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

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.

Targeting Argonaute to chromatin

“…Moreover, RDM1, a TGS protein in Arabidopsis, is a ssDNA-binding protein in vitro, inviting speculation that it might play a role in stabilizing single-stranded regions associated with Pol V transcription (Pikaard et al 2012). With these considerations in mind, the finding by Shimada et al (2016) that splicing of proximal intronic sequences reduces TGS strongly sug-gests that the base-pairing interactions are functionally occurring with the nascent RNA and not the associated DNA, where the intronic sequences are still present. One can argue that the results do not formally rule out the possibility that Ago-siRNA complexes initially bind to nascent RNA but subsequently engage the corresponding nontemplate DNA.…”

“…The results of Shimada et al (2016) also have important implications with regard to the possibility of siRNA-DNA base-pairing in TGS. In plants, the possibility of siRNA-DNA interactions has been considered to explain the precision of RNA-directed DNA methylation as well as the observation that artificially induced siRNAs can trigger DNA methylation of homologous transgene sequences in the absence of detectable transcription of the transgene (You et al 2013).…”

“…Studies in both organisms have demonstrated that target loci need to be transcribed in order to be silenced and that Ago can be coimmunoprecipitated in association with RNAs derived from silenced loci (Motamedi et al 2004;Irvine et al 2006;Wierzbicki et al 2009;You et al 2013). Shimada et al (2016) provide additional evidence that RNAs generated at target loci, and not just the act of transcription, are required for transcriptional gene silencing (TGS). They engineered the production of siRNAs that match intron sequences within a reporter transgene, showing that intronic sequences placed near the 3 ′ end of the transgene allow for efficient TGS.…”

“…One can argue that the results do not formally rule out the possibility that Ago-siRNA complexes initially bind to nascent RNA but subsequently engage the corresponding nontemplate DNA. Alternatively, one might reconcile the results from Shimada et al (2016) with models of direct siRNA-DNA interaction by envisioning that proximal intron splicing disrupts R-loop formation that is needed for direct siRNA-DNA base pairing to occur. However, the simplest explanation is that Ago-siRNA complexes are recruited to chromatin by binding solely to nascent RNA transcripts.…”

Age-dependent methylation in epigenetic clock CpGs is associated with G-quadruplex, co-transcriptionally formed RNA structures and tentative splice sites

“…Some sequences were predicted to form both pseudoknot and non-pseudoknot conformations of very similar stability (< 1 kcal/mol difference between the most stable non-pseudoknot conformation and the next less stable pseudoknot conformation, Table S1). These sequences will hereon be referred to as ‘ambivalent’ sequences (Amb), since the presence of methylation could potentially change the equilibrium between their pseudoknot and non-pseudoknot conformations [33]. The ambivalent pseudoknots are frequent among the epigenetic clock sequence ensemble (Figure 2(A)) and were further studied together with pseudoknots, unless otherwise noted.…”