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）途径如何调节裂殖酵母中的异染色质基因沉默。RNAi是一种广泛存在的沉默机制，其在转录后和转录水平上起作用，并且由被加工成小干扰RNA（称为siRNA）的双链RNA分子触发。siRNA通过效应复合物引导互补靶核酸的失活。我们的实验室已经纯化了RITS（RNA诱导的转录基因沉默）复合物，包含Ago 1，Chp 1和Tas 3蛋白，它们将RNAi途径与异染色质物理连接。此外，我们已经纯化了合成dsRNA的RNAi复合物，将其加工成siRNA，并帮助将siRNA加载到RITS复合物上。我们已经提供了证据表明，这些RNAi复合物定位到特定的染色体区域，通过siRNA和新生的非编码转录本之间的碱基配对相互作用。这种定位导致组蛋白H3甲基转移酶复合物的募集，称为CRC（Clr 4-Rik 1-Cul 4）复合物，其甲基化赖氨酸-9上的组蛋白H3并促进异染色质形成。我们最近的研究结果表明，顺式合成的dsRNA，在异染色质组装的网站，需要H3赖氨酸-9甲基化。该提案的目标是了解RITS和dsRNA合成如何招募CRC组蛋白甲基转移酶复合物以及新生RNA转录物如何控制异染色质的扩散。此外，我们还将对异染色质siRNA的形成过程进行生化分析。公共卫生相关性：本项目研究RNA干扰（RNAi）在细胞核基因沉默中的作用，RNAi是一种保守的RNA沉默途径。该途径的组分参与调节人类的细胞分化和癌症。对RNAi在基因沉默中的作用的基本理解不仅将为理解该过程如何失败提供框架，还将为设计基于干预的治疗策略提供基础和知识。

DESCRIPTION (provided by applicant): The long-term goal of this work is to understand how the RNA interference (RNAi) pathway regulates heterochromatic gene silencing in fission yeast. RNAi is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels and is triggered by double stranded RNA molecules that are processed to small interfering RNAs (called siRNAs). siRNAs guide the inactivation of complementary target nucleic acids by effector complexes. Our laboratory has purified the RITS (RNA-Induced Transcriptional Gene Silencing) complex, containing the Ago1, Chp1, and Tas3 proteins, which physically links the RNAi pathway to heterochromatin. In addition, we have purified the RNAi complexes that synthesize dsRNA, process it into siRNAs, and help load siRNAs onto the RITS complex. We have provided evidence that these RNAi complexes localize to specific chromosome regions via base pairing interactions between siRNAs and nascent non-coding transcripts. This localization leads to recruitment of a histone H3 methyltransferase complex, called the CRC (Clr4-Rik1-Cul4) complex, which methylates histone H3 on lysine-9 and promotes heterochromatin formation. Our recent results suggest that cis-synthesis of dsRNA, at sites of heterochromatin assembly, is required for H3 lysine-9 methylation. The goals of this proposal are to understand how RITS and dsRNA synthesis recruit the CRC histone methyltransferase complex and how the nascent RNA transcript controls the spreading of heterochromatin. In addition, we will perform a biochemical dissection of steps that are involved in the formation of heterochromatic siRNAs. PUBLIC HEALTH RELEVANCE: This project addresses the role of RNA interference (RNAi), a conserved RNA silencing pathway, in gene silencing in the nucleus. Components of this pathway are involved in regulation of cell differentiation and cancer in humans. A basic understanding of the role of RNAi in gene silencing will not only provide a frame work for understanding how the process can fail, but also provides the substrate and knowledge to design therapeutic strategies based on intervention.