International Research Fellowship Program: Isolating Novel Components and Identifying Endogenous Targets of RNA-directed Chromatin Modification

[502 . 1999]国际研究奖学金计划使美国科学家和工程师能够到国外进行3至24个月的研究。该计划的奖励为联合研究提供了机会，并利用独特或互补的设施、专业知识和国外的实验条件。该奖项将支持Rebecca a . Mosher博士与David C. Baulcombe博士在英国诺维奇的John Innes中心进行为期22个月的研究。RNA介导的基因沉默，也称为共抑制、平息或RNA干扰，是一种重要的遗传调控机制。RNA介导的沉默被研究得最好的形式是转录后基因沉默（PTGS），其中短干扰RNA （siRNA）引导RISC （RNA干扰特异性复合体）切割任何与siRNA序列同源的mRNA。sirna还指导一种鲜为人知的rna介导的基因沉默形式，rna定向染色质修饰（RDCM）。在RDCM过程中，与sirna同源的DNA被甲基化，导致转录减少。组蛋白H3在sirna的作用下也被甲基化，特别是在赖氨酸9 （K9）上，赖氨酸9通常是惰性异染色质的标记。通过染色质修饰和其他核事件，RDCM加强了PTGS，也可能具有发育功能。拟南芥基因筛选在发现驱动PTGS的机制方面取得了重大进展。相比之下，对RDCM的机制和相关蛋白的了解相对较少。在本提案的具体目标1中，PI将使用突变体分析来发现RDCM的新成分。使用PVX-GFP沉默系统的基因筛选发现了30个新的RDCM突变，它们至少属于5个互补组。这些突变将使用传统的基于地图的技术进行克隆。RDCM的天然作用也不清楚，并且很少有内源性靶点被确定。在具体目标2中，PI将使用改进的AFLP程序，然后进行siRNA分析和染色质免疫沉淀，鉴定RDCM的内源性靶点。Sainsbury实验室的Baulcombe小组一直是rna介导沉默领域的先驱，并成功地利用遗传分析发现了RDCM中的新元素。塞恩斯伯里实验室和大约翰英纳斯中心是一个世界级的设施，拥有设备和支持人员，使这个项目取得成功。该项目在基础生物学、进化生物学和基因治疗等多个领域具有重要的智力潜力。

0502199MosherThe International Research Fellowship Program enables U.S. scientists and engineers to conduct three to twenty-four months of research abroad. The program's awards provide opportunities for joint research, and the use of unique or complementary facilities, expertise and experimental conditions abroad.This award will support a twenty-two-month research fellowship by Dr. Rebecca A. Mosher to work with Dr. David C. Baulcombe at John Innes Centre in Norwich, United Kingdom.RNA-mediated gene silencing, also known as co-suppression, quelling, or RNA interference, is an important genetic regulatory mechanism. The best studied form of RNA-mediated silencing is post-transcriptional gene silencing (PTGS), in which short interfering RNA (siRNA) guide RISC (RNA interference specificity complex) to cleave any mRNA with sequence homology to siRNAs. siRNAs also direct a less understood form of RNA-mediated gene silencing, RNA-directed chromatin modification (RDCM). During RDCM, DNA with homology to siRNAs is methylated, causing a reduction in transcription. Histone H3 is also methylated in response to siRNAs, specifically at lysine 9 (K9), commonly the mark of inert heterochromatin. Through chromatin modification and perhaps other nuclear events, RDCM reinforces PTGS and may also have a developmental function. Genetic screens in Arabidopsis have made deep inroads toward discovering the machinery driving PTGS. In comparison, relatively little is known of the mechanism and associated proteins of RDCM. In specific aim 1 of this proposal, the PI will use mutant analysis to uncover novel components of RDCM. A genetic screen using the PVX-GFP silencing system has uncovered 30 new RDCM mutations falling into at least five complementation groups. These mutations will be cloned using traditional map-based techniques. The natural role of RDCM is also unclear and few endogenous targets have been identified. In specific aim 2, the PI will identify endogenous targets of RDCM using a modified AFLP procedure followed by siRNA analysis and chromatin immunoprecipitation.The Baulcombe group at the Sainsbury laboratory has been pioneers in the field of RNA-mediated silencing and have successfully used genetic analysis to uncover novel elements in RDCM. The Sainsbury Laboratory and greater John Innes Centre is a world-class facility with the equipment and support staff to make this project a success. This project has significant intellectual potential in a number of areas, including basic biology, evolutionary biology, and gene therapy.