REVIEWS Small RNA sorting: matchmaking for Argonautes Benjamin Czech and Gregory J. Hannon Abstract | Small RNAs directly or indirectly impact nearly every biological process in eukaryotic cells. To perform their myriad roles, not only must precise small RNA species be generated, but they must also be loaded into specific effector complexes called RNA-induced silencing complexes (RISCs). Argonaute proteins form the core of RISCs and different members of this large family have specific expression patterns, protein binding partners and biochemical capabilities. In this Review, we explore the mechanisms that pair specific small RNA strands with their partner proteins, with an eye towards the substantial progress that has been recently made in understanding the sorting of the major small RNA classes — microRNAs (miRNAs) and small interfering RNAs (siRNAs) — in plants and animals. RNase III protein The discovery of RNA interference (RNAi) in the late The biogenesis of most small RNA classes, includ- A member of a family 1990s sparked a renaissance in our understanding of ing microRNAs (miRNAs) and many small interfering of ribonucleases that RNAs as regulatory molecules. A growing number RNAs (siRNAs), requires the action of RNase III family process dsRNA, leaving 5` of small RNA classes has since emerged from studies of proteins (reviewed in REFS 1–3). Some small RNA classes, monophosphates and 2-nt 3` overhangs with hydroxyl ends. eukaryotic organisms, and these RNAs can be approxi- including Piwi-interacting RNAs (piRNAs) and second- Drosha and Dicer are examples mately divided into two groups: small RNAs that engage ary siRNAs in worms, however, are not derived from of such ribonucleases. RNAi-related machinery and those that do not. As yet, dsRNA precursors and are produced through alterna- we know very little about many newly discovered groups tive biogenesis mechanisms independently of RNase III RNA-induced silencing of small RNAs, but our understanding of the biogenesis enzymes4–8. complex A regulatory multi-protein and biological functions of RNAi-related small RNA Following their production, small RNAs are sorted complex containing an classes is growing rapidly. to confer association with specific Argonaute family Argonaute protein bound Small RNAs that engage RNAi-related pathways share proteins, which function as the core of the RNA-induced to a single-stranded small several characteristic features. They are mainly ~20–30 silencing complex (RISC). Argonaute proteins can be clas- RNA that regulates gene ` expression through sequence nucleotides (nt) in length, have 5 phosphate groups sified into three subgroups according to their sequence complementarity between and 3` hydroxyl (-OH) (although sometimes modified) relationships: the AGO subfamily, the Piwi subfamily and the guide RNA and the termini, and they associate with specific members of the worm-specific WAGO clade9–11. Piwi subfamily pro- target transcript. a large protein family — the Argonautes. The precise teins load small RNAs derived from single-stranded combination of a small RNA with a particular Argonaute precursors (piRNAs) and AGO clade proteins usually protein determines its biological function. Therefore, it associate with small RNA duplexes processed by RNase is crucial that these very similar species are appropri- III endonucleases (miRNAs and siRNAs; reviewed in ately sorted among closely related partners. Only then REFS 1,2). Small RNAs that occupy WAGO clade proteins can the target specificity conferred on Argonaute pro- are usually direct products of RNA synthesis6,7,9. Watson School of Biological teins by their small RNA guides enable their myriad Mature RISC consists of a single-stranded small RNA Sciences, Howard Hughes Medical Institute, Cold Spring important roles, which include the regulation of gene bound to an Argonaute protein. As some small RNAs are Harbor Laboratory, expression, modification of chromosome structure and generated as duplexes, only one strand (the guide strand) 1 Bungtown Road, Cold Spring protection from mobile elements. Conceptually, all small is retained and the other (passenger) strand is discarded Harbor, New York 11724, USA. RNA-mediated regulatory events can be considered as during RISC assembly12–14. AGO clade proteins are gen- Correspondence to G.J.H. the culmination of several consecutive steps: small RNA erally loaded with small RNA duplexes before RISC e-mail: [email protected] doi:10.1038/nrg2916 biogenesis, strand selection (in which dsRNA is the pre- maturation. Thus, it is of key importance to assemble Published online cursor), loading into Argonaute, target recognition and RISC in a manner that ensures that the appropriate 30 November 2010 effector function. guide strand is selectively