CHARACTERIZATION OF PROTEIN COMPLEXES INVOLVED IN RNA INDUCED TRANSCRIPTIONAL GE

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 转录基因沉默是由特定染色体位点的异染色质形成引起的。重要的染色体亚结构如着丝粒依赖于异染色质，并在基因组稳定性、细胞分裂和基因表达调控中发挥关键作用。因此，异染色质的不受控制的形成和维持与癌症和发育障碍等疾病密切相关。令人惊讶的是，发现着丝粒是通过与RNA干扰（RNAi）密切相关的途径建立和维持的。在粟酒裂殖酵母中，已经鉴定了三种主要的蛋白质复合物，它们是RITS -携带siRNA的效应物复合物-RNA依赖性RNA聚合酶复合物（RDRC）以及基于蛋白质Rikl的蛋白质复合物。我的研究将集中在RITS和Rik 1的功能和结构表征。通过在杆状病毒表达系统中重组异源表达来接近目标复合物的产生，随后对复合物进行结构和功能研究，旨在阐明它们在RNAi介导的转录基因沉默过程中的作用。蛋白质复合物在真核细胞培养中的表达和具有挑战性的功能以及高分辨率结构分析将有利地增加我以前在低分辨率结构工作中的经验。有了这个奖学金，我将得到一个极好的机会，在冷泉港实验室非常刺激和高度跨学科的环境中发展我的职业生涯，成为一名独立的研究人员。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Transcriptional gene silencing is brought about by the formation of heterochromatin at specific chromosomal loci. Important chromosomal substructures such as the centromere depend on heterochromatin and play a crucial role in genome stability, cell division and regulation of gene expression. Consequently, uncontrolled formation and maintenance of heterochromatin are closely linked to diseases such as cancer and developmental disorders. It has been quite surprising to find that centromeres are established and maintained by a pathway closely linked to RNA interference (RNAi). In Schizosaccharomyces pombe three major protein complexes have been identified which are RITS - the siRNA carrying effector complex - an RNA dependent RNA polymerase complex (RDRC) as well as protein complexes based on the protein Rik1. My research will focus on the functional and structural characterization of RITS and Rik1. Production of the target complexes will be approached by recombinant heterologuous expression in the Baculo virus expression system with subsequent structural and functional investigation of the complexes aimed at elucidating their role in the process of RNAi mediated transcriptional gene silencing. Expression of protein complexes in eukaryotic cell cultures and challenging functional as well as high resolution structural analysis will favourably add to my previous experience in low resolution structural work. With this fellowship, I will get an excellent opportunity to develop my career towards an independent researcher in the very stimulating and highly interdisciplinary environment of the Cold Spring Harbor Laboratories.