RNAi-mediated Targeting of Hetorochromatin 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一种这样的效应复合物，直接或间接靶向DNA组装表观遗传异染色质结构域。这种复合物，我们称之为RITS（RNA诱导的转录基因沉默），包含Ago 1，Chp 1和Tas 3蛋白，并将RNAi途径与异染色质物理连接。Ago 1亚基从酵母到人类都是保守的，其同源物形成另一种类型的效应复合物的核心亚基，该效应复合物使用siRNA靶向mRNA以使其失活。Chp 1是一种异染色质结构蛋白，与异染色质的保守标记物赖氨酸9-甲基化组蛋白H3相关。此外，该复合物含有与异染色质组装的着丝粒DNA重复序列相匹配的siRNA。本提案的目标是了解RITS如何靶向异染色质失活的特定染色体区域，并对RNAi介导的异染色质组装的机制进行生物化学解剖。 RNAi和异染色质蛋白的保守性表明，这里为裂变酵母复合物开发的原理将适用于其他环境。对RNAi在表观遗传染色质结构域组装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，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 wide spread 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 one such effector complex that either directly or indirectly targets DNA for assembly of epigenetic heterochromatin domains. This complex, which we have termed RITS (RNA-induced [nitiation of Transcriptional Gene Silencing), contains the Ago1, Chp1, and Tas3 proteins and physically links the RNAi pathway to heterochromatin. The Ago1 subunit is conserved from yeast to human and its homologs form the core subunits of another type of effector complex that uses siRNAs to target mRNA for inactivation. Chp1 is a heterochromatin structural protein that associates with lysine 9-methylated histone H3, a conserved marker of heterochromatin. In addition, the complex contains siRNAs that match centromeric DNA repeats where heterochromatin is assembled. The goals of this proposal are to understand how RITS targets specific chromosome regions for heterochromatic inactivation and to perform a biochemical dissection of the mechanism of RNAi-mediated heterochromatin assembly. The conservation of RNAi and heterochromatin proteins suggests that the principles developed here for the fission yeast complexes will apply in other settings. A basic understanding of the role of RNAi in assembly of epigenetic chromatin domain 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.