Abstract
Small RNAs regulate chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although these processes have been well studied, fundamental mechanistic aspects remain obscure. Specifically, it is unclear exactly how small RNA-loaded Argonaute protein complexes target chromatin to mediate silencing. Here, using fission yeast, we demonstrate that transcription of the target locus is essential for RNA-directed formation of heterochromatin. However, high transcriptional activity is inhibitory; thus, a transcriptional window exists that is optimal for silencing. We further found that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. However, the kinetics of pre-mRNA processing is critical. Introns close to the 5' end of a transcript that are rapidly spliced result in a bistable response whereby the target either remains euchromatic or becomes fully silenced. Together, our results discount siRNA-DNA base pairing in RNA-mediated heterochromatin formation, and the mechanistic insights further reveal guiding paradigms for the design of small RNA-directed chromatin silencing studies in multicellular organisms.
Highlights
RNAi broadly refers to silencing pathways that depend on conserved Argonaute family proteins to repress gene expression (Höck and Meister 2008)
We further show that pre-mRNA splicing is compatible with RNAi-directed heterochromatin formation and that intronic sequences can serve as binding sites for siRNAs that are acting in trans
Current models of RNAi-directed heterochromatin assembly propose that RNA-induced silencing complexes (RISCs)/RNA-induced transcriptional silencing (RITS) is targeted to chromatin via base-pairing interactions with nascent or chromatinbound RNAs (Motamedi et al 2004; Nakama et al 2012; Holoch and Moazed 2015)
Summary
RNAi broadly refers to silencing pathways that depend on conserved Argonaute family proteins to repress gene expression (Höck and Meister 2008). This suggests that RITS is targeted to chromatin through base pairing between siRNA and pre-mRNA followed by recruitment of RDRC and histone-modifying enzymes (Bühler et al 2006; Bühler and Moazed 2007). 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. Our results provide the first direct evidence for the nascent transcript model of small RNA-directed epigenetic gene repression and reveal guiding paradigms for the design of small RNA-directed chromatin silencing experiments in other organisms
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