxymethylbenzyl; SEQ ID NO:54) and a 5' adapter aligonucleotide (5' acggaattctcactAAA: uppercase, RNA; lowercase, DNA; SEQ ID NO:55) were ligated to the short HeLa cell RNAs. RT/PCR was performed with 3' primer (5' GACTAGCTGGAATTCGCGGTTAAA; SEQ ID NO:56) and 5' primer (5' CAGCCAACGGAATTCCTCACTAAA; SEQ ID NO:57), and followed by concatamerization after Eco RI digestion and T4 DNA -24 ligation (8). After ligation of concatamers into pCR2.1 TOPO vectors, about 100 clones were selected and subjected to sequencing. 15. 1. Schneider, J Embryol Exp Morphol 27, 353 (1972). 16. R. Feinbaum, V. Ambros, Deir. Biol. 210, 87 (1999). 17. S. M. Hammond, S. Boettcher, A. A. Caudy, R. Kobayashi, G. J. Hannon, Science 293, 1146 (2001). 18. A. A. Aravin et al., Curr. Biol. 11, 1017 (2001).. - 19. H. Tabara et al., Cell 99, 123 (1999). 20. M. Fagard, S. Boutet, J. B. Morel, C. Bellini, H. Vaucheret, Proc. Nati. Acad. Sci. USA 97; 11650 (2000). 21. C. Catalanotto, G. Azzalin, G. Macino, C. Cogoni, Nature 404, 245 (2000).' 22. S. R. Eddy, Curr. Opin. Genet. Dev. 9, 695 (1999). 23. J. Cavaille at al., Proc. Nati. Acad. Sci. USA 97, 14311 (2000). 24. A; Hfttenhofer et al., EMBO J. 20, 2943 (2001). 25. L. Argaman et al., Curr. Biol. 11, 941 (2001). 26. K. M. Wassarman, F. Repoila, C. Rosenow, G. Storz, S. Gottesman, Genes & Dev. 15, 1637 (2001). 27. Supplementary Web material is available on Science Online at www.sciencemag.org/cgi/content/full/xxx 28. D. H. Mathews, J. Sabina, M. Zuker, D. H. Turner, J. Mol. Biol. 288, 911 (1999). 29. E. Bernstein, A. A. Caudy, S. M. Hammond, G. J. Hannon, Nature 409, 363 (2001). 30. Graham, F.L. and van der Eb, A.J., (1973), Virol. 52, 456. 31. McCutchan, J.H. and Pagano, J.S., (1968), J. Nati. Cancer Inst. 41, 351. 32. Chu, G. et al., (1987), Nucl. Acids Res. 15, 1311. 33. Fraley, -R. et al., (1980), J. Biol. Chem. 255, 10431. 34. Capecchi, M.R., (1980), Cell 22, 479. 35. Feigner, P.L. et al., (1987), Proc. Nati. Acad. Sci USA 84, 7413.

-25 36. Lau N.C., Lim L.P., Weinstein E.G., Bartel D.P., (2001), Science 294, 858-862. 37. Lee R.C., Ambros V., (2001), Science 294, 862-864. 38. Ambros V., (2001), Cell 107, 823-826. 39. Ambros V., Horvitz H.R., (1984), Science 226, 409-416. -40. Wightman B., Ha I., Ruvkun G., (1993), Cell 75, 855-862. 41. Rougvie A.E., (2001), Nat. Rev.-Genet, 2,-690-701. 42. Ketting R.F., Fischer S.E., Bernstein E., Sijen T., Hannon G.J., Plasterk R.H., (2001), Genes & Dev. 15, 2654-2659. 43. Hallam S.J., Jin Y., (1998), Nature 395, 78-82. 44. Gauwerky C.E., Huebner K., Isobe M.; Nowell -P.C., Croce C.M. (1989), Proc. Natl. Acad. Sci. USA 86, 8867-8871. 45. P. Chomczynski, N. Sacchi, Anal Biochem 162, 156, (1987). 46. Mourelatos Z., Dostie J., Paushkin S., Sharma A.;, Charroux B., Abel L., J.R., Mann M., Dreyfuss G., (2002), Genes & Dev., in press. 47. Tam W., (2001), Gene 274, 157-167.

