A novel pathway that produces small regulatory RNAs in Drosophila

描述（由申请人提供）：近年来，人们对microRNAs （miRNAs）产生了浓厚的兴趣，这是一类广泛的小调控rna，源于独特的发夹前体转录物。在分析果蝇miRNA通路的过程中，我们发现了来自短内含子的新型小RNA基因，我们称之为miRtrons。尽管它们与miRNA基因具有某些共同的特征，但miRtron的生物发生与miRNA的生物发生有着根本的区别。具体来说，我们的工作表明miRtrons来源于发夹，其末端由剪接而不是由RNAse III切割决定。结构方面的考虑表明，miRtrons通过Dicer途径并转移到有效的效应复合物中。我们已经产生了实验和计算证据，证明miRtrons确实是功能性抑制rna，可以通过完美的sirna型靶标以及不完美的mirna型靶标进行操作。miRtrons的存在表明我们并不完全了解Dicer可用的细胞底物范围。我们对非典型miRNA和miRtron基因的克隆和功能验证进一步证明了这一事实。因此，我们完成的研究为已知小RNA途径打开了一扇以前未被识别的底物的大门。在这个应用中，我们建议使用我们成熟的实验和计算专业知识来分析miRtron通路。我们的主要目标是阐明miRtron生物发生的生物化学，分析miRtron对基因调控网络的影响，评估miRtron和microRNA途径可以加工的rna的宽度，并测试miRtron是否存在于其他物种中。综上所述，这些研究不仅将为果蝇产生小调控RNA的新途径提供开创性的知识，而且将拓宽我们对动物Dicer途径可用的RNA底物的认识。microRNAs是一种小的，约22个核苷酸的调控rna，它控制信使rna的活性，而信使rna是蛋白质合成的模板。事实上，microRNAs构成了现存最大的基因家族之一，并介导了一个调控相互作用的网络，该网络似乎涉及基因组编码的大多数信使rna。由于如此多的基因受到microRNA的影响，microRNA功能障碍导致疾病的可能性是巨大的。与此同时，利用microRNA途径和相关的调控RNA途径作为研究工具和治疗策略的潜在好处同样巨大。这些考虑强调了继续对小RNA途径的性质和功能进行基础研究的重要性。在这个提议中，我们描述了一种新的果蝇途径，它与microRNA途径相交，从而产生了以前未被表征的一类调控rna。由于这些新的rna直接来源于短内含子的剪接，我们把它们称为miRtrons。在我们的提案中，我们描述了miRtrons的研究，这些研究利用了我们在microRNA基因实验和计算分析方面的成熟专业知识。这些研究将阐明miRtron生物发生的生化途径，证明miRtron对基因调控网络的影响，评估miRtron和microRNA途径可加工的rna的宽度，并测试miRtron是否存在于其他物种中。

DESCRIPTION (provided by applicant): Recent years have witnessed intense interest in microRNAs (miRNAs), an extensive class of small regulatory RNAs that derive from distinctive hairpin precursor transcripts. In the course of analyzing the miRNA pathway in Drosophila, we uncovered novel small RNA genes derived from short introns that we term miRtrons. Although these share certain features with miRNA genes, miRtron biogenesis deviates fundamentally from that of miRNAs. Specifically, our work indicates that miRtrons derive from hairpins whose ends are defined by splicing, rather than by RNAse III cleavage. Structural considerations suggest that miRtrons transit the Dicer pathway and are transferred into active effector complexes. We have generated experimental and computational evidence that miRtrons are indeed functional inhibitory RNAs that can operate through perfect, siRNA-type targets as well as imperfect, miRNA-type targets. The very existence of miRtrons indicates that we do not fully understand the range of cellular substrates available to Dicer. This fact is made further evident by our cloning and functional verification of atypical miRNA and miRtron genes with unorthodox structures. Therefore, our completed studies open a door onto previously unrecognized substrates for known small RNA pathways. In this application, we propose to use our proven experimental and computational expertise to analyze the miRtron pathway. Our major goals are to elucidate the biochemistry of miRtron biogenesis, to analyze the effect that miRtrons have on gene regulatory networks, to assess the breadth of RNAs that can be processed by the miRtron and microRNA pathways, and to test whether miRtrons exist in other species. Taken together, these studies will not only provide pioneer knowledge of a novel pathway that produces small regulatory RNAs in Drosophila, but will broaden our appreciation of RNA substrates that are available to animal Dicer pathways. microRNAs are small, ~22 nucleotide regulatory RNAs that control the activity of messenger RNAs, which are the templates for protein synthesis. In fact, microRNAs constitute one of the largest gene families in existence, and mediate a network of regulatory interactions that appears to involve a majority of messenger RNAs encoded by the genome. Because so many genes are influenced by microRNAs, the potential for microRNA dysfunction to underlie disease is enormous. At the same time, the potential benefit of exploiting the microRNA pathway and related regulatory RNA pathways as research tools and therapeutic strategies is similarly vast. These considerations emphasize the importance of continued basic research into the nature and the functions of small RNA pathways. In this proposal, we describe a novel Drosophila pathway that intersects with the microRNA pathway, thereby producing a previously uncharacterized class of regulatory RNAs. Since these new RNAs derive directly from the splicing of short introns, we have termed these miRtrons. In our proposal, we describe studies of miRtrons that take advantage of our proven expertise in the experimental and computational analysis of microRNA genes. These studies will elucidate the biochemical pathway for miRtron biogenesis, demonstrate the effect that miRtrons have on gene regulatory networks, assess the breadth of RNAs that can be processed by the miRtron and microRNA pathways, and test whether miRtrons exist in other species.