New human and mouse microRNA genes found by homology search Michel J. Weber Laboratoire de Biologie Mole ´ culaire Eucaryote, UMR5099, CNRS and Universite ´ Paul Sabatier, IFR109, Toulouse, France MicroRNAs (miRNA) are  22 nucleotide-long RNAs that function in translational repression by base pair- ing with their target mRNA in a variety of pluricellu- lar organisms. They originate from long precursors (pri-miRNA) that, in animals, are cleaved by the Dro- sha endonuclease in the nucleus [1] to give  70 nuc- leotide-long miRNA precursors (pre-miRNAs) with a characteristic hairpin structure. In the plants, excision of pre-miRNAs is performed by DCL1, a Dicer homo- logue [2,3]. Following the export of pre-miRNAs to the cytoplasm by Exportin-5 [4,5], the loop region of the hairpin is removed by the Dicer endonuclease to produce a short double-stranded RNA (dsRNA), a strand of which, corresponding to the mature miRNA, is predominantly incorporated in the RNA-induced silencing complex (RISC). The RISC complex either inhibits translation elongation or triggers mRNA deg- radation, depending upon the degree of complementar- ity of the miRNA whith its target (for reviews, see [6–8]). Since the seminal identification of the first miRNAs, Caenorhabditis elegans lin-4 and let-7, using genetic approaches [9,10], hundreds of miRNAs have been characterized experimentally using various cloning strategies in plants, C. elegans, Drosophila melanogas- ter, zebrafish, pufferfish, mouse, rat and human [8]. More recently, algorithms have been developed to identify putative precursor miRNAs from sequenced genomes [11,12]. These approaches predict that the human genome might contain 200–255 miRNA genes Keywords antisense transcript; genomic localization; human genome; microRNA; mouse genome; mRNA degradation; RNA interference Correspondence M. Weber, LBME, 118 route de Narbonne, 31062 Toulouse Cedex, France Fax: +33 5 61 33 58 86 Tel: +33 5 61 33 59 56 E-mail: weber@ibcg.biotoul.fr (Received 4 June 2004, revised 24 August 2004, accepted 7 September 2004) doi:10.1111/j.1432-1033.2004.04389.x Conservation of microRNAs (miRNAs) among species suggests that they bear conserved biological functions. However, sequencing of new miRNAs has not always been accompanied by a search for orthologues in other spe- cies. I report herein the results of a systematic search for interspecies ortho- logues of miRNA precursors, leading to the identification of 35 human and 45 mouse new putative miRNA genes. MicroRNA tracks were written to visualize miRNAs in human and mouse genomes on the UCSC Genome Browser. Based on their localization, miRNA precursors can be excised either from introns or exons of mRNAs. When intronic miRNAs are anti- sense to the apparent host gene, they appear to originate from ill-character- ized antisense transcription units. Exonic miRNAs are, in general, nonprotein-coding, poorly conserved genes in sense orientation. In three cases, the excision of an miRNA from a protein-coding mRNA might lead to the degradation of the rest of the transcript. Moreover, three new exam- ples of miRNAs fully complementary to an mRNA are reported. Among these, miR135a might control the stability and⁄ or translation of an alter- native form of the glycerate kinase mRNA by RNA interference. I also dis- cuss the presence of human miRNAs in introns of paralogous genes and in miRNA clusters. Abbreviations miRNA, microRNA; pre-miRNAs, miRNA precursors; RISC, RNA-induced silencing complex; snoRNA, small nucleolar RNA. FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 59 [13]. However, a comprehensive list of these sequences is still not available. It was recognized that many miRNAs are evolu- tionarily conserved, some of them from worm to human [14]. MiRNA genes have been characterized experimentally from a variety of organisms and might have orthologues in other species, suggesting a powerful method to predict the existence of new miRNA genes. I report here on the results of a sys- tematic search for potential human orthologues of mouse miRNAs deposited in the miRNA Registry [15] and, vice-versa, of potential mouse orthologues of known human miRNAs. In addition, I searched for human orthologues of recently identified rat miRNAs [16]. This led me to identify potential orthologues of miRNAs that were described previ- ously in one species, but not in the other. After inclusion of these new data, the total number of human and mouse miRNAs deposited at the miRNA Registry now approaches the theoretical number of 255 predicted by Lim et al. [13]. Using this information, I wrote custom tracks that allow for the localization of the miRNA genes in the human and mouse genomes. These are now available on the UCSC Genome Browser [17]. Using