The Paf1 complex represses small-RNA-mediated epigenetic gene silencing

Abstract

RNA interference (RNAi) refers to the ability of exogenously introduced double-stranded RNA (dsRNA) to silence expression of homologous sequences. Silencing is initiated when the enzyme Dicer processes the dsRNA into small interfering RNAs (siRNAs). Small RNA molecules are incorporated into Argonaute protein-containing effector complexes, which they guide to complementary targets to mediate different types of gene silencing, specifically posttranscriptional gene silencing (PTGS) and chromatin-dependent gene silencing 1 . Although endogenous small RNAs play critical roles in chromatin-mediated processes across kingdoms, efforts to initiate chromatin modifications in trans by using siRNAs have been inherently difficult to achieve in all eukaryotic cells. Using fission yeast, we show that RNAi-directed heterochromatin formation is negatively controlled by the highly conserved RNA polymeraseassociated factor 1 complex (Paf1C). Temporary expression of a synthetic hairpin RNA in Paf1C mutants triggers stable heterochromatin formation at homologous loci, effectively silencing genes in trans. This repressed state is propagated across generations by continual production of secondary siRNAs, independently of the synthetic hairpin RNA. Our data support a model where Paf1C prevents targeting of nascent transcripts by the siRNA-containing RNA-induced transcriptional silencing (RITS) complex and thereby epigenetic gene silencing, by promoting efficient transcription termination and rapid release of the RNA from the site of transcription. We show that although compromised transcription termination is sufficient to initiate the formation of Reprints and permissions information is available at www.nature.com/reprints. A patent application has been filed.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#termsCorrespondence and requests for materials should be addressed to M.B. (marc.buehler@fmi.ch). * Correspondence to: marc.buehler@fmi.ch. Author Contributions Y.S., K.M.K., V.F., and J.B. generated strains and performed experiments; Y.S. performed the sms screen; genome-wide small RNA and gene expression data was analyzed by K.M.K.; M.B.S. designed and performed the computational analysis of the mutant genome resequencing data; M.B. designed experiments and prepared the manuscript. All authors discussed the results and commented on the manuscript.

Silencio/CG9754 connects the Piwi–piRNA complex to the cellular heterochromatin machinery

“…In this respect, it is interesting to note that tethering of Ago1 as well as other RITS (RNA-induced transcriptional silencing) complex components to nascent transcripts in S. pombe is very inefficient in initiating heterochromatin as well (Buhler et al 2006). A recent report indicates that impairing transcription elongation by loss of the Paf1 complex enables trans-silencing by RITS, presumably as the residence time of the nascent transcript at the target locus is increased (Kowalik et al 2015).…”

The RNA-induced transcriptional silencing complex targets chromatin exclusively via interacting with nascent transcripts

“…We recently discovered that siRNA-directed heterochromatin formation in S. pombe is inhibited by the RNA polymerase-associated factor 1 complex (Paf1C). Paf1C mutant strains are highly susceptible to de novo assembly of heterochromatin and stable gene silencing by synthetic, trans-acting primary siRNAs complementary to the coding sequence of protein-coding genes (Kowalik et al 2015). This has provided us with a unique tool to address these remaining fundamental mechanistic questions.…”

Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing

“…Therefore, the decelerated transcription kinetics that results from antisense transcription is the mechanism that allows ERI-1 to distinguish RNAs from disiRNA loci from non-disiRNA loci. In fision yeast, the Paf1 complex promotes efficient transcription termination and is a negative regulator of RNAi-directed heterochromatin formation (Kowalik et al 2015). Therefore, in both Neurospora and fission yeast, transcription kinetics plays an important role in determining the recognition of nascent transcripts by chromatin-modifying factors.…”