Annotation of the small RNA/microRNA component Drosophila genome

描述(申请人提供)：我们的长期目标是阐明真核小RNA网络及其调控靶点。我们这项努力的动机是越来越多的证据表明，在20-30个核苷酸范围内，不同类别的RNA具有基本的细胞活性。一类受到特别关注的是microRNAs，这是一个丰富的-22核苷酸抑制RNA家族，来自发夹前体转录本。据估计，至少有1000个人类microRNAs，它们共同调控着至少30%的人类基因。其他类别的加工小RNA包括内源性小干扰RNA(SiRNAs)、重复相关的siRNAs和Piwi相关的RNAs。在这里，我们提出了一套集中的实验和计算实验来表征果蝇基因组的小RNA成分。(1)我们将对多种文库进行高通量焦磷酸测序，以全面覆盖果蝇小RNA的表达。(2)我们将利用最近十几个测序苍蝇基因组的可用性，对电子计算机中的microRNA基因和microRNA靶标进行高度特异性的计算预测。(3)我们将使用微阵列技术和其他分子技术来验证microRNAs和其他新的小RNA基因的内源性表达。我们还将进行体内表型分析，以证明它们的生物活性。(4)我们将通过对这些暂时性物种进行遗传稳定，然后进行基因组平铺微阵列分析，来阐明初级microRNA转录本的结构。这项工作将在果蝇中对不同类别的加工的小RNA及其相关的前体转录本进行彻底的注释。除了果蝇，已经有充分的证据表明，小RNA途径的错误调节对所有真核物种的发育和生理都有巨大的不利后果。这种在苍蝇中发现小RNA基因的全面努力不仅将为人类基因组中小RNA基因的注释提供信息，还将为未来研究它们的正常和病理作用奠定基础。从这种遗传上易驯化的模式生物中获得的见解将与理解小RNA对人类疾病的功能贡献相关。

DESCRIPTION (provided by applicant): Our long-term objective is to elucidate networks of eukaryotic small RNAs and their regulatory targets. We are motivated in this endeavor by the growing body of evidence that demonstrates fundamental cellular activities for diverse classes of RNAs in the 20-30 nucleotide range. One class that has received particular attention are the microRNAs, an abundant family of -22 nucleotide inhibitory RNAs that derive from hairpin precursor transcripts. It is estimated that there at least one thousand human microRNAs, which collectively regulate at least 30% of human genes. Additional classes of processed small RNAs include endogenous small interfering RNAs (siRNAs), repeat associated siRNAs, and Piwi-associated RNAs. We propose herein a focused set of experimental and computational experiments to characterize the small RNA component of the Drosophila melanogaster genome. (1) We will perform high-throughput pyrosequencing of a broad variety of libraries to obtain comprehensive coverage of expressed Drosophila small RNAs. (2) We will exploit the recent availability of a dozen sequenced fly genomes to make highly specific computational predictions of microRNA genes and microRNA targets in silico. (3) We will use microarray profiling and other molecular techniques to validate the endogenous expression of microRNAs and other novel small RNA genes. We will also perform in vivo phenotypic assays to demonstrate their biological activity. (4) We will elucidate the structures of primary microRNA transcripts by genetically stabilizing these transient species, followed by genome tiling microarray analysis. This work will culminate in a thorough annotation of the different classes of processed small RNAs and their associated precursor transcripts in Drosophila. Going beyond Drosophila, it is well documented that misregulation of small RNA pathways has tremendous adverse consequences for the development and physiology of all eukaryotic species. This comprehensive effort to uncover small RNA genes in flies will not only inform the annotation of small RNA genes in the human genome, but also lay the foundation for future studies of their normal and pathological roles. Insights gained from this genetically tractable model organism will be relevant for understanding the functional contributions of small RNAs to human disease.