RNA-Guided Genome Rearrangement of Tetrahymena

RNA引导的四膜虫基因组重排

• 批准号: 0642162
• 负责人: Douglas Chalker
• 金额: --
• 依托单位: Washington University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0642162&HistoricalAwards=false
• 关键词: RNA; Guided; Genome; Rearrangement; Tetrahymena

RNA interference (RNAi)-related mechanisms participate in diverse epigenetic phenomena. Few are more extreme than the genome remodeling of the ciliate Tetrahymena thermophila. This organism eliminates nearly 15 megabases of its germline DNA from the somatic nucleus during its development. This project aims to understand the regulation of this massive genome reorganization and ultimately learn fundamental principles governing chromosome structure and stability. Understanding how ~6000 DNA segments, called internal eliminated sequences (IESs), are selectively excised is challenged by the fact that they share little common structure. The current model is built on the observations that bi-directional IES transcription leads to the generation of ~28 nucleotide RNA molecules (scan RNAs) that then target specific modification(s) to the homologous chromosomal location(s). The DNA rearrangement machinery recognizes this modified chromatin state and eliminates the targeted DNA segment. These studies will certainly provide fundamental insight into RNAi-related mechanisms that direct chromatin modifications that are critical for transcriptional gene silencing and heterochromatin formation in eukaryotes. The specific intellectual goals of this project are: 1) Identification of general cis-requirements for IES excision that should define basic constraints imposed on RNAi-directed chromatin modification. 2) Characterization of the specificity of germline, non-genic transcription that is postulated to be the initiating event in this remarkable genome reorganization. 3) Continued characterization of Dicer-like, RNAseIII homologues of Tetrahymena and their putative partner proteins. This will lead to elucidation of their roles in DNA rearrangement and/or other RNAi-related processes. The plan is to accomplish these goals through a combination of genetic and molecular biological approaches, taking specific advantage of tools available for studies in this model organism. Underlying this project is a goal to understand how RNA molecules can communicate genetic information between the parental and developing genomes, which has great potential to reveal novel roles for RNA in epigenetic programming. Additionally, because it is believed that many of the DNA segments targeted for elimination are important for germline chromosome structure, an increased understanding of how the cell specifically recognizes these sequences will contribute to the current knowledge base of mechanisms that ensure the chromosome stability that is essential to prevent aberrant rearrangements. The ability of RNAs with sequences complementary to genomic DNA to regulate the activity of the eukaryotic genome has been revealed by recent studies of diverse biological processes, including genome rearrangements of Tetrahymena. The unique biology of this organism offers an excellent context which with to uncover the fundamental mechanisms by which such RNAs elicit their action on the DNA. In addition to providing such insights into this important genetic regulatory mechanism, this project will serve to train undergraduates, post-baccalaureate laboratory technicians, and PhD students in hypothesis-driven research and prepare them for future scientific careers. Underrepresented minorities in science have been trained using prior support and this continued support enables future mentoring. The research is directly linked to the further development of a laboratory course in which undergraduate students engage in original, sophisticated research. This course's problem-based approach teaches students how to use current technologies in a model organism to generate a thorough understanding of the biological process being explored. Gene silencing vectors that will be developed in this project will be incorporated into the design of future offerings of this course, providing a broader impact of the project's intellectual pursuits allowing direct carryover into its educational goals. The research tools and curriculum generated by this project, such as these vectors for gene inhibition studies, will be extremely valuable reagents that will be distributed to the broader Tetrahymena research and education community. Thus these efforts will help make this important model organism more accessible to biologists from other fields.

RNA干扰（RNAi）相关机制参与多种表观遗传现象。 很少有比四膜虫（Tetrahymena thermophila）的基因组重塑更极端的。这种生物体在发育过程中从体细胞核中消除了近15兆碱基的生殖细胞DNA。 该项目旨在了解这种大规模基因组重组的调控，并最终了解控制染色体结构和稳定性的基本原则。 了解约6000个DNA片段（称为内部消除序列（IES））是如何被选择性切除的，这是一个挑战，因为它们几乎没有共同的结构。目前的模型建立在以下观察结果的基础上：双向IES转录导致产生约28个核苷酸的RNA分子（扫描RNA），然后将特异性修饰靶向同源染色体位置。 DNA重排机制识别这种修饰的染色质状态并消除靶向DNA片段。 这些研究肯定会提供基本的见解RNAi相关的机制，直接染色质修饰是转录基因沉默和异染色质形成的真核生物中的关键。本项目的具体智力目标是：1）确定IES切除的一般顺式要求，应定义对RNAi指导的染色质修饰施加的基本约束。2)种系特异性的表征，非基因转录被假定为这一显着的基因组重组的起始事件。3)四膜虫的Dicer样RNAseIII同源物及其推定伴侣蛋白的继续表征。这将导致阐明它们在DNA重排和/或其他RNAi相关过程中的作用。 该计划是通过遗传和分子生物学方法相结合来实现这些目标，具体利用可用于这种模式生物研究的工具。该项目的目标是了解RNA分子如何在亲本和发育基因组之间传递遗传信息，这对于揭示RNA在表观遗传编程中的新作用具有巨大潜力。 此外，由于人们认为许多靶向消除的DNA片段对种系染色体结构很重要，因此对细胞如何特异性识别这些序列的理解将有助于确保染色体稳定性的机制的现有知识基础，这对于防止异常重排至关重要。具有与基因组DNA互补的序列的RNA调节真核基因组活性的能力已经通过最近对包括四膜虫的基因组重排在内的多种生物过程的研究而被揭示。这种生物体的独特生物学提供了一个很好的背景，揭示了这些RNA引起它们对DNA作用的基本机制。 除了提供这种重要的遗传调控机制的见解，该项目将有助于培养本科生，学士后实验室技术人员和博士生在假设驱动的研究，并为他们未来的科学生涯做好准备。 利用先前的支持对科学领域代表性不足的少数群体进行了培训，这种持续的支持使未来的辅导成为可能。这项研究直接关系到实验室课程的进一步发展，在该课程中，本科生从事原创，复杂的研究。 本课程的问题为基础的方法教学生如何使用当前的技术在一个模式生物产生一个深入的了解正在探索的生物过程。将在本项目中开发的基因沉默载体将被纳入本课程未来课程的设计中，为项目的智力追求提供更广泛的影响，从而直接延续到其教育目标中。该项目产生的研究工具和课程，如用于基因抑制研究的载体，将是非常有价值的试剂，将分发给更广泛的四膜虫研究和教育界。 因此，这些努力将有助于使这一重要的模式生物更容易获得来自其他领域的生物学家。