-26 Table 1 D. melanogaster miRNAs. the sequences given represent the most abundant, and typically longest mIRNA sequence identified by cloning; mIRNAs frequently vary in length by one or two nucleotides at their 3' termini. From 222. short RNAs sequenced, 69 (31%) corresponded to miRNAs, 103 (46%) to already characterized -functional RNAs (rRNA, 7SL RNA, tRNAs), 30 (14%) to transposon RNA fragments, and 20 (10%) sequences with no database'6ntry. The frequency (freq.) for cloning a particular miRNA relative to all identified miRNAs is indicated in percent. Results of Northern blotting of total RNA isd6sted from staged populations of D. melanogaster are summarized. Ej embryo; L; larval stage; -P; pupae; A, adult; S2, Schneider-2 cells. The strength of the signal within each blot is represented from strongest (+ + +) to undetected (-). let-7 stRNA was probed as control. Genbank accession numbers and honiologs of miRNAs identified by database searching in other species are provided as supplementary material. nIRNA sequence (5' to 3') freq. E E LI+ L3 P A S2 (%) 0-3 h 0-6 h L2 miR-1I UGGAAUGUAAAGAAGUAUGGAG 32 + + ++ ++ ++ (SEQ ID NO:58) + + + - - + mR-2" UAUCACAGCCAGCUUUGAUGAGC 3 (SEQ ID NO:59) mFR--2b" UUCACAGCCAGCUUUGAGGAGC 3 ++ ++ ++ ++ +" + ++ (SEQ ID NO:60) + + mlR-3 UCACUGGGCAAAGUGUGUCUCA# 9 +++ +++ mi-4 AUAAAGCUAGACAACCAUUGA 6 .+ +++ - (SEQ ID NO:62) miR-5 AAAGGAACGAUCGUUGUGAUAUG 1 +++ +++ +/ +/ (SEQ ID No.63) R-6 UAUCACAGUGGCUGUUCUUUUU 13 ++ +++ +/ +/ - - (SEQ ID NO:64) ii7 UGGAAGACUAGUGAUUUUGUUGU 4 +++ ++ +/ +/ +- -7-' +/-. (SEQ ID 10:65) m|R-8 UAAUACUGUCAGGUAAAGAUGUC 3 + +/ ++ +4 + + (SEQ ID NO:66) + + + -27 m- UCUU AUCUAGCUGUAUGA +++ ++ + + + +/- (SEQ ID 30:67)- + + + ACCCUGUAGAUCCGAAUUU '"~' ++ ++ (SEQ ID NO:68) + mW-11 CAUCACAGUCUGAGUCUUGC 7 +++ ++4 ++ ++ ++ + (SEQ ID N0: 69) + + mlR-12 UGAGUAUUACAUCAGSUACOGGU T + + ++ ++r- + - + (SEQ ID NO:70) + iR-i3a* UAUCACAGCCAUUUGACGAGU T +++ +++ ++ ++ + ++. ++ (SEQ ID NO:71) + . + mIR-13b* UAUCACAGCCAUUU3UGAGU .- 0- (SEQ ID NO:72) miR-14 UCAGUCUUUUUCUCUCUCCUA 1 - - - (SEQ ID N0.73) let-7 UGAGGUAGUAGGUUUUAUAGUU 0 - - - - ++ + (SEQ ID N0.74) + + # (SEQ ID NO:61) *Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes.

-28 Table 2 Human miRNAs. From 220 short RNAs sequenced, 100 (45%) corresponded to miRNAs, 53 (24%) to already characterized functional RNAs (rRNA, snRNAs, tRNAs), and 67 (30%) sequences with no database entry. Results of Northern blotting of. total. RNA- isolated from different vertebrate species and S2 cells are indicated. For legend, see Table 1. miRNA sequence (5 to 3') fraq. HeLa . mouse adult - frog -. S2 (%) calls kidney fish ovary let-7i* UGAGGUAGUAGGUUGUAUAGUT# 10 +++ +++ +++ et-7b* UGAGUAGUAGGUUGUGUGG UU - '(SEQ ID NOi'76) ' - -.

