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)途径如何调控分裂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，在转录后和转录水平上都发挥作用，由双链RNA分子被加工成小干扰RNA(称为siRNAs)而触发。SiRNAs通过效应复合体引导互补的靶核酸失活。我们的实验室已经纯化了RITS(RNA诱导转录基因沉默)复合体，其中包含Ago1、Chp1和Tas3蛋白，它们在物理上将RNAi途径与异染色质联系在一起。此外，我们还纯化了合成dsRNA的RNAi复合体，将其加工成siRNAs，并帮助将siRNAs加载到RITS复合体上。我们提供的证据表明，这些RNAi复合体通过siRNAs和新生非编码转录本之间的碱基配对相互作用定位于特定的染色体区域。这种定位导致组蛋白H3甲基转移酶复合体的招募，称为CRC(CLR4-Rik1-CUL4)复合体，它将组蛋白H3甲基化在赖氨酸-9上，并促进异染色质的形成。我们最近的结果表明，H3赖氨酸-9甲基化需要在异染色质组装部位顺式合成dsRNA。这项建议的目标是了解RITS和dsRNA合成如何招募CRC组蛋白甲基转移酶复合体，以及新生RNA转录本如何控制异染色质的传播。此外，我们还将对形成异染色质siRNA所涉及的步骤进行生化剖析。与公共卫生相关：该项目探讨了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。该途径的组成部分参与了细胞分化和人类癌症的调节。对RNAi在基因沉默中作用的基本了解不仅将为理解该过程如何失败提供一个框架，而且还将为设计基于干预的治疗策略提供底物和知识。

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.