BioProject

Small RNAs are implicated in chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. More...

Small RNAs are implicated in chromatin modification and transcriptional gene silencing across the eukaryotic kingdom. Although well understood, fundamental mechanistic aspects of RNA-mediated chromatin regulation have remained obscure. It has not been possible to unambiguously distinguish whether small RNA loaded Argonaute proteins target the nascent transcript or the underlying DNA sequence. Here, we demonstrate that transcription of the target locus is indispensable for the formation of heterochromatin in fission yeast. Yet, high transcriptional activity opposes RNA-directed chromatin silencing. We further find that pre-mRNA splicing is compatible with RNA-directed heterochromatin formation. Small RNAs complementary to promoter distal introns trigger the formation of stable heterochromatin irrespective of pre-mRNA splicing. However, targeting a functional intron close to the 5’ end of a transcript results in a bistable chromatin state. Together, our results disprove siRNA-DNA base-pairing and provide mechanistic insights that will inform the experimental design of small RNA-directed chromatin silencing studies in multicellular organisms. Overall design: The study comprises of 8 small RNAs sequencing data sets from cells expressing a ura4-cox4 hairpin construct that gives rise to siRNAs against 4 engeneered target genes. Experiments were performed in wt and leo1 mutant cells. Less...