RNA-Guided Genome Rearrangement of Tetrahymena

RNA干扰（RNAi）相关机制参与多种表观遗传现象。很少有比纤毛虫嗜热四膜虫的基因组重塑更极端的了。这种生物在发育过程中从体细胞核中消除了近15兆碱基的生殖系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理。6000个被称为内部消除序列（IESs）的DNA片段是如何被选择性切除的，这一事实对它们几乎没有共同的结构这一事实提出了挑战。目前的模型是建立在双向IES转录导致产生约28个核苷酸RNA分子（扫描RNA）的观察基础上的，然后将特定修饰靶向到同源染色体位置。DNA重排机制识别这种修饰的染色质状态，并消除目标DNA片段。这些研究将为真核生物中转录基因沉默和异染色质形成至关重要的直接染色质修饰的rnai相关机制提供基础见解。该项目的具体智力目标是：1)确定IES切除的一般顺式要求，该要求应定义rnai定向染色质修饰的基本约束。2)生殖系非基因转录的特异性表征，该转录被认为是这一显著基因组重组的起始事件。3)继续鉴定四膜虫的dicer样、RNAseIII同源物及其推测的伴侣蛋白。这将有助于阐明它们在DNA重排和/或其他rna相关过程中的作用。该计划是通过遗传和分子生物学方法的结合来实现这些目标，利用可用于研究这种模式生物的工具的特定优势。本项目的目标是了解RNA分子如何在亲本和发育基因组之间传递遗传信息，这具有揭示RNA在表观遗传编程中的新作用的巨大潜力。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，因此增加对细胞如何特异性识别这些序列的理解将有助于当前确保染色体稳定性机制的知识库，这对于防止异常重排至关重要。近年来，包括四膜虫基因组重排在内的多种生物过程研究揭示了具有基因组DNA互补序列的rna调节真核生物基因组活性的能力。这种生物的独特生物学特性为揭示rna对DNA的作用的基本机制提供了一个极好的背景。除了对这一重要的基因调控机制提供见解外，该项目还将为本科生、学士后实验室技术人员和博士生提供假设驱动研究的培训，并为他们未来的科学事业做好准备。在科学领域未被充分代表的少数群体已经利用先前的支持进行了培训，这种持续的支持使未来的指导成为可能。这项研究与实验课程的进一步发展直接相关，在实验课程中，本科生从事原创的、复杂的研究。本课程以问题为基础的方法，教导学生如何在模式生物中使用当前的技术，从而对正在探索的生物过程产生透彻的理解。本项目开发的基因沉默载体将被纳入本课程未来课程的设计中，为本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。这个项目产生的研究工具和课程，如基因抑制研究的载体，将是非常有价值的试剂，将被分发到更广泛的四膜虫研究和教育界。因此，这些努力将有助于使其他领域的生物学家更容易接触到这种重要的模式生物。

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.