Understanding Complex Gene Editing Systems in Protists

描述（由申请人提供）：原生生物以前所未有的一系列遗传组织让分子生物学家感到惊讶，从自剪接RNA和端粒酶的发现，到基因大小的片段，程序化的DNA删除，以及纤毛虫的DNA重排。因此，这些生物体是集中进行程序化DNA重排的机械、进化和计算研究的自然场所。特别是，纤毛虫的“混乱基因”为研究复杂遗传和计算机制的起源和功能提供了一个独特的实验系统。从原生生物到人类，许多其他系统中都存在着混乱的基因。然而，很少有基因片段真的在DNA水平上“缝合”在一起，从所有片段中创造出一个连续的基因。在纤毛虫中发生的DNA剪接事件的复杂性使它们成为最有趣和最具挑战性的研究案例之一。此外，频繁的DNA缺失和其他广泛的基因组重排事件越来越多地与几种癌症相关，强调了研究此类生物事件的重要性。RNA通常被认为是基因表达的管道，最近的发现在纤毛原生动物中具有一种新的作用模式，这激发了本提案的研究。目标是研究纤毛虫的基因组重排，将一个近乎完整的基因组计划与最近的发现和工具结合起来，使Oxytricha成为一个强大的模型系统。例如，RNA模板既可以为DNA重排提供组织指南，也可以将自发的体细胞突变传递给下一代。rna引导的DNA修复对Oxytricha来说意义深远，因为这种生物通过整体重排破坏了95%的生殖系DNA，然后对剩余的片段进行分类和重新排序。本研究的具体目标都集中在基因组、进化或发育尺度上的混乱基因处理问题上。总体目标是利用该模型系统来探索微生物真核生物基因组中复杂的重写机制。具体目标包括：1。RNA模板能够编程DNA重排的最低要求是什么？2. 了解RNA模板核苷酸取代的表观遗传。3. 3 .小rna在棘毛虫及其相关纤毛虫柱头纲中的序列分布。木犀草大核发育相关候选基因的鉴定与检测。三叶草打乱基因的基因组分布及数据库的重大更新。6. 高度混乱的种系核糖体RNA位点的比较研究。

DESCRIPTION (provided by applicant): Protists have surprised molecular biologists with an unprecedented array of genetic organization, from the discoveries of self-splicing RNA and telomerase, to gene-sized pieces, programmed DNA deletion, and scrambled DNA rearrangements in ciliates. Therefore, these organisms are the natural place to focus a combined mechanistic, evolutionary, and computational study of programmed DNA rearrangements. In particular, the "scrambled genes" in ciliates offer a unique experimental system in which to study the origin and function of a complex genetic and computational mechanism. Scrambled genes exist in many other systems, ranging from protists to humans. Rarely, however, are the gene fragments actually 'sewn' back together at the level of DNA, to create a contiguous gene from all pieces. The complexity of DNA splicing events that take place in ciliates makes them one of the most interesting and challenging cases to study. Moreover, frequent DNA deletions and other extensive genome rearrangement events are increasingly associated with several cancers, underscoring the importance of studying such biological events. The recent discovery that RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa motivates the research in this proposal. The goal is to study genome rearrangements in the ciliate Oxytricha, pairing a nearly complete genome project with recent discoveries and tools that make Oxytricha a powerful model system. For example, RNA templates can provide both an organizing guide for DNA rearrangements and a template that can transmit spontaneous somatic mutations to the next generation. This opportunity for RNA-guided DNA repair is profound in Oxytricha, because this organism destroys 95% of its germline DNA through global rearrangements, and then sorts and reorders the remaining segments. The specific aims of this research all focus on questions about the processing of scrambled genes at the genomic, evolutionary, or developmental scale. The global aim is to use this model system to explore the complex rewriting mechanisms in microbial eukaryotic genomes. Specific aims include: 1. What are the minimal requirements for an RNA template to be able to program DNA rearrangement? 2. Understanding the epigenetic inheritance of nucleotide substitutions from an RNA template. 3. Small RNAs and their sequence distribution in Oxytricha and the related ciliate Stylonychia. 4. Identification and testing of candidate genes involved in macronuclear development in Oxytricha. 5. The genomic distribution of scrambled genes in O. trifallax, and significant updates to a database. 6. Comparative studies of the highly scrambled germline ribosomal RNA locus. PUBLIC HEALTH RELEVANCE: DNA rearrangements (deletions, inversions, and duplications) are a major factor contributing to genome instability associated with many human diseases, including cancer, with DNA translocations and gross deletions responsible for a significant portion of cancers and inherited diseases. Recombination often occurs between "hotspots", which may be short identical repeats that resemble "pointers" at DNA recombination junctions in Oxytricha, and such events can result in either deletion of tumor-suppressing genes, the formation of chimeric genes, or duplication and subsequent over-expression of genes that promote tumor stability. Because of its magnitude of DNA rearrangements and the recent demonstration that RNA templates can reprogram DNA rearrangements in vivo, Oxytricha is unparalleled as a model system to shed light on the complex events during DNA rearrangement and similar mechanism(s) responsible for cancer and genome instability in humans.