"Understanding Complex Gene Editing Systems in Protists"

描述(由申请人提供)： RNA通常被认为是基因表达的管道，但在纤毛虫中有一种新的作用模式，其中又长又小的非编码RNA协调了复杂的基因组重塑过程，而母体RNA模板既为DNA重排提供了组织指南，也为将自发的体细胞突变传递给下一代提供了模板。这项研究的多学科目标是了解导致基因组重排的不寻常的生物信息处理形式。与之前的更新一样，重点将主要放在纤毛虫上，这是该实验室目前的重点，将完整的体细胞和接近完整的生殖系基因组组装与最近的发现和工具相结合，这些发现和工具使Oxytricha trifalax成为研究基因组重排的强大模型。RNA引导的DNA修复的机会在Oxytricha是深刻的，它通过全球DNA重排删除了95%的生殖系基因组，这种重排严重破坏了它的染色体，然后对剩下的数十万个片段进行排序和重新排序。这项拟议的研究结合了不同的方法，共同关注纤毛虫大规模基因组重组的机制和潜在逻辑，范围从单基因功能实验到基因组范围和系统水平的分析。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。理解基因解读的本质是目前这一提议背后的推动力，拟议的实验将检查参与这些复杂重排事件的分子的性质以及它们背后的机制。从拟议的研究中可以获得的最直接的见解是，在单基因和微生物真核基因组的微调处理的规模上，了解细胞杂技和在枝毛纤毛虫中执行这些令人惊叹的基因组重排的组件。更长期的见解包括与类似的RNA引导机制的相关性，这些机制影响基因组完整性，保护真核细胞免受疾病状态的影响，或允许正常细胞和操纵细胞中RNA介导状态的表观遗传。具体目标包括：1.通过长配对文库改进Oxytricha生殖系基因组组装，2.测试长的非编码模板RNA与piRNAs之间相互作用的共注射实验，3.研究Oxytricha中的piRNA的生物发生，4.了解长的非编码模板RNA的产生及其相互作用的伙伴，5.建立一个综合的数据库，评估接合过程中RNA、DNA和蛋白质的可用性，6.鉴定和功能测试体细胞染色体中的调控基序，以及7.识别和测试参与Oxytricha基因组重排的候选基因。

DESCRIPTION (provided by applicant): RNA, normally thought of as a conduit in gene expression, has a novel mode of action in ciliated protozoa, where long and small noncoding RNAs orchestrate an intricate process of genome remodeling, and maternal RNA templates provide both an organizing guide for DNA rearrangements and a template that can transmit spontaneous somatic mutations to the next generation. The multidisciplinary goal of this research is to understand the unusual forms of biological information processing that lead to genome rearrangement. As in the previous renewal, the focus will be mainly on ciliates, the current emphasis of the lab, pairing a complete somatic and nearly-complete germline genome assembly with recent discoveries and tools that make Oxytricha trifallax a powerful model to study genome rearrangements. The opportunity for RNA-guided DNA repair is profound in Oxytricha, which deletes 95% of its germline genome through global DNA rearrangements that severely fragment its chromosomes and then sort and reorder the hundreds of thousands of pieces remaining. The proposed research combines different approaches that together focus on the mechanism and underlying logic of massive genome reorganization in ciliates, over a range of scales, from single gene functional experiments to genome-wide and systems-level analyses. The global aim is to use this model system to explore the complex rewriting mechanisms in microbial eukaryotic genomes. Understanding the nature of gene unscrambling is the motivating force behind the current proposal, and the proposed experiments will examine the properties of both the molecules that participate in these complex rearrangement events and the machinery underlying them. The most immediate insight to be gained from the proposed research is an understanding of the cellular acrobatics and the components that execute these stunning genome rearrangements in stichotrichous ciliates, on the scales of fine-tune processing of single genes and microbial eukaryotic genomes. Longer-term insights include relevance to similar RNA-guided mechanisms that influence genome integrity, protecting eukaryotic cells from a diseased state, or that permit epigenetic inheritance of RNA-mediated states, in normal and manipulated cells. Specific aims include: 1. Improving the Oxytricha germline genome assembly through long mate-paired libraries, 2. Co-injection experiments that test the interplay between long, noncoding template RNAs and piRNAs, 3. Studying piRNA biogenesis in Oxytricha, 4. Understanding long, noncoding template RNA production and their interacting partners, 5. Creating a comprehensive database assessing RNA, DNA, and protein availability during conjugation, 6. Identifying and functionally testing regulatory motifs in somatic chromosomes, and 7. Identifying and testing candidate genes involved in Oxytricha genome rearrangement.