The RNA Polymerase II-associated factor 1 complex represses small-RNA-mediated heterochromatin formation and gene silencing

Abstract

RNAi interference (RNAi) is a highly conserved regulatory mechanism employed by almost all Eukaryotes. With RNAi organisms can modulate the expression of endogenous genes and protect the integrity and identity of their genomes. All RNAi-based processes depend on a complex containing small non-coding RNAs (sRNA) associated with Argonaute proteins. In this sRNA-Argonaute complex, sRNA recognizes its sequence-specific target messenger RNA (mRNA) via a base-pairing interaction, and directs the Argonaute protein to it. Upon binding, the Argonaute protein can repress target gene expression at different stages. In the case of the most studied class of sRNAs, the microRNAs, the repression of gene expression occurs at the post-transcriptional level. MicroRNAs inhibit the translation of target mRNAs and promote their degradation in the cytoplasm of a cell. In contrast, nuclear RNAi-based processes have been implicated in directing chromatin modifications and repressing gene activity at the transcriptional level. RNAi-mediated chromatin modifications have been linked to epigenetic gene silencing across kingdoms but the mechanistic details of the small RNA-dependent transgenerational silencing remain uncovered. One of the obstacles in the way to understanding these regulatory processes is the fact that attempts to stably silence genes by ectopic small RNA mediated, locus-independent heterochromatin formation, have proven to be inherently difficult. By performing a mutagenesis screen we identified the highly conserved RNA Polymerase II-associated factor 1 (Paf1) complex as a repressor of sRNA-directed heterochromatin formation in the fission yeast Schizosaccharomyces pombe. We showed that small RNAs produced from a hairpin construct effectively silenced the expression of the target gene in trans, if the function of Paf1 complex was impaired. The induced repression was locus- and sequence-independent, and involved de novo formation of a functional heterochromatic domain. Importantly, we observed that the silent state could be transmitted through meiosis and was subsequently inherited through tens of generations, even in the absence of the primary siRNAs source. Thus, the Paf1 complex represses sRNA-induced heterochromatin formation in an epigenetic fashion. By performing a genetic analysis, we found that the Paf1 complex represses sRNA-mediated heterochromatin formation by contributing to efficient transcription termination and nascent transcript release. Thereby, we demonstrate that defective transcription termination exposes genes to sRNA-mediated repression. The findings described in this dissertation are not only an advancement to the mechanistic research on sRNA-directed transgenerational gene silencing. The ability to stably repress gene activity without changing the underlying DNA sequence may also provide important technological implications, in particular in plant biotechnology.