UGAGGUAGUAGGUUGUAUGGUU 3 (SEQ ID NO:77) let-7d* AGAGGUA~GUAGGUUGCAUAGU 2 +-+ +++ +++ (SEQ ID NO:78) let-7e* UGAGGUAGGAGGUUGUAUAGU 2 +++ +++ +++ - (SEQ ID NO:79) let-7f* UGAGGAGAGAUUGUAUAGUU 1 (SEQ ID-NO:80) miR-15 UAGCAGCACAUAAUGGUUUGUG 3 +++ ++ (SEQ ID No:81) mIR-16 UAGCAGCACGUAAAUAUUGGCG 10 +++ + + +/ (SEQ ID NO:982) miR-17 ACUGCAGUGAAGWCACUUGU 1 ++ - - (SEQ ID NO:83) m[R-18 UAAGGUGCAUCUAGUGCAGAUA 2 +++ (SEQ ID NO:84) mROS1*i UGUGCAAAUCUAUCAAAACUGA I ++ - - --- . (SEQ ID NO.85) miR-19* UGUGCAPAAUCCA~UGCAAAACUSA 3 (SEQ ID NO:86) mlR-20 UAAAGUGCU UAUAGuGCAGGUA +++ + (SEQ ID NO:87) miR-21 UAGCUUAUCAGACUGAUGUUGA 10 ++ r + r (SEQ ID NO:8) mlR-22 AAGCUGCCAUUGAGAACUGU 10 ++ + + (SEQ ID NO.89) mR-23 AUCACAUUCCCAGGGAUUUCC 2 + + +++ +r (SEQ ID NO:90) -29 mIR-24 UGCUCAGUUCAGG G 4 ++ +++ + (SEQ ID NO: 91) m-2 CAUUGCACUUGUUCGGUGA 3l +++ + ++ (SEQ ID 90:92) miR-26a* UUCAAGUAAuCcAGGAUAGCU 2 r ++ 92 +++ . (SEQ ID NO:93) mIR-26b* UUCAAGUAAUUCAGGAUAGGUU 1 (SEQ ID NO:94) mIR-27 UUCACAGUGGCUAAGUUCCGCU r 2.+ ++-. - . (SEQ ID X0:95) mIR-28 AAGGAGCUcCAGUCUAUUGAG 2 +++ +++ (SEQ ID NO: 96) . miR-29 CUAGCACAUCUGAAAUCGGUU 2 + +++ + (4E6 ID N40,97) miR-310 -C AGAU GUCAUUGCAGC - 2 +++ -+++ - (SEQ ID NO:98) miR-31 GGCAAGAUGCUGAAG7CUGC 2 +++ (SEQ ID NO:99) mIR--32 UAUUGCACAUJUAUAUUGC - (SEQ ID NO:100) miR-33 GUGCAUGUAGUUGCAUUG 1 - (SEQ ID NO:101) miR-1 UGAUGAAUAGAUUGGAG 0 + (SEQ ID NO:1032) iR-7 UGGAAGWAUCUAGUGAU GUUGU 0 :+ (SEO ID NO:1303) (SEQ ID NO: 104) miR-10 ACCCUGUAGAUdGAAUCUGU 0 - + - (SEQ ID 0:o105) # = (SEQ ID NO:75) *Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes.

