Silencing of Mobile Genetic Elements by Small RNAs

描述(由申请人提供)：本项目的目标是描述转座子沉默途径，特别是关注小RNA的内源性作用。自20世纪80年代末S以来，通过使用经典的遗传工具来询问与转座机制有关的问题，转座子在线虫中得到了很好的表征。1999年，人们发现内源小RNA途径调控转座子，并进行了几次筛选以分离种系中具有活跃转座子的突变体。然而，尽管在属于这一途径的基因中分离出了许多突变体，但对单个蛋白质的作用进行机械表征的工作还很少。此外，由于转座子移动的大多数分析是在高通量测序出现之前完成的，因此没有全基因组数据来检查转座子的移动。除了转座子外，许多其他类型的转录本都是通过内源RNA途径来处理的，包括异常转录本、重复和假设的基因以及非编码区，然而这些转录物被识别并进入这些途径的机制完全未知。通过探索这些途径的各个组成部分，研究它们与什么物种的RNA相关，以及它们的酶功能在什么过程中起作用，这个项目将实现对转座子沉默的更全面的理解。本实验旨在确定调节转座子动员的因素，包括基因组特征、宿主蛋白和环境刺激，目的是阐明这一途径的已知和新成分的作用机制。这项工作的结果将对理解活跃的转座子如何影响基因表达和改变基因组结构具有重要意义，这些转座子可以导致包括癌症在内的许多疾病。Ruvkun实验室是研究线虫RNAi机制调节转座子的理想场所。该实验室不仅在小RNA领域拥有广泛的专业知识，而且在应激反应途径、高通量基因筛选、蛋白质纯化和生物化学方面也拥有丰富的专业知识。实验室环境包含了一群对线虫生物学的基本方面感兴趣的极具积极性和科学多样性的研究人员。该实验室两侧是两个研究神经生物学和发病机制的优秀线虫实验室，并得到一个高度合作的部门的支持，该部门拥有在RNA生物学和染色质结构方面的专业知识。我计划在我剩余的博士后任期内利用所有这些资源。在我的整个研究生涯中，我一直专注于染色质和RNA生物学，这对了解和预防癌症至关重要。具体地说，对RNA沉默和转座子沉默的更多了解可能会导致癌症诊断或治疗的进步。我致力于学术研究的事业，并计划成立一个实验室，专注于这项提案中的目标。

DESCRIPTION (provided by applicant): The goal of this project is to characterize the transposon-silencing pathway, specifically focusing on the endogenous role of small RNAs. Since the late 1980's, transposons have been well characterized in C. elegans through the use of classical genetic tools to interrogate questions related to mechanisms of transposition. In 1999, it was discovered that endogenous small RNA pathways regulate transposons and several screens were performed to isolate mutants with active transposition in the germline. However, although many mutants have been isolated in genes attributed to this pathway, little work has been done to mechanistically characterize the roles of individual proteins. Additionally, because most analyses of transposon movement were done prior to the advent of high-throughput sequencing, there is no genome-wide data examining transposon mobilization. In addition to transposons, many other types of transcripts are processed through the endogenous RNA pathways, including aberrant transcripts, duplicated and hypothetical genes, and non-coding regions, however the mechanism by which these transcripts are recognized and routed into these pathways is completely unknown. By probing the individual components of these pathways, examining what species of RNA they associate with, and to what process their enzymatic functions contribute, this project will achieve a more complete understanding of transposon silencing. The proposed experiments herein aim to identify factors that regulate transposon mobilization, including genomic features, host proteins, and environmental stimuli with the goal of elucidating the mechanisms of action of the known and novel components of this pathway. The results of this work will have significant implications in understanding how active transposons affect gene expression and alter genome structure, which can result in many diseases including cancer. The Ruvkun lab is an ideal place to study regulation of transposons by RNAi machinery in C. elegans. The lab not only has extensive expertise in the small RNA field, but also in stress response pathways, high- throughput genetic screens, protein purification, and biochemistry. The laboratory environment contains an extremely motivated and scientifically diverse group of researchers interested in fundamental aspects of C. elegans biology. The lab is flanked by two excellent C. elegans labs studying neurobiology and pathogenesis and is supported by a highly collaborative department that houses labs with expertise in RNA biology and chromatin structure. I plan to utilize all of these resources during the rest of my post-doctoral tenure. Throughout my research career, I have been focused on chromatin and RNA biology, which are fundamentally important in the understanding and prevention of cancer. Specifically, greater understanding of RNA silencing and transposon quiescence may lead to advances in cancer diagnoses or therapeutics. I am committed to a career in academic research and plan to start a lab focused on the aims in this proposal.