this tool, I systematically determined the position and orientation of miRNA genes relative to known transcriptional units, examined the conservation of miRNA gene local- ization between the human and mouse genomes, and made a comprehensive list of miRNA clusters. This search led to several testable hypotheses concerning the transcription of miRNA genes, and to the predic- tion of new mRNA targets. Results and Discussion New potential human and mouse microRNA precursors The entire set of human and mouse precursor and mature miRNA sequences from the miRNA Registry (version 2.2) was submitted to a BLAT search against the human genome. The results (in BED format) were exported to excel TM to generate a table with 2873 ent- ries. A similar BLAT search was performed against the mouse genome, generating an excel TM table of a sim- ilar size. These tables were then ranked according to chromosome number and chromosome position and filtered for perfect and near-perfect matches. The cor- responding sequences were subsequently examined for a potential hairpin structure with mfold, and the results were compared to those of known miRNAs from the miRNA Registry. I only considered, as valid candidates, those poten- tial miRNA precursors that conformed to the empir- ical criteria proposed by Ambros et al. [18] in particular, a hairpin structure of the lowest free energy, as predicted by mfold , and a minimum of 16 nucleotides of the mature miRNA engaged in Watson– Crick or G ⁄ U base pairings (criterion C). The method used for searching the new miRNAs ensured their phy- logenetic conservation (criterion D). Moreover, the detection of the  22-nucleotide mature forms by Nor- thern blot (criterion A) and ⁄ or their identification in a cDNA library (criterion B) were checked for the sequences deposited in the Rfam2.2 miRNA Registry (Wellcome Trust Sanger Institute) for at least one species. In most cases, sequences of the new mature miRNAs were perfectly conserved between human and mouse, but differed by one nucleotide in few cases (see for example, mir-155). Further validation of these can- didates was performed on the basis of additional cri- teria, such as conservation of the host gene (see below) or position relative to known miRNA clusters. This led to the identification of 60 new potential miRNA precursors (15 for human and 45 for mouse) that were made available before publication in Rfam version 3 of the miRNA Registry of the Wellcome Trust Sanger Institute (Table S1). Moreover, with the collaboration of S. Griffiths-Jones (Wellcome Trust Sanger Institute), the names of several miRNAs depos- ited at the miRNA Registry have been changed, so that orthologous precursors, based on conservation of both sequences and synteny, have similar names in both human and mouse. This was particularly important when a mature miRNA had multiple, closely related, precursors (see for example mmu-mir-9-2, mmu-mir- 138-12 and mmu-mir-199a-1). Moreover, the coordinates of both new and previ- ously described miRNA precursors were used to write custom tracks that allowed their localization in the human and mouse genomes on the UCSC Genome Browser. A similar track was also written for the gen- ome of C. elegans. New human miRNA precursors predicted by homology with rat microRNAs I searched for potential human orthologues of recently cloned rat miRNAs [16]. The best BLAT hits were examined for conservation of both synteny in human and rodent genomes and the presence of a stem-loop structure. This allowed one to propose 20 new human microRNA precursors (Table 1). miR-151 was identified in the mouse [19], and miR- 151* in the rat [16]. In both cases, the same predicted New human and mouse microRNA gene M. J. Weber 60 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS Name hsa-miR-322 *hsa-miR-323 *hsa-miR-324-5p *hsa-miR-324-3p hsa-miR-325 *hsa-miR-326 *hsa-miR-328 hsa-miR-329 *hsa-miR-330 *hsa-miR-331 *hsa-miR-335 Mature miRNA Predicted precursor hairpin structure Note Differs from rno-miR322 by four nucleotides (italic) Same intron of the host gene of hsa-mir-299 (AK021542) Differs from rno-miR-325 by two nucleotides (italic) Differs from rno-miR-326 by one nucleotide (italic) Differs from rno-miR-329 by four nucleotides (italic) Differs from rno-miR-330 by one nucleotide (italic) Table 1. New human microRNAs predicted by homology with rat miR. The potential human orthologues of rat microRNA precursors described by Kim et al. [16] were localized in the human genome by BLAT on the UCSC Genome Browser server [54,57] or by examination of conserved sequences in the human genome in the regions orthologous to those that host the rat or mouse miRNAs. MiRNAs indicated by an asterisk have been deposited in the miRNA Registry (version 3.1). M. J. Weber New human and mouse microRNA gene FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 61 Name *hsa-miR-337 *hsa-miR-338 *hsa-miR-339 *hsa-miR-340 *hsa-miR-342 hsa-miR-345 hsa-miR-346 *hsa-miR-135b *hsa-miR-148b *hsa-miR-151* *hsa-miR-151 Mature miRNA Predicted precursor hairpin structure Note Differs from rno-miR-337 by one nucleotide (italic) Differs from rno-miR-345 by two nucleotides (italic) Differs from rno-miR-346 by three nucleotides (italic), and from mmu-miR-346 by two nucleotides hsa-miR-151 differs from rno-miR-151 by one nucleotide (italic) Table 1. (Continued). New human and mouse microRNA gene M. J. Weber 62 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS precursor encodes both mature miRNAs from its 5 0 (miR-151*) and 3 0 (miR-151) portions. This also holds true for hsa-mir-151, although the mature form of miR-151 differs from the rodent sequence by one nuc- leotide. This conservation reinforces the hypothesis that, in mammals, the same precursor gives rise to both miR-151 and miR-151*. From the predicted hairpin structure of the precursor, the energies of hybridization of the four nucleotides located at the 5 0 end of miR- 151* and miR-151 are 1.7 and 0.1 kcalÆmol )1 , respect- ively. As ‘RISC assembly favors the siRNA strand whose 5 0 end has a greater propensity to fray’ [20], it is expected that miR-151* will be more abundant than miR-151. Indeed, miR-151 was cloned only once among 913 miR sequences [19]. Although, to my know- ledge, neither miR-151, nor miR-151* were cloned in human, it is unlikely that the single base substitution compared to rodent sequence (at nt 10 of miR-151) could alter the balance between the two miRs. For certain rodent miRNA precursors, a BLAT search in the human genome produced no matches. However, a likely orthologue could be found by exploring the most likely (i.e. of conserved synteny) portion of human genome for conserved sequences; e.g. mmu-mir-345 resides upstream of the AK047628 RefSeq gene. Its human orthologue was found upstream of C14orf69, the best BLAT hit for AK047628. Such identification was made straightfor- ward by the examination of the ‘Human ⁄ Chimp ⁄ Mouse ⁄ Rat ⁄ Chicken Multiz Alignments & PhyloHMM Cons track’ of the human UCSC Genome Browser. Similarly, hsa-mir-329 and -322 were identified on the basis of their conserved stem-loop structure and conserved position relative to other miRNAs or Ref- Seq genes. However, the presumptive mature miRNAs hsa-miR-329 and -322 differ from their mouse and rat orthologues by four nucleotides (Fig. 1A,B). Most of these changes retained base-pairing in the precursor miRNA by forming G ⁄ U interactions or resided in un- paired positions (data not shown). Consequently, the folding free energy calculated using mfold was either little affected (mir-329) or even decreased in the case Fig. 1. Alignment of microRNA sequences from mammalian genomes. (A–C) Alignment of the sequences from mammalian mir-329 (A), mir-322 (B) and mir-346 (C). Abbreviations: hsa, Homo sapiens; mmu, Mus musculus; rno, Rattus norvegicus; pan, Pan troglodytes. (D) Alignment of the sequences of rodent mir-350 with the corresponding sequences from human and chimpanzee genomes. Rodent sequences were retrieved from The miRNA Registry. Human sequences were retrieved from the human genome sequence by examination of highly conserved regions in the syntenic segments. The sequence of pan-mir-350 was obtained by BLATing the human sequence against the chimpanzee genome. The sequences of mir-329 and mir-298 are 100% conserved between human and chimpanzee. The sequences of mature microRNAs are boxed. The antisense miR boxes indicate the portion of the hairpin precursor structure that is base-paired with the miR, as predicted by MFOLD. M. J. Weber New human and mouse microRNA gene FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 63 of mir-322 (DG ¼ )46.6 for human and )41.0 kcalÆ mol )1 for mouse). The same conclusion holds for hsa-mir-346, where the mature miR sequence differed from those of mouse and rat by two and three nucleotides, respectively (Fig. 1C). In this case, the folding free energy of the human and rat precursors remained comparable ()46.4 and )50.6 kcalÆmol )1 , respectively). Mutations in mature miARNs, particularly in their 3 0 portion, are compatible with their function as trans- lational repressors [21]. The same study, however, revealed that G:U wobble pairing in the 5 0 region of the miRNA had detrimental effects that could not be predicted on the basis of changes in the free energy of annealing with the target mRNA. Therefore, hsa-mir- 329, hsa-mir-322 and hsa-mir-346 require further experimental validation to be considered as bona fide miRNA precursors. It