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And Now Introducing Mammalian Mirtrons

2007; Elsevier BV; Volume: 13; Issue: 5 Linguagem: Inglês

10.1016/j.devcel.2007.10.010

1878-1551

Shih‐Peng Chan, Frank J. Slack,

RNA modifications and cancer

Mirtrons are short hairpin introns recently found in flies and nematodes that provide an alternative source for animal microRNA biogenesis and use the splicing machinery to bypass Drosha cleavage in initial maturation. The presence of mirtrons outside of invertebrates was not previously known. In the October 26 issue of Molecular Cell, Berezikov et al. expose a number of short mammalian introns as mirtrons. Mirtrons are short hairpin introns recently found in flies and nematodes that provide an alternative source for animal microRNA biogenesis and use the splicing machinery to bypass Drosha cleavage in initial maturation. The presence of mirtrons outside of invertebrates was not previously known. In the October 26 issue of Molecular Cell, Berezikov et al. expose a number of short mammalian introns as mirtrons. microRNAs (miRNAs) are noncoding small RNAs of ∼22 nucleotides (nts) that negatively regulate gene expression by binding to the 3′ untranslated region (3′UTR) of messenger RNA transcripts, triggering translational repression or cleavage of the target (reviewed by Bartel, 2004Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29415) Google Scholar). miRNAs are generally transcribed by RNA polymerase II as primary miRNAs (pri-miRNAs) that range from hundreds to thousands of nts in length and contain one or more extended hairpin structures (reviewed by Du and Zamore, 2005Du T. Zamore P.D. Development. 2005; 132: 4645-4652Crossref PubMed Scopus (670) Google Scholar). In animals, the nuclear RNase III enzyme Drosha, collaborating with DGCR8/Pasha/PASH-1, cleaves both strands near the base of the primary stem-loop and yields the precursor miRNA (pre-miRNA), an ∼65-nt stem-loop that harbors the miRNA in the 5′ or 3′ half of the stem (Lee et al., 2003Lee Y. Ahn C. Han J. Choi H. Kim J. Yim J. Lee J. Provost P. Rådmark O. Kim S. et al.Nature. 2003; 425: 415-419Crossref PubMed Scopus (3969) Google Scholar). The cleavage by Drosha defines one end of the mature miRNA and generates a 5′ phosphate and an ∼2-nt 3′ overhang. After being exported to the cytoplasm by Exportin-5/RAN, pre-miRNAs are further cleaved by the RNase III Dicer to define the other end of the mature miRNA and produce the double-stranded miRNA/miRNA∗ duplexes (Du and Zamore, 2005Du T. Zamore P.D. Development. 2005; 132: 4645-4652Crossref PubMed Scopus (670) Google Scholar). One strand of the miRNA/miRNA∗ duplex is then preferentially incorporated into the RNA-induced silencing complex (RISC) and can function to negatively regulate target genes (Du and Zamore, 2005Du T. Zamore P.D. Development. 2005; 132: 4645-4652Crossref PubMed Scopus (670) Google Scholar). In animals, most miRNAs either originate from their own transcription units, or derive from the exons or introns of other genes (Du and Zamore, 2005Du T. Zamore P.D. Development. 2005; 132: 4645-4652Crossref PubMed Scopus (670) Google Scholar). All of them seem to require both Drosha and Dicer for the two sequential cleavage events in maturation. Recently, the Bartel and Lai laboratories found that short hairpin introns in flies and nematodes can be alternative sources of miRNAs (Okamura et al., 2007Okamura K. Hagen J.W. Duan H. Tyler D.M. Lai E.C. Cell. 2007; 130: 89-100Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar, Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar). By high-throughput pyrosequencing, Bartel and colleagues identified 14 introns in D. melanogaster with a sequence and predicted stem-loop structure of a pre-miRNA, but without the lower stem structure and flanking single-stranded segments of the pri-mRNA (Figure 1), which mediate the recognition and cleavage by the DGCR8/Drosha complex (Han et al., 2006Han J. Lee Y. Yeom K.H. Nam J.W. Heo I. Rhee J.K. Sohn S.Y. Cho Y. Zhang B.T. Kim V.N. Cell. 2006; 125: 887-901Abstract Full Text Full Text PDF PubMed Scopus (1173) Google Scholar). In these cases, the 5′ and 3′ portions of the intron were inversely complementary to each other and the base-pairing capacity abruptly ended at the borders of the intron. The small RNAs obtained by pyrosequencing originated from the outer edges of the intron, and the most abundant ones, annotated as the mature miRNAs, derived from the 3′ arm of the predicted stem-loop structure (Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar). Because of their pre-miRNA and intronic characteristics, the Bartel laboratory named these introns "mirtrons." They also found three C. elegans mirtrons and reclassified a previously annotated miRNA gene, mir-62, as a mirtron due to its intron-edge location and lack of pri-miRNA structures (Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar). Lai and colleagues detected expression of D. melanogaster mirtrons in various tissues and developmental stages (Okamura et al., 2007Okamura K. Hagen J.W. Duan H. Tyler D.M. Lai E.C. Cell. 2007; 130: 89-100Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar). Both groups demonstrated that mirtrons are processed initially by the splicing machinery, enter the miRNA-processing pathway without Drosha-mediated cleavage, and produce functional small regulatory RNAs (Okamura et al., 2007Okamura K. Hagen J.W. Duan H. Tyler D.M. Lai E.C. Cell. 2007; 130: 89-100Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar, Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar). Mirtrons have only been found in flies and nematodes to date, and it was suggested that mirtrons might be more common in these species because they have a higher proportion of short introns with lengths typical of pre-miRNA hairpins than mammals do (Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar). Now, in