Production and actions of small V. Narry Kim and Mikiko C. Siomi Small (20–30 nt) RNAs are associated with members RNAs (endo-siRNAs) and -interacting RNAs of the (Ago) family, which comprises two (piRNAs). miRNAs control mRNA stability and subfamilies: Ago and Piwi. Based on their biogenesis by targeting cognate mRNAs. endo- MOLECULAR mechanism and the type of Argonaute proteins siRNAs suppress repetitive genes by cleaving their that they associate with, at least three classes of transcripts. Some piRNAs mediate RNA cleavage or small RNAs can be distinguished in eukaryotes: heterochromatin formation of transposons, although CELL BIOLOGY (miRNAs), endogenous small interfering the functions of most piRNAs are still unknown.

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&KEGT &KEGTKPȯKGU &KEGT &KEGTKPȯKGU 2TKOCT[ RTQEGUUKPI 64$2QT2#%6! 64$2QT2#%6! .QCFKPI &KEKPI 4&KPȯKGU .135KPȯKGU

/+9+ /+9+ /+9+ #)1s #)1KPȯKGU #)1s #)1s #)1KPȯKGU #)1 #)1 #)1KPȯKGU OK40# GZQUK40# GPFQUK40# /+.+ The figure shows the mammalian processing pathways with fly components other Ago proteins. piRNAs (24–31 nt) are processed from ssRNA precursors in brackets. miRNAs are ssRNAs of ~22 nt that are generated from that are transcribed from transposons or large piRNA clusters. piRNAs 5GEQPFCT[ transcripts that can fold into hairpin structures. Two RNase III type proteins, associate with Piwi-subfamily proteins; they are not dependent on either /+9+ RTQEGUUKPI /+.+ .QCFKPI Drosha and , mediate nuclear and cytoplasmic processing, respectively, Drosha or Dicer, although the requirement of Drosha has not been formally /+9+ thereby producing the miRNAs that subsequently associate with Ago- tested. They may instead use the activity of the Piwi proteins subfamily proteins (AGO1–4 in humans). In flies, most miRNAs are loaded themselves for their processing. piRNA biogenesis involves primary and /+.+ /+.+ onto AGO1, whereas miRNAs from highly base-paired precursors are sorted secondary processing mechanisms. The strand bias of piRNAs in secondary /+.+ into AGO2. exo-siRNAs are derived from experimentally introduced dsRNAs processing differs between mice and flies (for this reason, fly components are RK40# RK40# or viral RNAs. Human exo-siRNAs are loaded onto AGO1–4, but only AGO2 not shown). Primary processing and loading might occur in the cytoplasm has ‘slicer’ activity. endo-siRNAs are derived from cis or trans sense– because Piwi proteins MIWI and MILI are localized in the cytoplasm. Factors antisense dsRNAs or long hairpins. They are produced by Dicer and interact that are needed for primary processing are unknown. Not shown here are less with Ago-subfamily proteins. In humans, endo-siRNAs are thought to be abundant groups of miRNA-like RNAs that are derived from various sources loaded onto AGO2; it has not been determined whether they associate with of short hairpins in a Drosha-independent manner.

