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）途径如何调节裂变酵母中的异色基因沉默。RNAi是一种广泛存在的沉默机制，它在转录后和转录水平上都起作用，由双链RNA分子被加工成小干扰RNA（称为sirna）触发。sirna通过效应复合物引导互补靶核酸失活。我们的实验室已经纯化了一个这样的效应复合体，直接或间接地针对DNA组装表观遗传异染色质结构域。我们将这种复合物称为RITS （rna诱导的转录基因沉默起始），它包含Ago1、Chp1和Tas3蛋白，并将RNAi途径与异染色质物理连接起来。Ago1亚基从酵母到人类都是保守的，它的同源物形成了另一种效应复合物的核心亚基，这种效应复合物使用sirna靶向mRNA使其失活。Chp1是一种异染色质结构蛋白，与赖氨酸9-甲基化组蛋白H3（一种保守的异染色质标记）相关。此外，该复合体含有与异染色质组装的着丝粒DNA重复序列相匹配的sirna。本提案的目的是了解RITS如何针对特定的染色体区域进行异染色质失活，并对rnai介导的异染色质组装机制进行生化解剖。

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.