was however, surprising that, in the predicted hairpin structures of mir-329, -322 and -346, the anti- sense sequence of the miR was more conserved than the miR itself (Fig. 1A–C). Significantly, a mutation in the antisense sequence of miR-322 was accompanied by a compensatory change in the miR sequence, so that a G ⁄ C base pair in the mouse precursor was replaced by an A ⁄ U in the human one. The single change in the antisense sequence of miR-329 occurred at an unpaired position in both human and rodent precursor hairpin structures. As it is difficult to conceive that evolution- ary pressure might be higher on the antisense than on the sense strand [8], this may suggest that the antisense strand was cloned accidentally in certain cases [16]. In a few cases, the homology search allowed local- ization of human sequences similar to some rodent miRNA precursors but that had accumulated deleteri- ous mutations. For example, the human orthologous sequence of rodent mir-350 could be localized in an intron of the KARP-1-binding protein (KAB) gene. However, a nine base-pair deletion in the human and chimpanzee genomes removed the first seven nucleo- tides of the mature microRNA (Fig. 1D). It this case, it is noteworthy that the antisense strand accumulated mutations, possibly due to a lack of selective pressure after the inactivation of mir-350 by the deletion in the mature miRNA sequence. Human and mouse miRNA precursors reside in conserved regions of synteny Using the UCSC Genome Browser, I examined whether orthologous human and mouse miRNA precursors reside in conserved synteny regions. This proved to be the case for miRNAs located in known coding genes (see below). Many human miRNAs are located in introns of noncoding mRNAs. In general in these cases, the mRNA was not or poorly conserved in the mouse genome. When human miRNAs were in nonconserved genes, or outside characterized genes, I examined the known flanking genes. In all cases but three, human and mouse miRNAs were found to reside in conserved synteny regions. The three appar- ent discrepancies in the position of a miRNA in the human and mouse genomes were hsa-mir-9-3, hsa-mir- 339 and hsa-mir-326. As detailed in Appendix S1, these cases most probably originate from errors in the present assembly of the mouse genome. Orthologous human and mouse miRNAs thus reside either in introns of orthologous genes, and ⁄ or in conserved synteny with surrounding genes. Human miRNA precursors that reside in introns of known genes Eighty one human miRNA precursors were found to be located in an intron of a known gene, or of a gene defined by a complete cDNA sequence, in the sense orientation (Table S2). It is however, important to note that human miRNAs that were classified as located outside of known genes might in fact reside in still uncharacterized splicing variants. For example, hsa-mir-10b is located 972 nucleotides upstream of the HOXD4 gene (NM_014621). However, mmu-mir-10b resides in an intron of a long form of the mouse Hoxd4 pre-mRNA (NM_010469), but this alternative form has no documented human orthologue. According to current models, intronic miRNA pre- cursors that have the same orientation as their host gene might be produced upon cleavage of the spliced intron by the Drosha endonuclease. In certain cases, the miRNA sequence is included in intronless ESTs that are often members of a cluster of overlapping, also intronless, ESTs. It is striking that, often, no such EST cluster was found in the other introns of the miRNA host gene (e.g. hsa-mir-103–2 and hsa-mir-98) or only in adjacent introns. Due to the uncertainty in the orientation of intronless ESTs, it was not possible to assess the orientation of the clusters relative to that of the host gene, and that of the microRNA. Never- theless, this suggests that introns that host microRNAs might be particularly stable. Alternatively, certain of these intronic miRNAs might be produced by unchar- acterized transcription units embedded in the same orientation in the apparent host gene. However, this possibility appears unlikely, as to my knowledge, there is only two examples of such a situation in the human genome [22,23]. New human and mouse microRNA gene M. J. Weber 64 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS In addition, 17 miRNAs were located in an intron of a known gene, but in the antisense orientation (Table S3). Among these, 10 were in a miRNA cluster (see below). To determine how these miRNAs might be generated, their genomic context was carefully explored. As shown in Table S3, several miRNAs in this category are in fact in a transcription unit that has an orientation opposite that of the apparent host gene. In particular, hsa-mir-302 lies within an intron of the