- 30 Table 3 Mouse miRNAs. The sequences indicated represent the longest miRNA sequences identified by cloning. The 3'-terminus of mIRNAs is often truncated by one or two nucleotides. miRNAs that are more than 85% identical in sequence (i.e. share 18 out of 21 nucleotides) or contain I- or 2-nucleotide internal deletions are referred to by the same gene number -followed by a lowercase letter. Minor sequence variations between related miRNAs are generally found near the ends of the miRNA sequence and are thought to not compromise target RNA recognition. Minor sequence variations may also represent A to G and' C to U changes, which are accominodated as' G-U 'vdbble base pairs doring-thrgt recognition. miRNAs with the suffix -s or -as indicate RNAs derived from either the 5' half or the 3 '-half of a miRNA precursor. Mouse brains were dissected into midbrain, mb, cortex, cx, cerebellum, cb. The tissues analyzed were heart, ht; liver, Iv; small intestine, si; colon, co; cortex, ct; cerebellum, cb; midbrain, mb. miRNA sequence (5' to 3) Number of clones ht 1v sp si co ex ab Mnb let-7a UGAGGUAGUAGGUUGUAUAGUU 3 1 1 7 (SEQ ID NO:106) let-7b UGAGGUAGUAGGUUGUGUGGUU 1 1 2 5 (SEQ ID NO:107) let-7c UGAGGUAG=GUUGUGUrU 2 2 5 19 (SEQ ID NOs10) let-7d AGGGUAGUAGaGUUGCAUAGU 2 - 2 2 2 (sWQ In NO:109) let-7e UGGGUAGGAGGUUGIAMGU 1 2 (SEQ ID NO:110) let-7f UGAG=GUAAUUGUAUAGUU 2 3 3 (SEQ ID No:111) let-7g UGAGGUAUGUDUGUACAGUA 1 1 2 (SEQ ID] NO:112) let-7b UGAGGUAGUAGUGUGUACAGUU 1 1 (SEQ ID NO1113) - 31 let-7i UCeAGQUAaMUaUrUaueCU (EQ ID NO:13.4) xiif-lb usaUGaaemaoMUnLa 4 21 (SEQ ID 10:115) maiR-lc UGGAADUflAGUAMUQAC 7 (SSQ ID NO0,116) niiR-1d UcGhGMUGAAAGGAUGUAUU 16 (SEQ XD 310:-117) rniR-9 UCUOUUGOUCJAGCUGJAUGA 3 4 4 (SEQ ID 31:119) rniR-15a iAscAscacUauoOUzMu 1 2 (SEQ ID N0:119) niR-1 Sb UAGCAGCACAUCAUGOUUU&CAL I (SEQ-ID3 k0:120) miR-16- UASCACACGUAAAUAUUGGCG1W 1 2 3 (SEQ XD wa-la UAAGGUQCALJCUAGTICAMJ (SEQ ID 50:122) milt-I9b tJGUGCAATCC&UGCAACMU3 (SEQ ID NO: 2.23) miR-20 UAM;GUGCUJUAU CAGG (SEQ XD N10:124) mi-R-2 1 UAGCUUAUCAOACUGAUGtJUGA 1 1 2 (SEQ ID NO:125) milt-22 AAGCCAGUUmgAAACUQU 2 1 1 1 2 (SEQ ID NO:126) rniR-23a AUCACAtUSGCCAOGGGAUUTCC 1 (SEQ ID NO.127) miR-23b JWCCAUUOCCAGGGAULUACCAC (SEQ ID 310:129) nuR-24 UGGCUCAGUUCR=GCAACAO 1 (SEQ ID NOr139) mift-26a ULTAAGUAAJCCAGMtIGGC 3 2 (SEQ ID 31:130) miR-26b WUCAAGUAMMcAGSAUGMU 2 4 (SEQ ID N0:131) wiR-27a UUCACAGGGCAADUTJCCGCU 1 2 1 1 2 (SEQ ID 10: 132) miR-27b UUCZC~AUssConorrxCoUtI (SEQ IV3 31:133) mik-29a CUTAQCACCAUCUGAAAUCGGU (SEQ ID 310:134) milt-29b/rniK 102 UAGAOUUQACGUGM 1 5 3 (SEQ ID 310.135) miR-29c/ !JAGCACCAUUtIGRAASCGUUA 1 1 (SEQ ID NO.136) -32 