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Biogenesis of Small Rnas in Animals

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Biogenesis of Small Rnas in Animals REVIEWS POST-TRANSCRIPTIONAL CONTROL Biogenesis of small RNAs in animals V. Narry Kim*, Jinju Han* and Mikiko C. Siomi‡ Abstract | Small RNAs of 20–30 nucleotides can target both chromatin and transcripts, and thereby keep both the genome and the transcriptome under extensive surveillance. Recent progress in high-throughput sequencing has uncovered an astounding landscape of small RNAs in eukaryotic cells. Various small RNAs of distinctive characteristics have been found and can be classified into three classes based on their biogenesis mechanism and the type of Argonaute protein that they are associated with: microRNAs (miRNAs), endogenous small interfering RNAs (endo-siRNAs or esiRNAs) and Piwi-interacting RNAs (piRNAs). This Review summarizes our current knowledge of how these intriguing molecules are generated in animal cells. Heterochromatin The first small RNA, lin‑4, was discovered in 1993 by The best understood among the three classes, Highly condensed regions of genetic screens in nematode worms1,2. The number of miRNAs are generated from local hairpin structures by the genome in which known small RNAs has since expanded substantially, the action of two RNase III-type proteins, Drosha and Dicer transcription is generally mainly as a result of the cloning and sequencing of size‑ (BOX 2). Mature miRNAs of ~22 nt are then bound by limited. fractionated RNAs3–5. The recent development of deep‑ Ago‑subfamily proteins. miRNAs target mRNAs and 6,7 RNase III-type protein sequencing technologies and computational prediction thereby function as post‑transcriptional regulators. An endonuclease that cleaves methods8–11 has accelerated the discovery of less abun‑ The longest of the three classes, piRNAs (24–31 nt in double-stranded RNAs and dant small RNAs. The functions of small RNAs range length) are associated with Piwi‑subfamily proteins. creates 5′-phosphate and heterochromatin 3′-hydroxyl termini, leaving from formation to mRNA destabilization Intriguingly, the biogenesis of piRNAs does not depend 12,13 14 2-nucleotide 3′ overhangs. and translational control . Through such extensive on Dicer . piRNAs are highly abundant in germ cells patrolling across the genome and transcriptome, small and at least some of them are involved in transposon RNAs are involved in almost every biological process, silencing through heterochromatin formation or RNA including developmental timing, cell differentiation, cell destabilization. endo‑siRNAs have been studied mostly proliferation, cell death, metabolic control, transposon in Drosophila melanogaster, although they have also silencing and antiviral defence. been found in mouse oocytes and embryonic stem (ES) ‘Small RNA’ is a rather arbitrary term, because it was cells15–17. Despite their similarity with miRNAs in terms previously used for other non‑coding RNAs, such as small of their association with Ago‑subfamily proteins, endo‑ nuclear RNAs (snRNAs) and transfer RNAs (tRNAs). siRNAs differ from miRNAs in that they are derived *School of Biological Bacterial short regulatory RNAs have also been referred from long double‑stranded RNAs (dsRNAs) and are Sciences and Center for to as small RNAs, but they are not related to eukaryotic dependent only on Dicer and not on Drosha18–20. They National Creative Research, small RNAs. What distinguishes and defines eukaryo‑ are also slightly shorter (~21 nt) than miRNAs. At least Seoul National University, tic small RNAs in the RNA silencing pathway is their some of the endo‑siRNAs have been shown to function Seoul, 151-742, Korea. ‡Keio University School of limited size (~20–30 nucleotides (nt)) and their associa‑ as post‑transcriptional regulators that target RNAs. Medicine, 35 Shinanomachi, tion with Argonaute (Ago)‑family proteins (BOX 1; TABLE 1). This Review summarizes our current knowledge of Shinjuku-ku, Tokyo The Ago family can be grouped further into two clades: the biogenesis pathways of small RNAs. The focus of the 160-8582, and the Japan the Ago subfamily and the Piwi subfamily. At least three Review will remain on animal small RNAs, mainly in Science and Technology Agency (JST), Core Research classes of small RNAs are encoded in our genome, based mammals and flies. Several excellent reviews on small 21–24 for Evolutionary Science on their biogenesis mechanism and the type of Ago pro‑ RNAs in plants and yeast are available elsewhere . and Technology (CREST), tein that they are associated with: microRNAs (miRNA s), Saitama 332-0012, Japan. endo genous small interfering RNAs (endo‑siRNAs or microRNA biogenesis Correspondence to V.N.K. esiRNA s) and Piwi‑interacting RNAs (piRNAs). It should miRNAs are single‑stranded RNAs (ssRNAs) of ~22 nt and M.C.S. e-mails: [email protected]; be noted, however, that the recent discoveries of numerous in length that are generated from endogenous hair‑ 25 [email protected] non‑canonical small RNAs have blurred the boundaries pin‑shaped transcripts . miRNAs function as guide doi:10.1038/nrm2632 between the classes. molecules in post‑transcriptional gene regulation by 126 | FEBRUARY 2009 | VOLUME 10 www.nature.com/reviews/molcellbio © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Box 1 | Argonaute proteins and their associated small RNAs miRNA genes and their transcription. At present, the miRNA database contains 154 Caenorhabditis elegans, The Argonaute (Ago) a PAZ MID PIWI 152 D. melanogaster, 337 Danio rerio (zebrafish), 475 Gallus family can be classified N C gallus (chicken), 695 human and 187 Arabidopsis thal- into two subclades: the iana miRNAs. miRNAs are even present in simple multi‑ Ago subfamily and the b cellular organisms, such as poriferans (sponges) and Piwi subfamily (TABLE 1). Catalytic site PIWI 27 bilaterian The Ago proteins are cnidarians (starlet sea anemone) . Many of the expressed ubiquitously, animal miRNA s are phylogenetically conserved; ~55% interact with microRNAs of C. elegans miRNAs have homologues in humans, (miRNAs) or small MID which indicates that miRNAs have had important roles interfering RNAs PAZ throughout animal evolution28. Animal miRNAs seem (siRNAs), and function as 3′ end of to have evolved separately from those in plants because post-transcriptional guide strand 5′ end of their sequences, precursor structure and biogenesis regulators. The Piwi 29,30 guide strand mechanisms are distinct from those in plants . proteins are abundantly Most mammalian miRNA genes have multiple iso‑ expressed in germ cells and function in transposon silencing, together with forms (paralogues) that are probably the result of gene Piwi-interacting RNAs (piRNAs). Nature Reviews | Molecular Cell Biology Ago-family proteins are composed of three characteristic domains: the PAZ, MID duplications. For instance, the human genome has and PIWI domains (see the figure, part a). The PAZ domain serves as a docking site for 12 loci for let‑7‑family miRNAs. Paralogues often have the 3′ end of small RNA181–184, whereas the MID domain anchors the 5′ terminal identical sequences at nucleotide positions 2–7 relative nucleotide183–187 (see the figure, part b). Recent studies have determined the structure to the 5′ end of the miRNA. Because these six nucleo‑ of Thermus thermophilus Ago with a guide strand and target strand duplex183,184. The tides (called seed) are crucial in base pairing with the PIWI domain has a structure that is similar to RNase H, which cuts the RNA strand of target mRNA, the paralogues are thought to act redun‑ an RNA–DNA hybrid. Indeed, the PIWI domain of some Ago proteins can cleave the dantly. However, because the 3′ sequences of miRNAs target RNA bound to small RNA: this is called slicer activity. Of the four human Ago also contribute to target binding and because the expres‑ proteins (AGO1–4; also known as EIF2C1–4), only AGO2 has slicer activity, whereas in sion patterns of these sister miRNAs are often different Drosophila melanogaster all Ago and Piwi proteins possess slicer activity. Apart from from each other, members of the same seed family might the endonucleolytic cleavage that is mediated by the PIWI domain, the Ago proteins 31 can induce translational repression and exonucleolytic mRNA decay through have distinct roles in vivo . interaction with other protein factors13. Approximately 50% of mammalian miRNA loci The fly Piwi proteins Aubergine (AUB) and AGO3 can cleave target mRNAs, are found in close proximity to other miRNAs. These resulting in the silencing of retrotransposons, other intergenic repetitive elements clustered miRNAs are transcribed from a single poly- and protein-coding genes, such as Stellate (also known as SteXh)145,148. PIWI might cistronic transcription unit (TU)32, although there may function differently from AUB and AGO3, as it associates with chromatin and be exceptional cases in which individual miRNAs are interacts with HP1a (heterochromatin protein 1a), which is involved in derived from separate gene promoters. Some miRNAs heterochromatin formation. Thus, PIWI might contribute to the epigenetic control of are generated from non‑coding TUs, whereas others are the fly genome188. Consistently, AUB and AGO3 are localized in the cytoplasm, encoded in protein‑coding TUs (FIG. 1). Approximately whereas PIWI is found in the nucleus145–148. The expression patterns are also different; 40% of miRNA loci are located in the intronic region of PIWI, but not AUB and AGO3, was detected in the soma of ovaries, indicating that PIWI has a function outside of germ cells. Epigenetic changes were observed non‑coding transcripts, whereas
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