HDCMA18P gene in the antisense orientation, but in an intron of ESTs BG207228 and BU565001 in the sense orientation (Fig. 2A). These two ESTs largely overlap the HDCMA18P transcription unit in the opposite orientation. Similarly, hsa-let-7d resides in the gene defined by the BC045813 mRNA in the opposite orientation (Fig. 2B). In addition, it is located in a cluster of  50 unspliced ESTs that spans about 2-kb and over- laps the intronless BC064349 mRNA. The orientation of this cluster is opposite that of the BC045813 mRNA, as shown by the polyA tails of the BC064349 mRNA and of several ESTs (Fig. 2B). These data thus strongly suggest that hsa-let-7d, and possibly hsa-let-7f-1 and hsa-let-7a-1, are part of an intronless transcript antisense to the BC045813 transcription unit. A third example is hsa-mir-142 that lies within an intron of the AK090885 gene in the antisense orienta- tion, but also in the antisense, intronless and polyaden- ylated AX721088 mRNA (1.6kb). Interestingly, a natural chromosome translocation fuses the 5 0 portion of hsa-mir-142 to a truncated c-Myc gene in aggressive B-cell leukemia [19,24]. This translocation most prob- ably fuses the AX721088 transcription unit to the c-Myc gene. In several cases, the miRNA was embedded in a cluster of intronless ESTs, the orientation of which could not be ascertained. This however, cannot be con- sidered as indicative of an antisense transcript, as a similar observation was made for some miRNAs that reside in introns in the same orientation as the host gene (see above). Taken together, these observations suggest that when miRNA genes are located in introns of known genes in the antisense orientation, they might in fact be part of transcription units in opposite orientation to the presumptive host gene. In the case of hsa-mir- 302, the microRNA is clearly located in an intron of Fig. 2. Antisense intronic miRNAs. (A) Localization of hsa-mir-302. This miRNA resides in an intron of the HDCMA18P gene in the antisense orientation but in an intron of ESTs BG207228 and BU565001 in the sense orientation. In all figures, miRNA genes are colored in green when they reside on the upper strand, and in magenta when on the lower strand. (B) Localization of hsa-let-7d. This miRNA resides in an intron of pre-mRNAs BC045813 and BC036695 in the antisense orientation. The last exon of these two mRNAs overlaps the intronless BC064349 mRNA in the antisense direction. The asterisks indicate the positions of the polyA tails of mRNAs and ESTs. The miRNA hsa-let-7d resides in a cluster of several intronless ESTs, only some of which are shown. The orientation of this cluster is antisense to that of the BC045813 mRNA, as indicated by the presence of a polyA tail in the sequence of mRNA BC0644349 and of two ESTs. The corres- ponding transcription unit might also contain hsa-let-7a-1 and hsa-let-7f-1 in the sense orientation. Figures 2 and 3 are adapted from windows of the UCSC Genome Browser. M. J. Weber New human and mouse microRNA gene FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 65 the antisense transcript (Fig. 2A). In other cases, like hsa-mir-142, hsa-mir-133a-1 ⁄ mir-1-2 and possibly hsa- let-7d ⁄ let )1 -7f-1 ⁄ let )1 -7a-1, the microRNA is located in an exon of the antisense transcript. Human miRNA precursors that overlap with exons of known genes or ESTs After intersecting the new human miRNA table of the UCSC Genome Browser with the chrN_EST and chrN_mRNA tables, I examined miRNAs that are included in, or overlap with, exons. The miRNAs that were only included in intronless ESTs were for the most part not further examined due to the uncertainty in EST orientation. In addition, the localization of sev- eral miRNAs in an exon possibly corresponded to intron retention events (hsa-mir-126, mir-25 and -mir- 224). Accordingly, these miRNAs were classified as intronic. Except for the four examples discussed below (hsa- mir-135a-1, hsa-mir-99b, hsa-let-7e, hsa-mir-125a), miRNAs that are embedded in, or overlap with exons of known transcripts, are always in the same orienta- tion. The corresponding genes were generally noncod- ing, except for hsa-mir-198, which resides in the 3 0 -UTR of the FSTL1 (follistatin-like) gene, and for hsa-mir-133a-2, located in the C20orf166 gene that encodes a potential 117 amino acid protein. In addi- tion, hsa-mir-21 probably resides in the sense orienta- tion in an alternative form of the 3 0 -UTR of the VMP1 gene, characterized by mRNA BC053563. Accordingly, mmu-mir-21 is located in the 3 0 -UTR of the mouse VMP1 ortholog, 4930579A11Rik, also in the sense orientation. These three cases are particularly intriguing, as exci- sion of the miRNA precursor from the host mRNA by the Drosha endonuclease would probably