the October 26 issue of Molecular Cell, Berezikov et al., 2007Berezikov E. Chung W.J. Willis J. Cuppen E. Lai E.C. Mol. Cell. 2007; 28: 328-336Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar report the discovery of mammalian mirtrons. They found three mirtrons, sblock2, sblock3, and sblock4, that generate orthologous small RNAs in human, macaque, chimpanzee, rat, and mouse by analyzing 25,935 introns, 50–200 nt in length, from the UCSC Genome Browser Database (Kuhn et al., 2007Kuhn R.M. Karolchik D. Zweig A.S. Trumbower H. Thomas D.J. Thakkapallayil A. Sugnet C.W. Stanke M. Smith K.E. Siepel A. et al.Nucleic Acids Res. 2007; 35: D668-D673Crossref PubMed Scopus (244) Google Scholar). Small RNAs from sblock4 were recently cloned by Tuschl and colleagues independently and annotated as the canonical miRNA mir-877 (Landgraf et al., 2007Landgraf P. Rusu M. Sheridan R. Sewer A. Iovino N. Aravin A. Pfeffer S. Rice A. Kamphorst A.O. Landthaler M. et al.Cell. 2007; 129: 1401-1414Abstract Full Text Full Text PDF PubMed Scopus (3007) Google Scholar). To identify additional mammalian mirtrons, Berezikov et al., 2007Berezikov E. Chung W.J. Willis J. Cuppen E. Lai E.C. Mol. Cell. 2007; 28: 328-336Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar sequenced a set of small RNA libraries from human and rhesus macaque brains. Brains were chosen since high-throughput pyrosequencing of D. melanogaster head samples has given the highest diversity of mirtrons and canonical miRNAs, possibly to satisfy the high complexity of neuronal translational regulation. They identified 16 primate-specific mirtrons, and classified 46 additional hairpin introns from human (23 loci), macaque (16 loci), chimpanzee (3 loci), and mouse (4 loci) as mirtron candidates. Several lines of evidence argued that these sequences are not intron degradation products, but rather, mammalian mirtron products that are likely generated via the miRNA-processing pathway, as in flies and nematodes. There are several differences between mammalian and invertebrate mirtrons (Figure 1). Invertebrate mirtrons predominantly generate small RNAs from their 3′ portion, and these 3′ species preferentially start with a 5′ "U" residue, consistent with the bias for the 5′ nt of canonical miRNAs (Lau et al., 2001Lau N.C. Lim L.P. Weinstein E.G. Bartel D.P. Science. 2001; 294: 858-862Crossref PubMed Scopus (2680) Google Scholar). Indeed, the Bartel and Lai groups have shown that several of these species are functional miRNAs (Okamura et al., 2007Okamura K. Hagen J.W. Duan H. Tyler D.M. Lai E.C. Cell. 2007; 130: 89-100Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar, Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar). In contrast, several of the most highly expressed mammalian mirtrons predominantly produce small RNAs from the 5′ portion, which carries a 5′ "G" residue from the conserved splice site. Furthermore, these mirtrons exhibit a stem structure with single nt overhangs at both ends, instead of the typical 2-nt 3′ overhang found in most highly expressed invertebrate mirtrons. To explain this structural constraint, Berezikov et al., 2007Berezikov E. Chung W.J. Willis J. Cuppen E. Lai E.C. Mol. Cell. 2007; 28: 328-336Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar surveyed miRbase and showed that there are many deduced pre-miRNA hairpins lacking perfect 3′ 2-nt overhangs. It is possible that the pre-miRNA processing pathway accepts a range of hairpin structures, or that other factors participate. In addition, unlike in invertebrates, where mirtrons and bulk short introns exhibit similar GC content, in mammals mirtrons exhibit higher GC content than bulk short introns. The GC content of mammalian mirtrons is also higher than those of invertebrate and mammalian canonical miRNAs. The existence of several well-conserved mirtrons among diverse mammals, as well as in Drosophilids and nematodes (Okamura et al., 2007Okamura K. Hagen J.W. Duan H. Tyler D.M. Lai E.C. Cell. 2007; 130: 89-100Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar, Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar), indicates their relatively ancient incorporation into regulatory pathways, and their retention for beneficial reasons. However, since flies, nematodes, and mammals have completely different sets of mirtrons, it is possible that different animals evolved mirtrons independently. On the other hand, most D. melanogaster mirtrons are preserved within species of the melanogaster subgroup (Okamura et al., 2007Okamura K. Hagen J.W. Duan H. Tyler D.M. Lai E.C. Cell. 2007; 130: 89-100Abstract Full Text Full Text PDF PubMed Scopus (755) Google Scholar, Ruby et al., 2007Ruby J.G. Jan C.H. Bartel D.P. Nature. 2007; 448: 83-86Crossref PubMed Scopus (1129) Google Scholar), suggesting that mirtrons are possibly fast-evolving in nature. This idea is supported by the observation that a number of mammalian mirtrons are restricted to the primates, with some presenting conserved hairpin structures restricted to one or few primate subsets. The observation of many newly-evolved mirtrons suggests an easy way for the birth of new regulatory RNAs along with the pre-existing canonical pre-miRNA pathway and highlights their likely contribution to animal evolution. Mammalian Mirtron GenesBerezikov et al.Molecular CellOctober 26, 2007In BriefMirtrons are alternative precursors for microRNA biogenesis that were recently described in invertebrates. These short hairpin introns use splicing to bypass Drosha cleavage, which is otherwise essential for the generation of canonical animal microRNAs. Using computational and experimental strategies, we now establish that mammals have mirtrons as well. We identified 3 mirtrons that are well conserved and expressed in diverse mammals, 16 primate-specific mirtrons, and 46 candidates supported by limited cloning evidence in primates. Full-Text PDF Open Archive