RNA interference-mediated co-transcriptional gene silencing in fission yeast

In the last decade or so, RNA interference (RNAi) has gained unanticipated recognition in the fields of RNA biology and gene regulation. It exists in a wide variety of eukaryotic organisms, and various forms of RNAi are involved in diverse biological processes. At its core, RNAi comprises small non-coding RNAs (sRNAs) in association with Argonaute proteins. The sRNAs are usually produced by cleavage of long double-stranded RNA by the endoribonuclease Dicer enzymes. The sRNAs guide Argonautes to target transcripts via complementary base-pairing, resulting in repression that can occur at various stages of the RNA production process. Perhaps the most well-studied mechanisms of RNAi-mediated repression are those occurring in the cytoplasm at a post-transcriptional level, whereby the target transcript is subject to degradation and/or inhibition of translation. However, well-characterised examples of nuclear RNAi also exist, and usually involve RNAi-mediated chromatin modification such as DNA methylation in plants and histone methylation in protozoa and fungi. These modifications can contribute to heterochromatin formation and inhibit RNA production at the level of transcription. In addition to mediating post-transcriptional and transcriptional gene silencing, recent evidence from several organisms suggests that RNAi can mediate co-transcriptional gene silencing (CTGS), whereby physical association of the RNAi machinery with chromatin can promote degradation of the nascent transcripts and/or inhibit transcription. Such a mode of silencing was first proposed in the fission yeast Schizosaccharomyces pombe (S. pombe), where the RNAi machinery is thought to repress heterochromatic RNA at a transcriptional and co-transcriptional level. During my PhD, I focused on the association of the RNAi machinery with chromatin in S. pombe. Using a sensitive chromatin profiling technique called DamID, I was able to provide the first direct evidence that S. pombe Dicer functions in cis on chromatin. Secondly, I uncovered a novel role for RNAi in gene regulation outside of the well-studied heterochromatic regions. The evidence presented here shows that the S. pombe RNAi machinery is concentrated at nuclear pores where it acts to co-transcriptionally degrade euchromatic RNAs, particularly those from retrotransposon long-terminal repeats, non-coding RNAs and stress response genes bound by the activating transcription factor Atf1. This may keep such features ‘poised’ for expression, allowing more rapid upregulation under inducing conditions. Of particular note, Argonaute is not required for targeting the other RNAi components to euchromatin, suggesting that in this case guidance by the sRNA is not responsible for recognition of substrates. I discuss the implications of these results, particularly in the context of RNAi in other eukaryotes

HP1(Swi6) mediates the recognition and destruction of heterochromatic RNA transcripts

HP1 proteins are major components of heterochromatin, which is generally perceived to be an inert and transcriptionally inactive chromatin structure. Yet, HP1 binding to chromatin is highly dynamic and robust silencing of heterochromatic genes can involve RNA processing. Here, we demonstrate by a combination of in vivo and in vitro experiments that the fission yeast HP1(Swi6) protein guarantees tight repression of heterochromatic genes through RNA sequestration and degradation. Stimulated by positively charged residues in the hinge region, RNA competes with methylated histone H3K9 for binding to the chromodomain of HP1(Swi6). Hence, HP1(Swi6) binding to RNA is incompatible with stable heterochromatin association. We propose a model in which an ensemble of HP1(Swi6) proteins functions as a heterochromatin-specific checkpoint, capturing and priming heterochromatic RNAs for the RNA degradation machinery. Sustaining a functional checkpoint requires continuous exchange of HP1(Swi6) within heterochromatin, which explains the dynamic localization of HP1 proteins on heterochromatin