The RNA polymerase II subunit RPB-9 recruits the integrator complex to terminate Caenorhabditis elegans piRNA transcription.

PIWI-interacting RNAs (piRNAs) are genome-encoded small RNAs that regulate germ cell development and maintain germline integrity in many animals. Mature piRNAs engage Piwi Argonaute proteins to silence complementary transcripts, including transposable elements and endogenous genes. piRNA biogenesis mechanisms are diverse and remain poorly understood. Here, we identify the RNA polymerase II (RNA Pol II) core subunit RPB-9 as required for piRNA-mediated silencing in the nematode Caenorhabditis elegans. We show that rpb-9 initiates heritable piRNA-mediated gene silencing at two DNA transposon families and at a subset of somatic genes in the germline. We provide genetic and biochemical evidence that RPB-9 is required for piRNA biogenesis by recruiting the Integrator complex at piRNA genes, hence promoting transcriptional termination. We conclude that, as a part of its rapid evolution, the piRNA pathway has co-opted an ancient machinery for high-fidelity transcription.

RNA helicases are hubs that orchestrate exosome-dependent 3'-5' decay.

The RNA exosome is a conserved complex of proteins that mediates 3'-5' RNA processing and decay. Its functions range from processing of non-coding RNAs such as ribosomal RNAs and decay of aberrant transcripts in the nucleus to cytoplasmic mRNA turnover and quality control. Ski2-like RNA helicases translocate substrates to exosome-associated ribonucleases and interact with the RNA exosome either directly or as part of multi-subunit helicase-containing complexes that identify and target RNA substrates for decay. Recent structures of these helicases with their RNA-binding partners or the RNA exosome have advanced our understanding of a system of modular and mutually exclusive contacts between the exosome and exosome-associated helicase complexes that shape the transcriptome by orchestrating exosome-dependent 3'-5' decay.

Strategies for Generating RNA Exosome Complexes from Recombinant Expression Hosts.

The eukaryotic RNA exosome is a conserved and ubiquitous multiprotein complex that possesses multiple RNase activities and is involved in a diverse array of RNA degradation and processing events. While much of our current understanding of RNA exosome function has been elucidated using genetics and cell biology based studies of protein functions, in particular in S. cerevisiae, many important contributions in the field have been enabled through use of in vitro reconstituted complexes. Here, we present an overview of our approach to purify exosome components from recombinant sources and reconstitute them into functional complexes. Three chapters following this overview provide detailed protocols for reconstituting exosome complexes from S. cerevisiae, S. pombe, and H. sapiens. We additionally provide insight on some of the drawbacks of these methods and highlight several important discoveries that have been achieved using reconstituted complexes.

Reconstitution of the Human Nuclear RNA Exosome.

We describe procedures to clone, express, and reconstitute an active human nuclear RNA exosome. Individual recombinant subunits are expressed from E. coli and successfully reconstituted into the nuclear complex, which contains the noncatalytic nine-subunit exosome core, the endoribonuclease and exoribonuclease DIS3, the distributive exoribonuclease EXOSC10, the cofactors C1D and MPP6, and the RNA helicase MTR4.

SnapShot: The RNA Exosome.

The RNA exosome is a 3' to 5' ribonuclease that plays a fundamental role in maturation, quality control, and turnover of nearly all types of RNA produced in eukaryotic cells. Here, we present an overview of the structure, composition, and functions of the RNA exosome, including various cytoplasmic and nuclear exosome co-factors and associated protein complexes. To view this SnapShot, open or download the PDF.

Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA Exosome-MTR4 Complex.

The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.

piRNAs: from biogenesis to function.

Distinguishing self from non-self plays a crucial role in safeguarding the germlines of metazoa from mobile DNA elements. Since their discovery less than a decade ago, Piwi-interacting RNAs (piRNAs) have been shown to repress transposable elements in the germline and, hence, have been at the forefront of research aimed at understanding the mechanisms that maintain germline integrity. More recently, roles for piRNAs in gene regulation have emerged. In this Review, we highlight recent advances made in understanding piRNA function, highlighting the divergent nature of piRNA biogenesis in different organisms, and discussing the mechanisms of piRNA action during transcriptional regulation and in transgenerational epigenetic inheritance.

PRDE-1 is a nuclear factor essential for the biogenesis of Ruby motif-dependent piRNAs in C. elegans.

Piwi-interacting RNAs (piRNA) are small regulatory RNAs with essential roles in maintaining genome integrity in animals and protists. Most Caenorhabditis elegans piRNAs are transcribed from two genomic clusters that likely contain thousands of individual transcription units; however, their biogenesis is not understood. Here we identify and characterize prde-1 (piRNA silencing-defective) as the first essential C. elegans piRNA biogenesis gene. Analysis of prde-1 provides the first direct evidence that piRNA precursors are 28- to 29-nucleotide (nt) RNAs initiating 2 nt upstream of mature piRNAs. PRDE-1 is a nuclear germline-expressed protein that localizes to chromosome IV. PRDE-1 is required specifically for the production of piRNA precursors from genomic loci containing an 8-nt upstream motif, the Ruby motif. The expression of a second class of motif-independent piRNAs is unaffected in prde-1 mutants. We exploited this finding to determine the targets of the motif-independent class of piRNAs. Together, our data provide new insights into both the biogenesis and function of piRNAs in gene regulation.

piRNAs can trigger a multigenerational epigenetic memory in the germline of C. elegans.

Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.

Function, targets, and evolution of Caenorhabditis elegans piRNAs.

Piwi-interacting RNAs (piRNAs) are small RNAs required to maintain germline integrity and fertility, but their mechanism of action is poorly understood. Here we demonstrate that Caenorhabditis elegans piRNAs silence transcripts in trans through imperfectly complementary sites. Target silencing is independent of Piwi endonuclease activity or "slicing." Instead, piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes and tend to overlap the start and end of transposons in sense and antisense, respectively. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNA interference in C. elegans.