trigger the degradation of the rest of the mRNA. A similar mech- anism is documented in E. coli, where RNase III cleaves its own mRNA at a stem-loop structure and triggers its degradation [25]. Similarly, RNase III cleaves the polycistronic metY-nusA-infB RNA, to release the metY tRNA and initiate the decay of the nusB-infB protein-coding mRNA [26]. Whether such a mechanism also operates in higher eucaryotes remains speculative. Of note, no rodent orthologue of hsa-mir- 198 could be found by a BLAT search in the mouse and rat genomes. Four miRNAs (hsa-mir-34b, hsa-mir-205, hsa-mir- 133a-2 and hsa-mir-99b) overlap a splicing site. In the three first cases, it is not clear how the miRNA precur- sor is produced. It might originate either from an exon, or from an intron of uncharacterized alternative forms of the host gene mRNA. The case of hsa-mir- 99b is further discussed below. MiRNAs complementary to expressed sequences and potential regulation of glycerate kinase gene expression by miR-135a The mouse mmu-mir-135a-1 miRNA resides in the antisense orientation in an alternative, long form of the 3 0 -UTR of the 6230410P16Rik gene, the ortho- logue of the human GLYCTK (glycerate kinase) gene (Fig. 3A). Therefore, mmu-miR-135a is perfectly com- plementary to the alternative form of Glyctk mRNA and might regulate its stability by an siRNA mechan- ism. Interestingly, mmu-mir-135a-1 also resides in an intron of the spliced AK051019 mRNA in the sense orientation; the latter might thus be the actual miRNA host gene. These two transcriptional units are largely overlapping, so that several genomic segments are exonic on both strands (Fig. 3A). A similar situation holds for the human genome: hsa-mir-135a-1 is located 741-base pairs downstream of the GLYCTK gene, in the antisense orientation. This miRNA is embedded in a cluster of 10 overlap- ping intronless ESTs that might be part of a longer, alternative form of the GLYCTK 3 0 -UTR (Fig. 3B). This hypothesis is supported by the fact that several ESTs of this cluster (AI493054, AI380271, AW204878, AW207007 and BM555864) are polyadenylated. In addition, hsa-mir-135a-1 resides, in the sense orienta- tion, in an intron of EST AI936688. Based on these observations, it is tempting to speculate that, in both mouse and human, mir-135a-1 is produced from an intron of the host gene (AK051019 and AI936688 in mouse and human, respectively) and can direct the degradation of a long form of the glycerate kinase mRNA by an RNA interference mechanism. Accord- ingly, a switch from GLYCTK to AI936688 gene tran- scription would be accompanied by the production of hsa-miR-135a, which could base-pair with pre-existing GLYCTK long mRNAs and trigger their degradation. This mechanism would thus block glycerate kinase production at both the transcriptional and transla- tional levels, while the shorter form of glycerate kinase mRNA, which results from the use of an alternative polyA site, would not be affected. In addition, hsa- miR-135a could be produced from its second precur- sor, hsa-mir-135a-2. As noted above, hsa-let-7e and hsa-mir-125a are located in the first exon of mRNA AK125996, in the antisense orientation, whereas hsa-mir-99b overlaps the splicing donor site (Fig. 3C). Interestingly, the first exon of mRNA AK125996 overlaps that of the antisense New human and mouse microRNA gene M. J. Weber 66 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS mRNA AY358799 over 113 nucleotides. Although the overlapping region is outside of the miRNA-containing segment, it is tempting to speculate that these three miRNAs reside, in the sense orientation, in a longer form of the AY358799 mRNA, and might regulate the translation and ⁄ or stability of the mRNA AK125996. Fig. 3. Antisense exonic miRNAs. (A) Localization of mmu-mir-135a-1. This miRNA resides in the antisense orientation in an alternative lon- ger form of the 3 0 -UTR of the mouse 6230410P16Rik mRNA. This gene is the orthologue of the human GLYCTK gene, as shown by BLATing the sequence of the human protein (see BLAT track). The miRNA mmu-mir-135a-1 is also located in an intron of the antisense AK051019 mRNA, indicated in red. (B) Localization of hsa-mir-135a-1. This miRNA resides in a cluster of ESTs downstream of the GLYCTK gene, in the opposite orientation. The asterisks indicate the localization of the polyA tail of some of these ESTs. In addition, hsa-mir-135a-1 resides in the sense orientation in an intron of EST AI936688 (indicated in red). (C) Localization of hsa-mir-99b, hsa-let-7e and hsa-mir-125a. The latter two miRNAs are in the first exon of the AK125996 mRNA, in the antisense orientation, whereas hsa-mir-99b overlaps the splicing donor site. Note that the AK125996 mRNA overlaps in the antisense