RNAi-mediated Targeting of Hetorochromatin Assembly

RNAi 介导的异染色质组装靶向

• 批准号: 6857655
• 负责人: DANESH MOAZED
• 金额: $ 31.79万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857655
• 关键词: RNA interference; Schizosaccharomyces pombe; amidohydrolases; chromatin; fungal genetics; gene induction /repression; helicase; heterochromatin; histones; methyltransferase; molecular assembly /self assembly; nucleosomes; protein protein interaction; replicase; small interfering RNA

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.

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 是一种广泛传播的沉默机制，在转录后和转录水平上发挥作用，并由被加工成小干扰 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失活。我们的实验室已经纯化了一种这样的效应复合物，它直接或间接靶向DNA以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的启动），包含 Ago1、Chp1 和 Tas3 蛋白，并将 RNAi 途径与异染色质物理连接。 Ago1 亚基从酵母到人类都是保守的，其同源物形成另一种效应复合物的核心亚基，该复合物使用 siRNA 来靶向 mRNA 进行失活。 Chp1 是一种异染色质结构蛋白，与赖氨酸 9-甲基化组蛋白 H3（异染色质的保守标记）相关。此外，该复合物还含有与组装异染色质的着丝粒 DNA 重复序列相匹配的 siRNA。该提案的目标是了解 RITS 如何针对特定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剖析。 RNAi 和异染色质蛋白的保守性表明，这里为裂殖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了解不仅可以为理解该过程如何失败提供框架，而且还可以为设计基于干预的治疗策略提供基础和知识。