miR-30a-s/hiR-97 UGUAMcAUccUcGACUGGAAGC (SEQ IM NO:137) miR-30a-as cDuUcAGucGGAwuuuGcAGc (SEQ ID NO:138) miR-30b UGUAAAcAUCCUACACUCAGC 1 2 (SEQ ID NO:139) mURc UGUAAAcACCUACACUCJCAGC 2 2 (SEQ ID N0:140) miR-30d UGAAAAUCCCcGACEGGAAO. (SEQ ID NO:141) miR-99a/iR-99 ACCCGUAGAUCCGAUCUUGU (SEQ ID NO:142) miR-99b C&CCCGUAAGACCGCCU YCG (SEQ ID NO10143) miR-101 UACAGUACUGJUGAAACUGA 2 1 (SEQ ID NO:144) niR-122a UGGAGUGUGACAWGGUGUUUGU 3 (SEQ ID NO0145) miR-122b UGGAGUGUGACAAUGGUGUUUGA 11 (SEQ ID NO:146) miR-122ab UGGAPiUGUGACAAUGGUUUG 23 (SEQ ID NO:147) miR-123 CAUUAUUACUUUEGGUAWCG .1 2 (SEQ ID NO:148) niiR-124a UUAAGGACGCGG-UGAAUGCCA 1 37 41 24 (SEQ ID NO:149) niR-124b UUAAGOCACGCGGGUGAAUGC 1 3 (SEQ .ID NO:150) miR-125a UCcCUGAGACCCUUUAACCUGUG 1 1 (SEQ ID NO:1351) miR-125b UCCCUGAGACCCU- -AACUUGUGA I (SEQ ID NO;152) nfR-126 UcQuAccGUGGUAUAAUGC 4 1 (SEQ ID NO:153) miR-127 UCGGAUCCGUcUGAGCUUGGCU (SEQ ID NO:154) niR-128 UcAcAGUOAACCGGUCUCUUU 2 2 2 (SEQ ID 0:3155) niR-129 cUUUWUcGGUcUGGGCUUUc 1 (SEQ ID NO: 156) minR-130 CAGUGCAAUGUUAAAAGGC (SEQ ID NO:157) niR-131 UAAGCUAGAUAACcOAAGU (SEQ ID NO:158) miR-132 UACAGUcUAcAGCAUGQUCGU (SEQ ID NO:159) - 33 miR-133 UUGSUCCWmCACCGU 4 (SQ D 0:60) miR-134 UtGCGUACCA;AGCIGA (SEQ ID 10:-161) miR-135 UAUGGCMUUwaUcCUuuGAAM (SQ .D.0:162) mIR-136 ACUCCAUCJUGDUUUGAUAUGGA (SEQ ID N6:163) ptiR-137 UAUUGCUTJAAGAUACGCGTJAG (SEQ 3ID 1*0:164) iniR-138 AGCWGGUGZUUWUGAAJC (SEtQ ID 1*:165) zniR-139 UCUACGASUGC&C;VCU (SEQ ID W.3.166) miR-140 AQUGGUtUtACCWjAUGGTAG - (SEQ ID 10:167) rrdR-141 . AACACEMGUGYOUAAAGAUGG (SEQ ID 1*0:169) miR-] 42-s CAUAAAGtAhDMAGCACUACx (SEQ IDl 10:169) miR-142-at' UGUAGtJQUUUCCUACUUUAUQO1 6 (SEQ ID 1*0u170) m1R4143 * ~ UGAGAUGAGCACt3GUAGCUCA 3 7 2 (SEQ ID 10:171) miR-144 UACIIGADmAGUGUACUAG 2 (SEQ ID N0:172) miR- 145 GUCCAGUIUUUCCCAUQAAUCCCUI I (SEQ ID 1*0:173) miR-146 UQAGAACUGUT3CCAUGGUMt I (SEQ ID 1*0:174) mik-147 SUQUGGAAAmUGCUrJCUGCC (SEQ ID 1*0:175) miR-148 uac~k~cIhcAaAcmsUGU (SRQ XD NO*0:176) miR-149 ucuoCeuccswGucuucac (SEQ ID X0.177) inI-1 50 uwUCCChacccuuGUAccaoUGU (SEQ ID 1*0:179) niiR-151 CUAWLCUGAGGCUCCUUGACGU (SEQ ID Pt0:179) miR-152 UCAGGCUGACAGMACtRIGG (SEO ID 10:190) rniR-153 UYUGCAUAOOCACAMAGUSA (SEQ ID:1a 11) rniR-154 UAGGUUAUCCGUWUtIGCCUUCG; (SEQ IV NO.182) -34 Wil-i 55 UUAAUGCUAUUGUGADAGGGQ 1 (SEQ ID NO:193) t The originaly described miR-30 was renamed -td miR-30a-as in order to distninish it from the miRNA derived from the Opposite strand- of the precursor encoded by the mir30a gene. miR-30a-s is equivalent to miR-97 [46]. 'A 1-nt length heterogeneity is found on both 5' and'3 nd. the 22-nt rmiR sequence is shown, but only 21-nt milNA wee cloned.