direction with the BC041134 and AY358799 mRNAs. M. J. Weber New human and mouse microRNA gene FEBS Journal 272 (2005) 59–73 ª 2004 FEBS 67 Only two other vertebrate miRNAs have been previ- ously shown to be fully complementary to a cellular mRNA: mmu-mir-127 and mmu-mir-136 reside in the intronless Rtl1 gene, in the opposite orientation. Whereas the Rtl1 gene is paternally expressed, the two miRNAs are only expressed from the maternal chro- mosome [27]. This reciprocal imprinting suggests that mmu-miR-127 and mmu-miR-136 regulate Rtl1 gene expression by an siRNA mechanism. This situation probably also holds for the human genome, as both hsa-mir-127 and hsa-mir-136 reside in the inverse ori- entation within the presumptive human orthologue of Rtl1 (XM_352144). Therefore, this study uncovers new cases where a miRNA might regulate the stability of a cellular mRNA through an siRNA mechanism. In all cases dis- cussed above, the miRNA resides on the opposite strand relative to its target mRNA. This situation dif- fers from that of miR-196a, which is fully complement- ary to the 3 0 -UTR of HOXB8 mRNAs, except for a G ⁄ U wobble [28]. In that case, the miRNA gene resides at distance from its target gene, although in the same HOX locus. MicroRNAs in gene families As shown in Table 2, many related human precursor miRNAs reside in corresponding introns of paralogous genes. The mature miRNAs are either identical (miR- 15, miR-218), closely related (miR-199a and b, miR26a and b), or display significant homology (hsa- mir-148b and -152, hsa-mir-107, -103-1 and -103-2). Therefore, the sequences of these intronic miRNAs have been largely conserved after gene duplications, raising the possibility that their function might have been conserved as well. This also suggests that addi- tional genes in the families shown in Table 2 might contain still uncharacterized miRNAs. Intriguingly, hsa-mir-211 and -204 are located within an intron of the TRPM1 and TRPM3 genes, respectively, that bracket paralogous exons. These localizations are con- served in the mouse genome. However, these two miR- NAs have no sequence similarity. In this case, it is thus possible that the presence of miRNAs in the introns of the TRPM1 and TRPM3 genes is posterior to the expansion of the TRPM gene family. This hypothesis is reinforced by the fact that the other members of the family, TRPM2 and TRPM4 through TRPM8, do not host known miRNAs. The large dif- ference in the size of the introns of the TRPM1 and TRPM3 genes that host mir-211 and mir-204 (3 and 44 kb, respectively) also suggests extensive rearrange- ments posterior to gene duplication. It is also intriguing to note that hsa-mir-199b, -199a-1 and -199a-2 reside in introns of the DNM1, DNM2 and DNM3 genes, respectively, but in the opposite orientation (Table 2). As discussed above, the DNM genes are thus probably not the actual hosts for these miRNAs. Indeed, hsa-mir-199a-2 is embedded in a large cluster of ESTs antisense to the DNM3 gene that also contains hsa-mir-214 (Table S3). Similarly, mmu-mir-199a-2 and mmu-mir-214 reside in the opposite orientation in an intron of the dynamin 3 gene (9630020E24Rik) and are embedded in a large cluster of antisense mRNAs and ESTs (not shown). Although there is no clear evidence for tran- scripts antisense to DNM1 and DNM2 genes, these observations suggest that the conservation of highly related miRNAs in the DNM gene family might result from the expansion in the vertebrate genomes of antisense transcripts in addition to the DNM genes themselves. Clusters of microRNAs The existence of miRNA clusters has been already noted [14,29,30], but a precise definition of a cluster Table 2. MicroRNA that reside in introns of paralogous gene families. MicroRNA Host gene Function Notes hsa-mir-199b DNM1 Dynamins hsa-mir-199a-1 DNM2 hsa-mir-199a-2 DNM3 hsa-mir-26b CTDSP1 CTD RNA polII phosphatases hsa-mir-26a-1 CTDSPL hsa-mir-26a-2 CTDSP2 hsa-mir-153–1 PTPRN Protein tyrosine phosphatases a hsa-mir-153–2 PTPRN2 hsa-mir-218–1 SLIT2 Slit homologs, axonal guidance b hsa-mir-218–2 SLIT3 hsa-mir-107 PANK1 Pantothenate kinases c hsa-mir-103–2 PANK2 hsa-mir-103–1 PANK3 hsa-mir-211 TRPM1 Transient receptor potential cation channels (melastatins) d hsa-mir-204 TRPM3 hsa-mir-148b COPZ1 Coatamer protein complex, subunits zeta e hsa-mir-152 COPZ2 a mmu-mir-153 is located in the Ptprn2 gene. No documented miRNA resides in the mouse Ptprn gene. b No documented miRNA resides in the human SLIT1 gene. In the October 2003 freeze of the mouse gen- ome, the Slit2 gene, as evidenced by BLATing the human Slit2 protein, comes in two parts, located on chr5_random (aa 133–538) and chr5 (aa 539–1529). Mmu-mir-218–1 resides in the chr5_random part. c hsa-mir- 103–2 and mir-103–1 are closely related to hsa-mir-107 (87 and 91% identity, respectively). d hsa-mir-211 and )204 have no sequence homol- ogy. e hsa-mir-148b and )152 display significant homology (77% identity over 66 nucleotides). New human and mouse microRNA gene M. J. Weber 68 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS [...]