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RNA Interference in the Nucleus: Roles for Small Rnas in Transcription, Epigenetics and Beyond

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RNA Interference in the Nucleus: Roles for Small Rnas in Transcription, Epigenetics and Beyond REVIEWS NON-CODING RNA RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond Stephane E. Castel1 and Robert A. Martienssen1,2 Abstract | A growing number of functions are emerging for RNA interference (RNAi) in the nucleus, in addition to well-characterized roles in post-transcriptional gene silencing in the cytoplasm. Epigenetic modifications directed by small RNAs have been shown to cause transcriptional repression in plants, fungi and animals. Additionally, increasing evidence indicates that RNAi regulates transcription through interaction with transcriptional machinery. Nuclear small RNAs include small interfering RNAs (siRNAs) and PIWI-interacting RNAs (piRNAs) and are implicated in nuclear processes such as transposon regulation, heterochromatin formation, developmental gene regulation and genome stability. RNA interference Since the discovery that double-stranded RNAs (dsRNAs) have revealed a conserved nuclear role for RNAi in (RNAi). Silencing at both the can robustly silence genes in Caenorhabditis elegans and transcriptional gene silencing (TGS). Because it occurs post-transcriptional and plants, RNA interference (RNAi) has become a new para- in the germ line, TGS can lead to transgenerational transcriptional levels that is digm for understanding gene regulation. The mecha- inheritance in the absence of the initiating RNA, but directed by small RNA molecules. nism is well-conserved across model organisms and it is dependent on endogenously produced small RNA. uses short antisense RNA to inhibit translation or to Such epigenetic inheritance is familiar in plants but has Post-transcriptional gene degrade cytoplasmic mRNA by post-transcriptional gene only recently been described in metazoans. silencing silencing (PTGS). PTGS protects against viral infection, In this Review, we cover the broad range of nuclear (PTGS). Silencing achieved by prevents transposon mobilization and regulates endo­ RNAi pathways that have been discovered across organ- the degradation and/or prevention of translation of a genous genes. Three classes of small RNA can regulate isms so that readers can appreciate the conservation transcript targeted by small genes by targeting transcripts in the cytoplasm. These between these pathways while being aware of important RNAs. are: microRNAs (miRNAs), which are hairpin-derived differences. As such, we can only scratch the surface of RNAs with imperfect complementarity to targets and pathways in each organism, so readers are directed to that cause translational repression; small interfering other articles for an in‑depth analysis when appropri- RNAs (siRNAs), which have perfect complementarity ate. We concentrate on the siRNA and piRNA pathways to targets and cause transcript degradation; and PIWI- because their nuclear roles are best understood. intereacting RNAs (piRNAs), which target transposon To begin, we outline the biogenesis of small RNAs, transcripts in animal germ lines. Traditionally, the term focusing on the subcellular localization of these pro- RNAi has been used to describe siRNA pathways; how- cesses. Next, a mechanistic understanding of nuclear ever, the mechanistic details of diverse small RNA path- RNAi is described in model systems in which it has been ways are converging, so in this Review we use RNAi as elucidated. Nuclear small RNAs function in the germ an umbrella term to describe silencing that is dependent lines of a broad range of organisms in which mechanis- 1Watson School of Biological Sciences, Cold Spring Harbor on small RNA. tic details are not yet known, so we discuss the biologi- Laboratory, New York 11724, In plants and fungi, RNAi pathways in the nucleus cal importance of their roles, including in transposon USA. can repress target genes at the transcriptional level by regulation, epigenetic inheritance and developmental 2Howard Hughes Medical guiding epigenetic modification of chromatin by, for gene regulation, and we suggest parallels that can be Institute–Gordon and Betty example, histone and DNA methyltransferases. At first, drawn to well-understood mechanistic models. Finally, Moore Foundation. Correspondence to R.A.M. these pathways were thought to be absent from metazo- we look forwards by exploring the newly emerging rela- e‑mail: [email protected] ans, but recently a parallel mechanism has been found tionship between nuclear RNAi, genome maintenance doi:10.1038/nrg3355 in the germ line of several metazoans. These findings and DNA repair. 