Annotation of the small RNA/microRNA component Drosophila genome

描述（由申请人提供）：我们的长期目标是阐明真核小rna网络及其调控靶点。越来越多的证据表明，在20-30个核苷酸范围内，不同种类的rna具有基本的细胞活性，这促使我们进行这项努力。其中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，人类至少有1000个microrna，它们共同调节着至少30%的人类基因。加工小rna的其他类别包括内源性小干扰rna （sirna）、重复相关sirna和piwi相关rna。我们在此提出了一套重点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，以获得表达的果蝇小rna的全面覆盖。(2)我们将利用最近获得的12个苍蝇基因组测序，在计算机上对microRNA基因和microRNA靶点进行高度特异性的计算预测。(3)我们将使用微阵列分析和其他分子技术来验证microRNAs和其他新型小RNA基因的内源性表达。我们还将进行体内表型分析，以证明其生物活性。(4)我们将通过基因稳定这些瞬时物种来阐明初级microRNA转录本的结构，然后进行基因组平铺微阵列分析。这项工作将在果蝇中不同种类的加工小rna及其相关前体转录物的全面注释中达到高潮。除了果蝇之外，有充分的证据表明，小RNA途径的错误调控对所有真核生物物种的发育和生理都有巨大的不利影响。这项全面揭示果蝇小RNA基因的工作不仅将为人类基因组中小RNA基因的注释提供信息，也将为未来研究其正常和病理作用奠定基础。从这种遗传上易于处理的模式生物中获得的见解将有助于理解小rna对人类疾病的功能贡献。

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.