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真正编码蛋白质，但最近的研究表明，大多数基因组仍然从DNA转录成RNA，而后者的大部分功能未知。这些非编码区的一部分产生短RNA片段(microRNA或miRNA和piwi相关或piRNA)，这些片段似乎在基因转录沉默中发挥着以前被低估的普遍作用。在过去的几年里，这些miRNAs与130多种不同的人类疾病有关，包括多种形式的癌症。尽管它们在细胞功能中起着关键作用，但我们几乎对这些短调控RNA的自然变异以及这可能如何影响其mRNA靶标的变异一无所知。在这里，我们建议利用利用模式线虫线虫所取得的关于miRNA功能的基本发现，通过对密切相关的物种雷曼线虫内的小RNA变异和功能进行全面的基因组分析，这更适合于自然变异的研究。我们的目的是(1)确定导致miRNA功能起源和分化的进化力量，并测试这些进化模式与miRNAs功能特性的关系；(2)确定miRNA调控对mRNA进化的影响；(3)直接检验miRNA进化与功能分化之间的关系。我们将通过使用下一代技术来对来自两个不同种群的32个剩余毛虫品系和来自最近发现的密切相关的早期物种的另外3个品系的基因组进行测序，并通过使用分子种群遗传学中成熟的方法以及新颖的“SNP足迹”方法来分析这种变异，从而实现这些目标。我们还将使用miRNA系统免疫下拉来全面识别miRNAs的mRNA靶标。最后，我们使用miRNA缺失系来测试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。这项研究使用自然系统遗传学方法，将对微观进化过程的分析与我们对细胞内调控网络结构和功能的不断加深的理解相结合，以产生单个转录调控因子的全基因组视角--通过精细的SNP变异进行解剖--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.