100 | FEBRUARY 2013 | VOLUME 14 www.nature.com/reviews/genetics © 2013 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | Overview of small RNA classes and their functions in the nucleus Small Size Nuclear Biogenesis factors Genomic origin Nuclear function RNA class effector* Schizosaccharomyces pombe siRNA 21–23 nt Ago1 Dicer: Dcr1; RdRP: Rdp1 Heterochromatic repeat regions Centromere function; heterochromatin formation and spreading; RNA Pol II regulation Arabidopsis thaliana siRNA 24 nt AGO4; AGO6; Dicer: DCL3; RdRP: RDR2 Heterochromatic repetitive Systemic RdDM; reproductive AGO9 regions enriched for transposons strategy (meiosis or apospory) and retroelements Caenorhabditis elegans 22G RNA 22 nt NRDE‑3 Primary siRNA biogenesis: ERGO‑1; Disperse genomic loci Systemic heterochromatin (siRNA) Dicer: DCR‑1; RdRP: RRF‑3 formation; RNA Pol II regulation (primary), RRF‑1 (secondary) 21U RNA 21 nt WAGO‑9; PRG‑1 loaded with 21U for 22G Two clusters on chromosome IV Heritable heterochromatin (piRNA) WAGO‑10 production formation with 22G for TGS Drosophila melanogaster siRNA ~ 22 nt AGO2 Dicer: DCR2 Transposons, repetitive elements, Heterochromatin formation; convergent transcription units chromosome segregation; RNA and structured loci Pol II regulation piRNA 23 to PIWI Ping-pong cycle: PIWI, AUB Primary: heterochromatic piRNA Chromosome segregation; 29 nt (primary), AGO3 (secondary), clusters antisense to transposons, heterochromatin formation; Zucchini maternally deposited piRNAs; telomere homeostasis secondary: sense transcripts of active transposons Mus musculus siRNA 21 to AGO2 Dicer: DCR1 Dispersed naturally occurring Speculative 22 nt dsRNAs, pseudogenes piRNA 24 to MIWI2 Ping-pong cycle: MILI (primary), Primary: sense transcripts of De novo DNA methylation 31 nt MIWI2 (secondary), Maelstrom active transposons; secondary: antisense transcripts *Listed are only the Argonaute proteins that act as the eventual nuclear effectors; other Argonaute proteins may also be involved in these pathways. AGO, Argonaute; AUB, aubergine; DCR, Dicer; DCRL, Dicer-like; dsRNA, double-stranded RNA; nt, nucleotide; piRNA, PIWI-interacting RNA; Pol, polymerase; PTGS, post-transcriptional gene silencing; RdDM, RNA-directed DNA methylation; RdRP, RNA-directed RNA polymerase; siRNA, small interfering RNA; TGS, transcriptional gene silencing. Biogenesis of nuclear small RNAs overhangs4 and 5ʹ monophosphates4,5. Dicer-independent The siRNA and piRNA pathways differ in the source of mechanisms of siRNA production have also been pro- the primary RNA that elicits a response and the mecha- posed in Neurospora crassa6, Schizosaccharomyces pombe7 nism by which small RNA is subsequently produced and and C. elegans8. The cellular location in which dsRNA Argonaute Transcriptional gene amplified. The family of proteins are the effec- processing occurs has implications for how siRNA bio- silencing tors of RNAi, and this family consists of two subclades: genesis and nuclear effects are regulated. In S. pombe, (TGS). Silencing achieved by AGO proteins, which are ubiquitously expressed and transcription, processing, amplification by an RNA- the formation of a repressive bind miRNAs and siRNAs, and PIWI proteins, which dependent RNA polymerase (RdRP) and AGO-mediated chromatin environment at a were originally discovered in the germ line and bind target cleavage are all intimately linked in the nucleus4,9–12 locus targeted by small RNA, 1–3 TABLE 1 (FIG. 1a) making it inaccessible to piRNAs . summarizes the characteristics of . In animals, siRNA processing was originally 3 transcriptional machinery. the nuclear siRNA and piRNA pathways for some thought to occur in the cytoplasm ; however, recent stud- of the organisms described in this Review, highlighting ies in D. melanogaster have shown that Dicer 2 (DCR2) Argonaute the nuclear effector, size class and proteins involved in is predominantly found in the nucleus, challenging this The effector proteins of RNA 13 inteference that are composed biogenesis. view . This is in contrast to C. elegans, in which cyto- of three characteristic domains, plasmic processing for many siRNA pathways has been a PAZ domain and a MID siRNA biogenesis. dsRNAs are thought to be the trigger confirmed14 (FIG. 1b). domain, which bind the 3ʹ and for most if not all siRNA biogenesis and can be gener- After they have been generated, the siRNA duplexes 5ʹ ends of small interfering ated by several means (FIG. 1). When dsRNAs are available, are loaded into an appropriate effector Argonaute pro- RNA respectively, and a PIWI domain, which may possess the biogenesis of siRNA requires action of the RNase-III- tein. The subcellular location in which Argonaute load- RNase-H-like slicer activity if the like Dicer family of enzymes. Dicer cleaves dsRNAs into ing takes place is not yet fully understood across model protein is catalytically active. 20­–25‑nucleotide (nt) siRNA duplexes with 2 nt 3ʹ OH organisms. In Arabidopsis thaliana, AGO4 loading is NATURE REVIEWS | GENETICS VOLUME 14 | FEBRUARY
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