RNAi-Mediated Heterochromatin Assembly

描述（由申请人提供）：这项工作的长期目标是了解RNA干扰（RNAi）途径如何指导沉默异染色质DNA结构域的形成。异染色质是真核生物染色体的一个保守特征，在酵母和人类等生物的基因表达调控和染色体稳定性维持中起着重要作用。在分裂酵母Schiozosaccharomyces pombe中，RITS （rna诱导的转录沉默）复合物通过与新生非编码rna的碱基配对相互作用，利用小干扰rna （sirna）靶向特定的染色体区域。这种靶向与组蛋白H3赖氨酸9 （H3K9）甲基化和介导沉默的蛋白质募集相结合。RITS还通过招募RNA依赖的RNA聚合酶复合物（RDRC）来促进双链RNA的合成，从而介导siRNA扩增，双链RNA被Dicer核糖核酸酶切割成siRNA。目前尚不清楚非编码RNA如何产生启动RNAi的小RNA触发器，RNA如何参与组蛋白H3K9甲基转移酶复合物（CLRC）的募集，以及下游因子如HP1蛋白的募集如何导致转录基因沉默。在这个建议中，我们将使用体内方法和生化分析相结合的方法来研究(1)着丝粒rna中的功能元件及其触发RNAi的相关因子，(2)sirna介导的染色质甲基化机制，(3)异染色质依赖的转录基因沉默的分子机制。这些研究的最终目标是对基因沉默的完整分子理解。

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how the RNA interference (RNAi) pathway directs the formation of silent heterochromatic DNA domains. Heterochromatin is a conserved feature of eukaryotic chromosomes and plays important roles in regulation of gene expression and maintenance of chromosome stability in organisms ranging from yeast to human. In the fission yeast Schiozosaccharomyces pombe, the RITS (RNA-induced transcriptional silencing) complex uses small interfering RNAs (siRNAs) to target specific chromosome regions by base pairing interactions with nascent noncoding RNAs. This targeting is coupled to histone H3 lysine 9 (H3K9) methylation and the recruitment of proteins that mediate silencing. RITS also mediates siRNA amplification by recruiting the RNA-dependent RNA polymerase complex (RDRC) to promote the synthesis of double stranded RNA, which is cleaved into siRNAs by the Dicer ribonuclease. It has remained unclear how noncoding RNAs give rise to small RNA triggers that initiate RNAi, how RNA participates in the recruitment of histone H3K9 methyltransferase complex (CLRC), and how the recruitment of downstream factors such as HP1 proteins leads to transcriptional gene silencing. In this proposal we will use a combination of in vivo approaches and biochemical assays to investigate (1) the functional elements in centromeric RNAs and their associated factors that trigger RNAi, (2) the mechanism of siRNA-mediated chromatin methylation, and (3) the molecular mechanism of heterochromatin-dependent transcriptional gene silencing. The ultimate goal of these studies is a complete molecular understanding of gene silencing.