Person: Yu, Ruby
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Publication Local and Genomic Determinants of siRNA-Mediated Heterochromatin Formation and Silencing in Fission Yeast(2016-03-10) Yu, Ruby; Wu, Chao-ting; Kennedy, Scott; Young, RichardIn eukaryotes, 20-30 nt small RNAs (sRNAs) regulate many cellular processes by a process called RNA interference (RNAi). sRNAs serve as adaptable specificity factors to direct silencing at either the post-transcriptional or transcriptional level. On one hand, the ability to target genome-wide is a valuable feature; however, this can potentially be a double-edged sword, as spurious expression of sRNAs could lead to inappropriate silencing. Fission yeast have developed mechanisms to combat inappropriate sRNA-mediated silencing. In fission yeast, small interfering siRNAs (siRNAs) and silenced heterochromatin are mutually dependent at pericentromeric repeat sequences, and are required for proper centromere function. Euchromatic regions of the genome typically do not generate siRNAs and are refractory to RNAi-mediated silencing and heterochromatin formation. This dissertation aims to identify mechanisms by which the cell protects itself against inappropriate RNAi-induced silencing or heterochromatin formation. Specifically, we seek to define 1) factors that determine whether a locus can produce siRNAs and 2) factors that determine whether a locus can be targeted by siRNAs for silencing or heterochromatin formation. Using high throughput sequencing of small RNAs in cells overexpressing Dicer, I show that sites of overlapping transcription including convergent genes and centromeric repeats are potential substrates for Dicer activity, but at endogenous levels of Dicer only centromeric repeats generate siRNAs. RT-PCR and ChIP-seq experiments reveal that, at euchromatic loci, generation of siRNAs in cis does not correlate with reduced transcript levels or with methylation of H3K9, a conserved marker for heterochromatin. Thus, there are features of euchromatic loci that protect them from siRNA-mediated silencing. Genetic studies involving deletion or mutation of the 3’ UTR of an endogenous reporter gene in cells expressing complementary siRNAs identify transcriptional cleavage and polyadenylation signals as one of these protective factors. Direct sequencing of polyadenylated transcripts reveals a divergence in cleavage patterns between centromeric and mRNA-coding loci, supporting the idea that the ability of a nascent transcript to be targeted for silencing by siRNAs correlates with inefficient 3’ end processing. Finally, I show that Mlo3, a gene associated with 3’ ends of ORFs and involved in mRNA export, antagonizes siRNA-mediated heterochromatin formation genome-wide. ChIP experiments and growth assays show that mlo3∆ cells are capable of generating siRNA- mediated H3K9 dimethylation at a locus in trans, but surprisingly, this rarely correlates with silencing of the targeted locus. Enrichment in H3K9 dimethylation that is not associated with silencing is not heritable through meiosis. However, once siRNA-induced silencing is established, heterochromatin is stable and transmissible through meiosis even in the absence of the driver siRNA-producing locus. Silencing is better correlated with H3K9me3 than H3K9me2, and we propose that H3K9me3 is required for formation of a silenced and heritable heterochromatic state. Together, these results demonstrate that the cell has evolved several mechanisms to protect itself from spurious siRNA-mediated silencing or heterochromatin formation. First, limiting concentrations of Dicer restrict siRNA generation to repetitive sequences surrounding the centromeres. However, if siRNAs mapping to mRNAs are expressed, factors involved in efficient 3’ end processing antagonize silencing and H3K9 methylation directed by siRNAs, These factors include transcript cleavage and polyadenylation sequences and mRNA export factor mlo3+.