Functional and population genomics of small RNA regulation

描述（由申请人提供）： 虽然只有一小部分动物基因组实际上编码蛋白质，但最近的研究表明，大部分基因组仍然从DNA转录成RNA，其中大部分功能未知。这些非编码区的一部分产生短RNA片段（微小或miRNA和piwi相关或皮尔纳），其似乎在基因转录物沉默中发挥先前未被充分认识和普遍存在的作用。在过去的几年里，这些miRNAs与130多种不同的人类疾病有关，包括许多形式的癌症。尽管它们在细胞功能中起着关键作用，但我们对这些短调控RNA的自然变异以及这如何影响其mRNA靶点的变异几乎一无所知。在这里，我们建议利用关于miRNA功能的基本发现，已经使用模式线虫秀丽隐杆线虫进行了全面的基因组分析的小RNA变异和功能的密切相关的物种C。remanei，这是更适合自然变异的研究。我们的目标是：（1）确定负责miRNA功能起源和分化的进化力量，并测试这些进化模式与miRNA功能特性的关系;（2）确定miRNA调控对mRNA进化的影响;（3）直接测试miRNA进化和功能分化之间的关系。我们将利用下一代技术对32 C基因组进行测序，以实现这些目标.从两个不同的群体中提取的remanei品系和来自最近发现的密切相关的初期物种的3个另外的品系，并且通过使用来自分子群体遗传学的已建立的方法以及新的“SNP足迹”方法来分析这种变异。我们还将使用miRNA-system immuno pull-downs来全面鉴定miRNAs的mRNA靶点。最后，我们使用miRNA缺失线来测试miRNA自然变异的功能后果。除了解决具体的科学假设外，该项目还将开发一套对更广泛的科学界具有独特价值的遗传和基因组资源。本研究采用自然系统遗传学方法，将微进化过程的分析与我们对细胞内调控网络结构和功能的新兴理解相结合，以产生一个全基因组视图-用精细尺度SNP变异解剖-单个转录调控因子，这在其他主要模型系统中是不可能的。

DESCRIPTION (provided by applicant): Although only a small fraction of animal genomes actually code for proteins, recent work has shown that the majority of the genome is still transcribed from DNA into RNA, with much of the later being of unknown function. A portion of these non-coding regions generate short RNA fragments (micro or miRNA and piwi- associated or piRNA) that appear to play a previously-underappreciated and ubiquitous role in gene transcript silencing. Over the last few years, these miRNAs have been implicated in more than 130 different human diseases including numerous forms of cancer. Despite their critical role in cellular function, we know virtually nothing about natural variation in these short regulatory RNAs and how this might influence variation in their mRNA targets. Here we propose to capitalize on the fundamental discoveries regarding miRNA function that have been made using the model nematode Caenorhabditis elegans by performing a comprehensive genomic analysis of small RNA variation and function within the closely related species C. remanei, which is much better suited for studies of natural variation. We aim (1) to determine the evolutionary forces responsible for the origin and divergence of miRNA function and to test the relationship of these evolutionary patterns with the functional properties of the miRNAs; (2) to determine the influence of miRNA regulation on the evolution of mRNA; and (3) to directly test the relationship between miRNA evolutionary and functional divergence. We will accomplish these aims by using next generation technology to sequence the genomes of 32 C. remanei lines drawn from two different populations and 3 additional lines from a recently discovered closely-related incipient species, and by analyzing this variation using established methods from molecular population genetics in addition to a novel "SNP footprinting" approach. We will also use miRNA-system immuno pull-downs to comprehensively identify mRNA targets of the miRNAs. Finally, we use miRNA deletion lines to test the functional consequences of miRNA natural variation. In addition to addressing specific scientific hypotheses, this project will develop a set of genetic and genomic resources that will be uniquely valuable to the broader scientific community. This research uses a natural systems genetics approach that integrates an analysis of micro-evolutionary process with our emerging understanding of regulatory network structure and function within the cell to generate a genome-wide view-dissected with fine- scale SNP variation-of individual transcriptional regulators that is not possible in other major model systems.