Understanding Complex Gene Editing Systems in Protists

描述(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，从发现自剪接RNA和端粒酶，到基因大小的片段，程序性DNA缺失，以及纤毛虫中的DNA重排。因此，这些生物体是关注程序性DNA重排的联合机制、进化和计算研究的自然场所。特别是，纤毛虫中的“杂乱基因”为研究复杂的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。杂乱的基因存在于许多其他系统中，从原生生物到人类。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。纤毛虫中发生的DNA剪接事件的复杂性使其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有关，这突显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，但最近的发现是，RNA在纤毛虫中具有一种新的作用模式，这促使了这一提议的研究。目标是研究纤毛虫Oxytricha的基因组重排，将一个几乎完整的基因组计划与最近的发现和工具配对，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，也可以提供将自发的体细胞突变传递给下一代的模板。这种RNA引导的DNA修复机会在Oxytricha中是深刻的，因为这种有机体通过全球重排破坏了95%的生殖系DNA，然后对剩余的片段进行排序和重新排序。这项研究的具体目的都集中在基因组、进化或发育尺度上关于杂乱基因处理的问题上。全球的目标是使用这个模型系统来探索微生物真核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.了解从RNA模板进行核苷酸替换的表观遗传。3.棘毛虫及其相关纤毛虫的小RNA及其序列分布。4.棘豆大核发育相关候选基因的鉴定和检测。5.杂乱基因的基因组分布，以及数据库的重大更新。6.高混杂种系核糖体RNA基因座的比较研究 与公共卫生相关：DNA重排(缺失、倒置和复制)是导致与包括癌症在内的许多人类疾病相关的基因组不稳定的主要因素，DNA易位和大体缺失是相当大一部分癌症和遗传性疾病的原因。重组经常发生在“热点”之间，这些“热点”可能是短的相同重复序列，类似于Oxytricha中DNA重组连接处的“指针”，这种事件可能导致肿瘤抑制基因的缺失，嵌合基因的形成，或者促进肿瘤稳定性的基因的复制和随后的过度表达。由于其DNA重排的规模和最近证明RNA模板可以在体内重新编程DNA重排，Oxytricha作为揭示DNA重排过程中的复杂事件和导致人类癌症和基因组不稳定的类似机制(S)的模型系统是无与伦比的。

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.