- 35 Table 4 Mouse and human miRNAs. The sequences indicated represent the longest miRNA sequences identified by cloning. The 3' terminus of miRNAs is often truncated by one or two nucleotides. miRNAs-that are morse than 85% Identical in sequence (i.e. share 18 out of 21 nucleotides) or contain 1- or 2-nucleotide internal deletions are referred to -by the same gene number followed- by a lowercase letter. Minor sequence variations between related mIRNAs are generally found near the ends of the miRNA sequence and are thought to not. compromise target -RNA recognition. Minor sequence variations may also represent A to G and C to U changes; which are accommodated as G-U wobble, base pairs .during target recognition. Mouse brains were dissected into midbrain, mb, cortex, cx, cerebellum, cb. The tissues analyzed were lung, In; liver, lv; spleen, sp; kidney, kd; skin, sk;-testis, ts; ovary, ov; thymus, thy; eye, ey; cortex, ct; cerebellum, cb; midbrain, mb. The human osteosarcoma cells SAOS-2 cells contained an inducible p53 gene (p53-, uninduced p53; p53+, induced p53); the differences in miRNAs identified from induced and uninduced SAOS cells were not statistically significant.

36 a as a a ia s cc coCO cC ~m m0 c e ~ 6 CYC YCYC C a CCYC AA *4C4 aa UU 91 C4 2: J5 0 Y 2 -.~ - - 'NEli i Y9 a ass sesa as aqa 373 s~e ,- e 00 00 0 00 00 00 00 - 38 Table 5 D. melanogaster miRNA sequences and genomic location. The sequences given represent the most abundant, and typically longest mIRNA sequences identified by cloning. It was frequently observed that miRNAs vary in length by one or two nucleotides at their 3'-terminus. From 222 short RNAs sequenced; 69 (31 %) corresponded to miRNAs, 103 (46%) to already characterized functional RNAs (rRNA, 7SL RNA, tRNAs), 30 (14%) to transposon RNA fragments, and 20 (10%) sequences with no database entry. RNA sequences with a 5' guanosine are likely to be underrepresented due to the cloning procedure (8). miRNA homologs found in othe- species are'indicated. Chromosomal location (chr.) and GenBank accession numbers (acc. nb.) are indicated. No ESTs matching miR-1 to miR-14 were detectable by database searching. RiRNA sequence (5' to 3') chr., ac. nb. remarks miR-1 UGGAWGUAAAGAAGUAUGQAG 2L, AE003667 homologs: C. briggsae, G20U, (SEQ ID NO:58) A87074; Celegans G20U, U97405; mouse, G20U, G22U, AC020867; human, chr. 20, G20U, G22U, AL449263; ESTs zebrafish, G20U,. G22U, BF157 601: cow, G20U, 022U, BE722 224; human, G20U, G22U, A1220268 miR-2a AUacaCaocCAGCuOUGAUGAGC 2L, AE003663 2 precursor variants clustered (SEQ ID NO:59) with a copy of m.r-2b mIR-2b UaUaCAQCCAQCUUUGAGGAGC 2L, AE003620 2 precursor variants (SEQ ID NO:60) 2L, AE003663 miR-3 CACUGGGCAAAGQUGUCUCA 2R, AE003795 in cluster mir-3 to mir-6 (sEQ ID NO:61) miR-4 AUMACUAGACAACAUUGA 2R, AE003795 in cluster mir-3 to mir-6 (SEQ ID NO:62) - 39 miR-5 AUGA ACAUCGUUGUGAUAUG 2R, AE003795 in cluster mir3 to mir*6 (Big ID 3o:63) miR-6 UAUACALGDIGCUGOUCUaUUU 2R, AE003795 in cluster mir3 to mir-6 with 3 (SEQ ID NO: 64) Variants miR-7 UGAsaeAcUAGusAUuUeUuou 2R, AE003791 homnologsi hunan, cir. 19 (SEQ ID NO: 65) AC006537, EST BF373391; mouse chr. 17 AC026385, EST AA881786 miR-8 UAAUACIQUCAGQUAAAGA1GUC 2R, AE003805 (SEQ ID NO: 66) mIR-9 UcDUUGGUuAUCUAGcUGUAUGA 3L, AE003516 homologs: mouse, chr. 19, (SEQ ID NO:67) AF155142; human, cbr. 5, AC026701, chr. 15, AC00531 miR-10 ACCCUGUAQAUCCGAAUUUGU AE001574 homologs: mouse, chr 11, (SEQ ID NO: 6 8) AC011194; human, chr. 17, AF287967 mIR-11 CAUCASCUGAGUUUGC 3R, AE003735 intronic location (SEQ ID NO: 69) miR-12 uGAGUcAcucaGUAcuGGcU X, AE003499 Intronlo location (SEQ ID NO:7 0) miR-13a UAUcAc&GCCUUUG&cGAGY 3R, AE003708 mr-13a clustered with