REVIEWS Small silencing RNAs: an expanding universe Megha Ghildiyal and Phillip D. Zamore Abstract | Since the discovery in 1993 of the first small silencing RNA, a dizzying number of small RNA classes have been identified, including microRNAs (miRNAs), small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). These classes differ in their biogenesis, their modes of target regulation and in the biological pathways they regulate. There is a growing realization that, despite their differences, these distinct small RNA pathways are interconnected, and that small RNA pathways compete and collaborate as they regulate genes and protect the genome from external and internal threats. 2 Argonaute The defining features of small silencing RNAs are their both plants and nematodes, spreading from cell to cell . Argonaute proteins are the short length (~20–30 nucleotides), and their association In C. elegans, RNAi is also heritable: silencing can be effectors of small RNA-directed with members of the Argonaute family of proteins, which transferred to the progeny of the worm that was origi- silencing. Small RNAs guide they guide to their regulatory targets, typically resulting in nally injected with the trigger dsRNA3. Viral infection, Argonautes to their RNA targets; Argonautes carry out reduced expression of target genes. Beyond these defining inverted-repeat transgenes or aberrant transcription the regulation. Argonaute features, different small RNA classes guide diverse and products all lead to the production of dsRNA. dsRNA proteins are characterized by complex schemes of gene regulation. Some small silencing is converted to siRNAs that direct RNAi. siRNAs were two domains — Piwi (a RNAs, such as small interfering RNAs (siRNAs), derive discovered in plants4 and were later shown in animal ribonuclease domain) and PAZ (a ssRNA-binding module). from dsRNA, whereas others, such as Piwi-interacting extracts to serve as guides that direct endonucleolytic RNAs (piRNAs), do not. These different classes of regu- cleavage of their target RNAs5,6. siRNAs can be classified latory RNAs also differ in the proteins required for their according to the proteins involved in their biogenesis, biogenesis, the constitution of the Argonaute-containing their mode of regulation or their size. In this Review, we complexes that execute their regulatory functions, their differentiate the major types of siRNAs according to the modes of gene regulation and the biological functions in molecules that trigger their production — a classification which they participate. New small RNA classes and new scheme that best captures the biological distinctions examples of existing classes continue to be discovered, among small silencing RNAs. such as the recent identification of endogenous siRNAs (endo-siRNAs) in flies and mammals. Here, we provide siRNAs derived from exogenous agents. Early examples an overview of small silencing RNAs from plants and of RNAi were triggered by exogenous dsRNA. In these metazoan animals. For each class, we describe its bio- cases, long exogenous dsRNA is cleaved into double- genesis, function and mode of target regulation, provid- stranded siRNAs by Dicer, a dsRNA-specific RNase III ing examples of the regulatory networks in which each family ribonuclease7 (FIG. 1). siRNA duplexes produced by participates (TABLE 1). Finally, we highlight several exam- Dicer comprise two ~21 nucleotide strands, each bear- ples of unexpected, and often unexplained, complexity in ing a 5′ phosphate and 3′ hydroxyl group, paired in a way Department of Biochemistry the interactions between distinct small RNA pathways. that leaves two-nucleotide overhangs at the 3′ ends5,8,9. The and Molecular strand that directs silencing is called the guide, whereas the Pharmacology and Howard The discovery of RNAi Hughes Medical Institute, other strand, which is ultimately destroyed, is the passen- University of Massachusetts In 1998, Fire and Mello established dsRNA as the silenc- ger. Target regulation by siRNAs is mediated by the RNA- Medical School, 364 ing trigger in Caenorhabditis elegans1. Their experiments induced silencing complex (RISC), which is the generic Plantation Street, Worcester, overturned the contemporary view that antisense RNA name for an Argonaute–small RNA complex6. In addi- Massachusetts 01605, USA induced silencing by base pairing to its mRNA coun- tion to an Argonaute protein and a small RNA guide, the Correspondence to P.D.Z. e-mail: phillip.zamore@ terpart, thereby preventing its translation into protein. RISC might also contain auxiliary proteins that extend or umassmed.edu In worms and other animals, siRNA-mediated silenc- modify its function; for example, proteins that redirect the doi:10.1038/nrg2504 ing is known as RNAi. Remarkably, RNAi is systemic in target mRNA to a site of general mRNA degradation10. 