stabilized, as loading of the NATURE REVIEWS | GENETICS VOLUME 12 | JANUARY 2011 | 19 © 2011 Macmillan Publishers Limited. All rights reserved REVIEWS passenger strand would obviously misdirect RISC a multiprotein complex called the Microprocessor21–24. towards inappropriate targets. Small RNAs guide mature This complex recognizes the duplex character of the pri- RISC through complementary base pairing to its targets, miRNA, although the precise RNA–protein interactions with the most common outcome being target repression that select pri-miRNAs as Microprocessor substrates (reviewed in REFS 15–17). and how the cleavage site is determined by these inter- The knowledge of the mechanisms that guide a par- actions are matters of ongoing work. The pri-miRNA ticular small RNA strand into a specific Argonaute family is cleaved by Drosha to liberate a ~60–70-nt precursor member is crucial. It impacts our ability to predict the miRNA (pre-miRNA) from the primary transcript25. The biological function of a small RNA and to effectively use nuclear export protein Exportin 5 recognizes the 2-nt small RNAs as experimental tools or therapeutics. This single-stranded 3` overhang of the pre-miRNA (char- Review focuses on our understanding of small RNA sort- acteristic of RNase III-mediated cleavage) and actively ing in plants and animals. We consider biogenesis as a transports it in a Ran–GTP-dependent manner to the starting point as this affects the nature of small RNAs and, cytoplasm26–28. Additional factors, including the nuclear in some cases, the complexes which the small RNAs join. export receptor Exportin 1 (XPO1), the cap-binding Next, we discuss the small RNA-intrinsic determinants of complex (CBC) and the Arabidopsis thaliana SERRATE sorting, followed by RISC loading and maturation. Finally, homologue, ARSENITE-RESISTANCE PROTEIN 2 we briefly cover the implications of sorting for Argonaute (ARS2), were recently suggested to play a part in the function. We do not extensively discuss the effector mech- transition from pri- to pre-miRNA29–31. anisms of mature RISC, but instead refer the reader to Once in the cytoplasm, the pre-miRNA is cleaved several excellent recent reviews on this topic15–17. into a ~22–23-nt miRNA:miRNA* duplex by Dicer32–35. For this purpose, the sole mammalian Dicer partners Small RNA biogenesis with the dsRBD protein TAR RNA-binding protein 2 In effect, the first step of small RNA sorting is biogen- (TARBP2, also known as TRBP)36,37, whereas the esis, as this determines the small RNAs that are available Drosophila melanogaster miRNA-generating Dicer 1 for RISC loading. Moreover, the precise enzymes that (DCR1) similarly interacts with a specific isoform of liberate small RNAs from their precursor transcripts or its dsRBD protein partner Loquacious (LOQS-PB)38–42. generate them de novo seem to impact the choice of their Small RNA duplexes generated by Dicer (and its pro- ultimate Argonaute partner. Therefore, it is important tein partner) exhibit 2-nt single-stranded 3` overhangs to begin with an introduction to the varied mechanisms at both ends, a signature of RNase III cleavage. that can produce small RNAs. Several unconventional miRNAs that are defined by Small RNA duplexes from partial or perfect dsRNA their use of alternative maturation strategies have now precursors are generated by RNase III family enzymes been noted. For example, mirtrons have been found in through sequential endonucleolytic cleavage events. flies and mammals43–45. Mirtrons bypass the Drosha These enzymes often partner with dsRNA binding processing step and instead use the splicing machin- domain (dsRBD) proteins, which serve to increase sub- ery to generate pre-miRNAs. Mirtrons are very short Guide strand strate specificity and affinity, leading to increased activity. introns and are excised, debranched and refolded into During RISC loading, one strand The resulting products are duplex ~20−24-nt small short stem–loop structures that mimic pre-miRNAs of an siRNA is selected and RNAs consisting of two strands (the guide or miR and and are processed into mature miRNAs by Dicer. A stabilized. This is termed the passenger or miR* strands). These small RNAs feature 5` few recently discovered mirtrons in flies are initially guide strand, and it confers monophosphates and 2-nt overhangs that have hydroxyl generated with extended 3` tails that must be resected target specificity. miRNA guide ` strands are termed miR strands. groups at their 3 termini. by the exosome to form a pre-miRNA suitable for Dicer processing46. Passenger strand Animal miRNA processing. miRNAs are ubiquitous in The non-incorporated strand animal genomes and are often transcribed as separate miRNA biogenesis in plants. Plant miRNAs are tran- of the siRNA duplex that