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Table | Eukaryotic small RNAs are associated with Argonaute-family proteins Subfamily Ago-family protein Class of small RNA* Length of small RNA Origin of small RNA‡ Mammals Ago AGO1–4 miRNA 21–23 nt miRNA genes Translational repression, mRNA degradation, mRNA cleavage and heterochromatin formation? endo-siRNA§ 21–22 nt Intergenic repetitive genes, pseudogenes and endo-siRNA clusters mRNA cleavage? Piwi MILI (PIWIL2 in humans) Pre-pachytene piRNA and 24–28 nt Transposons and piRNA clusters Heterochromatin formation (DNA methylation) pachytene piRNA MIWI (PIWIL1 in humans) Pachytene piRNA 29–31 nt piRNA clusters ? MIWI2 (PIWIL4 in humans) Pre-pachytene piRNA 27–29 nt Transposons and piRNA clusters Heterochromatin formation (DNA methylation) (PIWIL3 in humans) ? ? ? ? Drosophila melanogaster Ago AGO1 miRNA 21–23 nt miRNA genes Translational repression and mRNA degradation AGO2 endo-siRNA ~21 nt Transposons, mRNAs and repeats RNA cleavage exo-siRNA ~21 nt Viral genome Viral RNA cleavage Piwi AUB piRNA 23–27 nt Transposons, repeats, piRNA clusters and Su(Ste) locus RNA cleavage AGO3 piRNA 24–27 nt Transposons and repeats (unknown in testis) RNA cleavage PIWI piRNA 24–29 nt Transposons, repeats and piRNA clusters Heterochromatin formation? Schizosaccharomyces pombe Ago Ago1 endo-siRNA ~21 nt Outer centromeric repeats, mating-type locus and Heterochromatin formation subtelomeric regions Arabidopsis thaliana|| Ago AGO1 miRNA 20–24 nt miRNA genes mRNA cleavage and translational repression endo-siRNA (tasiRNA including TAS3) 21 nt TAS genes mRNA cleavage exo-siRNA 20–22 nt Viral genome Viral RNA cleavage AGO4 and AGO6 rasiRNA 24 nt Transposons and repetitive elements Heterochromatin formation AGO7 miR-390 21 nt miRNA gene Cleavage of TAS3 RNA *Small RNAs that are the main partners of a given Ago protein are listed. ‡miRNAs, as a class, are expressed in all cell types, whereas endo-siRNAs and piRNAs are expressed abundantly in germline cells and contribute to germline development. §So far, only AGO2 has been shown to be required for endo-siRNAs. ||Plants have ten Ago proteins, but only those with known small RNA partners are shown. Components of the RISC complex at Abcam Abbreviations Contact information Abcam is able to deliver a comprehensive portfolio of all the All our antibodies to the components of the RISC complex are Ago, Argonaute; AUB, Aubergine; CCR4, C-C chemokine type 4; DGCR8, V. Narry Kim is at the School of Biological Sciences and Center for very best and most up to date antibodies to components of highly characterized and our datasheets provide a library of DiGeorge syndrome critical region gene 8; DNMT, DNA methyltransferase; National Creative Research, Seoul National University, Seoul, 151-742, the RISC complex. We are constantly increasing the number relevant information. dsRNA, double-stranded RNA; endo-siRNA; endogenous small interfering South Korea. | Mikiko C. Siomi is at Keio University School of Medicine, of ChIP-grade and batch tested antibodies, enabling the RNA; exo-siRNA, exogenous small interfering RNA; H, histone; 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, and at the Japan Science Investigate these and other chromatin and RNAi antibodies and Technology Agency (JST), Core Research for Evolutional Science and research community to deconstruct epigenetic pathways. HMT, histone methyltransferase; HP1, heterochromatin protein 1; further at www.abcam.com/RISC LOQS, Loquacious; m7G, 7-methylguanosine; Me, methyl; miRNA, Technology (CREST), Saitama 332-0012, Japan. Three of our very best Dicer and Ago antibodies: The Abpromise microRNA; nt, ; piRNA, Piwi-interacting RNA; Pol II, RNA e-mails: [email protected]; [email protected] • Dicer 1 (ab13502) If our antibodies do not perform as described on the polymerase II; PACT, PKR-activating protein; pre-miRNA, precursor miRNA; • Ago1 (Drosophila) (ab5070) datasheet, notify us within 4 months of delivery so we can pri-miRNA, primary miRNA; rasiRNA, repeat-associated small interfering • Ago2 (Drosophila) (ab5072) RNA; ssRNA, single-stranded RNA; Su(Ste), Suppressor of Stellate; TAS, tasi For further reading, see www.nature.com/nrm/posters/smallrnas help you or offer a replacement or a refund. gene; tasiRNA, trans-acting siRNA; TRBP, HIV-1 TAR RNA-binding protein. Two of our very best RISC antibodies: Linked review article • TRBP (ab42018) Poster design by Vicky Askew, edited by Arianne Heinrichs, Kim, V. N., Han, J. & Siomi, M. C. Biogenesis of small RNAs in animals. • DDX6 (ab40684) copyedited by Simon Bishop. © 2009 Nature Publishing Group. Nature Rev. Mol. Cell Biol. (doi:10.1038/nrm2608).

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