mlr-13b (bEQ ID NO:71) X; AE003446' 'on chr. 3R miR-13b uAucACAGCCADUIUCGUQ&GU 3R, A003708 mir-13 clustered with mir-13b (SEQ ID NO m72) on chr. 3R mIR-14 UcAGUcUUUDUUCUCEICcUA 2R, AE003833 no signal by Northern analysis (SEQ ID No:73) -40 Table 6 Human mIRNA sequences and genomic location. From 220 short RNAs sequenced, 100 (45%) corresponded to miRNAs, 53 (24%) to already characterized functional RNAs (rRNA, snRNAs, tRNAs), and 67 (30%) 5 sequences with no database entry. For legend, see Table 1. mIRNA sequence (5' to 3) chr. or EST, remarks* acc. nb. Iet-7a UGAGG1GUAGGUGUAUAGU 9, ACO7924,, sequences of chr 9 arnd 17 F. (SEQ ID NO:75) I, AP001359, Identical and clustered with let-74, 17, AC087784, hornologs: C. elegans, AF274345 22, AL049853 C briggsae, AF210771, D. melanogaster, AE003659 let-7b UGAGQUAGUAGGUUGUUGUU 22, AL049853t, homologs: mouse, EST A1481799; (SEQ ID NO:76) ESTs, A1382133, rat, EST, BE120662 AW028822 let-7C UGAGGUAGUAGQGGUUAUQGU 21, AP001667 Honologs: mouse, EST, (SEQ ID NO:77) AA575575 let-7d AGAGOUAGUAGUUGCAUAGU 17, AC087784, Identical precursor sequences (gEQ ID No78) 9, AC007924 let-7e UGAGSGGLAGGUAUAGU 19, AC018755 (SEQ ID NO.:79) let-7f UalGAUafAgUAUGUAUAGDU 9, AC007924, sequences of chr 9 and 17 (SEQ ID N:O80) 17, AC087784, identical and clustered with let-7a X, AL592046 rniR-15 UAGCAGCUAAUGGUUUG 13, AC069475 In cluster with mir-16 homolog (SEQ ID NOs81) miR-16 UAGCACACGUAAAuAUUGG 13, AC069475 In cluster with mir-15 homolog (SEQ ID NO82) -41 miR-17 ACUGCGUGAAGCUUGU 13, AL138714 in cluster with mir-17 to mir-20 (SEQ ID NO:83) miR-18 UAAGuGCAUCOAUGCAGAUA 13, AL138714 In cluster with mlr17 to mir-20 (SEQ ID NO:84) miR--19a UGUGCAAAUCUCAAACUG 13, AL138714 in cluster with inr17 to mir20 A (SEQ ID NO985) mIR-19b UGuOCaAUccAvCAAMCUG 13, AL138714, in cluster-with mir-17 to mir-20 A (SEQ ID NO:86) X, AC002407 mlR-20 UAaeGauaD~UAGUSCAGGUA 13, AL138714 in cluster with m/r17 to mir-20 (SEQ ID NO:87) mlR-21 UAGcuAucATGACGAUGUUGA 17, AC004686, -homologs: mouse, EST, (SEQ ID NO:88) EST, BF326048 AA209594 miR-22 AAGCUGCCAGDGGAAGA&CUGU fESTs, human ESTs highly similar; (SEQ ID NO. 89) AW961681t, homologs: mouse, ESTs, e.g. AA456477, AA823029; rat, ESTs, e.g. A1752503, BF843690 BF030303, HS1242049 mirR-23 AUCACAuUGecCwGGAUIIUC 19, AC020916 homologs: mouse, EST, (SEQ ID NO.90) AW124037;rat, EST, BF402515 mIR-24 UGscucauucacAGGA&CW 9, AF043896, homologs: mouse, ESTs, (SEQ ID N0191) 19, AC020916 AA11146, A1286629; pie, EST, BE030976 miR-25 CAUUGCAcuOGUcUCGGUCUG& 7, AC073842, human chr 7 and EST identical; (SEQ ID NO:92) EST, BE077684 highly similar precursors In mouse ESTs (e.g. A1595464); fish precursor different STS: 046757 mIR-26a UUcAQUAUCcHAAUAACU 3, AP000497 (SEQ ID NO93) H:\aaincmocn\NRPortb]\DCC\AAR\5 881146_ .DOC-10/12/2013 -42 miR-26b UUCAAGUAAUUCAGGAUAGGUU 2, AC021016 (SEQ ID NO : 94) miR-27 UUCACAGUGGCUAAGWCCGCU 19, AC20916 U22C mutation in (SEQ ID NO : 95) human genomic sequence miR-28 AAGGAGCUCACAGUCUAWGAG AC063932 (SEQ ID NO : 96) miR-29 CUAGCACCAUCUGAAAUCGGUU AF017104 (SEQ ID NO : 97) miR-30 CUUUCAGUCGGAUGUUUGCAGC 6, AL035467 (SEQ ID NO: 98) miR-31 GGCAAGAUGCUGGCAUAGCUG 9, AL353732 (SEQ ID NO : 99) miR-32 UAUUGCACAWACUAAGWGC 9, AL354797 not detected by (SEQ ID NO : 100) Northern blotting miR-33 GUGCAUUGUAGUUGCAUUG 22, Z99716 not detected by (SEQ ID NO: 101) Northern blotting * If several ESTs were retrieved for one organism in the database, only those with different precursor sequences are listed. 5 t Precursor structure shown in Figure 4. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will 10 be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or 15 admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (25)