... known rodent microRNA precursors in the human genome has led to the identification of 35 new miRNA genes Similarly, 42 new mouse miRNAs were found by orthology with known human miRNA precursors Added to those deposited in the miRNA registry, about 200 miRNA genes are now localized in both human and mouse genomes, a number close to the total number (255) predicted by Lim et al [13] Among the 196 human miRNA... S1 Human and mouse miRNA precursors reside in conserved regions of synteny Fig S1 miRNAs gene candidates clustered with known miRNAs genes Table S1 New microRNA precursors deposited in The miRNA Registry (version 3.0), at the Wellcome Trust Sanger Institute Table S2 Human miRNAs that reside in introns of known genes in the sense orientation Table S3 Human miRNAs that reside in introns of known genes. .. between human and mouse: miR-323, miR-134, miR-154, miR-323 and miR-299, while hsamiR-329 differs from its rodent orthologue by four nucleotides (Table 1) Moreover, the human orthologue of mmu-mir-300 could not be found by a BLAT search The reason for such a differential conservation of miRNAs within this cluster will probably be understood when their function is elucidated Prediction of miRNA candidates... has also been published [53] Methods The sequences of human and mouse mature (miR) and precursor (mir) miRNAs were obtained from The miRNA Registry (version Rfam2.2), and the entire set of sequences FEBS Journal 272 (2005) 59–73 ª 2004 FEBS M J Weber was subjected to a BLAT search [54] in the human and mouse genomes (UCSC version hg16, July 2003 and UCSC version mm4, October 2003, respectively) on the... miRNA candidates near known miRNA genes The analysis of highly conserved regions with a hairpin structure near known miRNAs can reveal numerous new miRNA candidates For example, such a sequence resides 94 base pairs from hsa-mir-144 (Figure S1A) The orthologous mouse sequence is located 101 base 69 New human and mouse microRNA gene pairs from mmu-mir-144 Two other candidates are located near hsa-mir-195,... the human, chimpanzee, mouse and rat genomes (Figure S1D) and might thus contain additional candidates As pointed out in reference [8], there are more conserved hairpin structures in the human genome than they are miRNAs However, restricting the infomatic search of conserved hairpin structures to the vicinity of known miRNAs will certainly unravel many interesting candidates Conclusions A systematic search. .. hsa-miR-23 ⁄ 27 ⁄ 24-2 and hsa-miR17 ⁄ 18 ⁄ 19a ⁄ 20 ⁄ 19b-1 clusters [32] A comprehensive list of 37 human miRNA clusters is presented in Table S4 All of them are conserved in the mouse genome, although some mouse clusters contain additional members, for which no human orthologue could be detected (mmu-mir-300, -329 and -341) Among the 37 human clusters, 19 are located in RefSeq genes, known genes, or mRNAs... (2004) MicroRNAs: genomics, biogenesis, mechanism, and function Cell 116, 281–297 FEBS Journal 272 (2005) 59–73 ª 2004 FEBS New human and mouse microRNA gene 7 Carrington JC & Ambros V (2003) Role of microRNAs in plant and animal development Science 301, 336–338 8 Lai EC (2003) microRNAs: runts of the genome assert themselves Curr Biol 13, R925–R936 9 Lee RC, Feinbaum RL & Ambros V (1993) The C elegans... Bartel DP (2004) MicroRNA- directed cleavage of HOXB8 mRNA Science 304, 594–596 29 Mourelatos Z, Dostie J, Paushkin S, Sharma A, Charroux B, Abel L, Rappsilber J, Mann M & Dreyfuss G (2002) miRNPs: a novel class of ribonucleoproteins containing numerous microRNAs Genes Dev 16, 720–728 30 Lagos-Quintana M, Rauhut R, Meyer J, Borkhardt A & Tuschl T (2003) New microRNAs from mouse and human RNA 9, 175–179... computational-experimental approach predicts human microRNA targets Genes Dev 18, 1165– 1178 52 Rodriguez A, Griffiths-Jones S, Ashurst JL & Bradley A (2004) Identification of mammalian microRNA host genes and transcription units Genome Res 14, 1902– 1910 53 Seitz H, Royo H, Bortolin ML, Lin SP, Ferguson-Smith AC & Cavaille V (2004) A large imprinted microRNA gene cluster at the mouse Dlk1-Gtl2 domain Genome Res . New human and mouse microRNA genes found by homology search Michel J. Weber Laboratoire de Biologie Mole ´ culaire Eucaryote, UMR5099, CNRS and Universite ´ Paul. predic- tion of new mRNA targets. Results and Discussion New potential human and mouse microRNA precursors The entire set of human and mouse precursor and mature