CHARACTERIZATION OF PROTEIN COMPLEXES INVOLVED IN RNA INDUCED TRANSCRIPTIONAL GE

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 转录基因沉默是由特定染色体上异染色质的形成引起的。着丝粒等重要的染色体亚结构依赖于异染色质，在基因组稳定、细胞分裂和基因表达调控中起着至关重要的作用。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，着丝粒的建立和维持是由一条与RNA干扰(RNAi)密切相关的途径来实现的。在裂殖酵母中已经鉴定出三种主要的蛋白质复合体，即RITS-携带siRNA的效应复合体-RNA依赖的RNA聚合酶复合体(RDRC)以及基于蛋白Rik1的蛋白质复合体。我的研究将集中在RITS和Rik1的功能和结构特征上。目的复合体的产生将通过在杆状病毒表达系统中的重组异源表达来实现，随后对复合体的结构和功能进行研究，旨在阐明它们在RNAi介导的转录基因沉默过程中的作用。在真核细胞培养中表达蛋白质复合体以及具有挑战性的功能和高分辨率结构分析将有助于增加我以前在低分辨率结构工作方面的经验。有了这笔奖学金，我将获得一个极好的机会，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.