94 | FEBRUARY 2009 | VOLUME 10 www.nature.com/reviews/genetics © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | Types of small silencing RNAs Name Organism Length (nt) Proteins Source of trigger Function Refs miRNA Plants, algae, animals, 20–25 Drosha (animals only) Pol II transcription Regulation of mRNA 93–95, viruses, protists and Dicer (pri-miRNAs) stability, translation 200–202,226 casiRNA Plants 24 DCL3 Transposons, repeats Chromatin 38,44,51, modification 52,61–63 tasiRNA Plants 21 DCL4 miRNA-cleaved RNAs from Post-transcriptional 64–68 the TAS loci regulation natsiRNA Plants 22 DCL1 Bidirectional transcripts Regulation of 71,72 induced by stress stress-response genes 24 DCL2 21 DCL1 and DCL2 Exo-siRNA Animals, fungi, protists ~21 Dicer Transgenic, viral or other Post-transcriptional 4,5,8,227 exogenous dsRNA regulation, antiviral Plants 21 and 24 defense Endo-siRNA Plants, algae, animals, ~21 Dicer (except secondary Structured loci, convergent Post-transcriptional 75–79,82, fungi, protists siRNAs in C. elegans, and bidirectional regulation of 83,86,87, which are products of transcription, mRNAs transcripts 200,201, RdRP transcription, paired to antisense and transposons; 228 and are therefore not pseudogene transcripts transcriptional gene technically siRNAs) silencing piRNA Metazoans excluding 24–30 Dicer-independent Long, primary transcripts? Transposon regulation, 157, Trichoplax adhaerens unknown functions 163–169, 177,202 piRNA-like Drosophila 24–30 Dicer-independent In ago2 mutants in Unknown 76 (soma) melanogaster Drosophila 21U-RNA Caenorhabditis elegans 21 Dicer-independent Individual transcription of Transposon regulation, 114, piRNAs each piRNA? unknown functions 173–175 26G RNA Caenorhabditis elegans 26 RdRP? Enriched in sperm Unknown 114 ago2, Argonaute2; casiRNA, cis-acting siRNA; DCL, Dicer-like; endo-siRNA, endogenous small interfering RNA; exo-siRNA, exogenous small interfering RNA; miRNA, microRNA; natsiRNA, natural antisense transcript-derived siRNA; piRNA, Piwi-interacting RNA; Pol II, RNA polymerase II; pri-miRNA, primary microRNA; RdRP, RNA-dependant RNA polymerase; tasiRNA, trans-acting siRNA. Mammals and C. elegans each have a single Dicer duplex. AGO2 can then cleave the passenger strand as that makes both microRNAs (miRNAs) and siR- if it were a target RNA27–31. AGO2 always cleaves its NAs11–14, whereas Drosophila species have two Dicers: RNA target at the phosphodiester bond between the DCR-1 makes miRNAs, whereas DCR-2 is specialized nucleotides that are paired to nucleotides 10 and 11 for siRNA production15. The fly RNAi pathway defends of the guide strand8,9. Release of the passenger strand against viral infection, and Dicer specialization might after its cleavage converts pre-RISC to mature RISC, reduce competition for Dicer between precursor miR- which contains only single-stranded guide RNA. In NAs (pre-miRNAs) and viral dsRNAs. Alternatively, flies, the guide strand is 2′-O-methylated at its 3′ end DCR-2 and Argonaute2 (AGO2) specialization might by the S-adenosyl methionine-dependent methyltrans- reflect the evolutionary pressure on the siRNA pathway ferase HEN1 (also known as piRNA methyltransferase, to counter rapidly evolving viral strategies to escape PIMET), completing RISC assembly32,33. In plants, RNAi. In fact, dcr-2 and ago2 are among the most both miRNAs and siRNAs are terminally methylated, rapidly evolving Drosophila genes16. C. elegans might a modification that is crucial for their stability34–36. achieve similar specialization with a single Dicer by Plants exhibit a surprising diversity of small RNA using the dsRNA-binding protein RNAi defective types and the proteins that generate them. The diver- (RDE-4) as the gatekeeper for entry into the RNAi sification of RNA silencing pathways in plants might pathway17. However, no natural virus infection has reflect the need of a sessile organism to cope with biotic been documented in C. elegans18. By contrast, mammals and abiotic stress. The number of RNA silencing pro- might not use the RNAi pathway to respond to viral teins can vary enormously among animals too, with infection, having evolved an elaborate, protein-based C. elegans producing 27 distinct Argonaute proteins immune system19–21. compared with 5 in flies. Phylogenetic data suggest that The relative thermodynamic stabilities of the 5′ ends nearly all of these ‘extra’ C. elegans Argonautes act in the of the two siRNA strands in the duplex determines the secondary siRNA pathway37, perhaps because endog- identity of the guide and passenger strands22–24. In flies, enous secondary siRNAs are so plentiful in worms. this thermodynamic difference is sensed