1. An isolated nucleic acid molecule having a length of from 18 to 25 nucleotides comprising a nucleotide sequence which has an identity of at least 80% to a sequence shown in SEQ ID No:183 (miR-155) or a complement thereof.

2. The nucleic acid molecule of Claim 1, which has a length of from 19 to 24 nucleotides.

3. The nucleic acid molecule of Claim 1 or Claim 2 comprising a nucleotide sequence as shown in SEQ ID No:183, or comprising the complement of a nucleotide sequence as shown in SEQ ID No:183.

4. The nucleic acid molecule of Claim 3, which consists of a nucleotide sequence as shown in SEQ ID No:183.

5. The nucleic acid molecule of Claim 3, which consists of the complement of a nucleotide sequence as shown in SEQ ID No:183.

6. An isolated nucleic acid molecule having a length of 60 to 80 nucleotides comprising a nucleotide sequence which has an identity of at least 80% to a sequence shown in SEQ ID No:183 or a complement thereof.

7. The nucleic acid molecule of Claim 6 comprising a precursor structure as shown in Figure 7, or comprising the complement of a precursor structure as shown in Figure 7.

8. The nucleic acid molecule of Claim 7, which consists of a precursor structure as shown in Figure 7. 1: \aar\Intrwovn\NRPortbl\DCC\AAR\5049059 I doc-5 04 2013 -44

9. The nucleic acid molecule of Claim 1 or Claim 6, wherein the nucleic acid molecule comprises a nucleotide sequence which has an identity of at least 95%.

10. The nucleic acid molecule of any one of Claims 1-9, which is single-stranded.

11. The nucleic acid molecule of any one of Claims 1-9, which is at least partially double-stranded.

12. The nucleic acid molecule of any one of Claims 1-11, which is selected from RNA, DNA or nucleic acid analog molecules.

13. The nucleic acid molecule of Claim 12, which is a molecule containing at least one modified nucleotide analog.

14. The nucleic acid molecule of Claim 13, wherein the at least one modified nucleotide analog is a sugar-modified ribonucleotide.

15. The nucleic acid molecule of Claim 13, wherein the at least one modified nucleotide analog is a backbone-modified ribonucleotide.

16. The nucleic acid molecule of Claim 13, wherein the at least one modified nucleotide analog is a locked nucleic acid.

17. A recombinant expression vector comprising a nucleic acid molecule of Claim 1 or 6.

18. A pharmaceutical composition containing as an active agent at least one nucleic acid molecule of any one of Claims 1-17 and optionally a pharmaceutically acceptable carrier. 1: \aar\Intrwovn\NRPortbl\DCC\AAR\5049059 I doc-5 04 2013 - 45

19. The composition of Claim 18 when used for a diagnostic application.

20. The composition of Claim 18 when used for a therapeutic application.

21. The composition of Claim 18 when used as a marker or a modulator for developmental or pathogenic processes.

22. The composition of Claim 18 when used as a marker or modulator of gene expression.

23. The composition of Claim 18 when used as a marker or modulator of developmental disorders, particularly cancer, such as B-cell chronic leukemia.

24. The composition of Claim 23 when used as a marker or modulator of the expression of a gene, which is at least partially complementary to said nucleic acid molecule.

25. A nucleic acid molecule according to any one of Claims 1 to 16 or a recombinant expression vector of Claim 17 or a composition of any one of Claims 18 to 24 substantially as herein described with reference to the Figures and/or Examples.