Non-canonical miRNA biogenesis mechanisms in Drosophila and mammals

MicroRNAs(MiRNAs)是一类丰富的小RNA，起源于独特的 发夹前体转录本。它们的生物发生通常涉及DROSHA和 DICER RNaseIII酶和由此产生的成熟miRNAs介导了广泛的 转录后调控。在分析果蝇miRNA途径的过程中，我们 揭示了来自短发夹内含子的非典型miRNAs，我们称之为镜像。他们的 生物发生用DROSHA RNaseIII酶取代看似必需的加工事件 用拼接机器。我们描述了镜像的生物起源和进化范围 使用生化、分子、遗传和计算方法，跨越四个动物物种。 这项工作导致了一系列意想不到的途径，进一步扩大了非 规范的miRNAs，包括一种杂交途径，它将剪接与RNA外切体结合起来，以 生成用作DICER底物的发夹，以及生成 在脊椎动物中，miRNAs存在于非依赖于Dester的机制中。此外，这部作品还作为一种 这是我们对miRNA功能的生物学研究的基础。 在我们的续订应用中，我们选择了几个了解酶的方向 以及介导Mirtron和Dester无关miRNAs生物发生的机制。我们 为我们提出的目标提供大量未公布的数据，包括发现数百个 包括哺乳动物中一种新的3‘尾巴亚型，这是第一次展示 果蝇非依赖于DICER的miRNA功能的研究及体外检测方法的建立 不依赖于DICER的miRNA生产。这些初步数据是个体基因的基础 研究以了解详细的机制，我们将扩展到全基因组的角度。 此外，我们希望将这些调查结果与它们对其他小岛屿发展中国家的更广泛影响联系起来。 RNA(即规范的miRNAs)和新底物的发现。最后，我们关注的是 将我们的研究与更大的进化问题联系起来，包括 MiRNAs和RNAi本身，并利用我们的机械知识来推动RNA 沉默作为一种技术工具。

microRNAs (miRNAs) are an abundant class of small RNAs that derive from distinctive hairpin precursor transcripts. Their biogenesis typically involves cleavage by the Drosha and Dicer RNase III enzymes, and the resultant mature miRNAs mediate extensive networks of post-transcriptional regulation. In the course of analyzing the miRNA pathway in Drosophila, we uncovered atypical miRNAs derived from short hairpin introns that we term mirtrons. Their biogenesis substitutes a seemingly obligate processing event by the Drosha RNase III enzyme with the splicing machinery. We characterized the biogenesis and evolutionary range of mirtrons across four animal species, using biochemical, molecular, genetic, and computational methods. This work led to a series of unexpected avenues that further expanded the diversity of non- canonical miRNAs, including a hybrid pathway that marries splicing with the RNA exosome to generate hairpins that serve as Dicer substrates, as well as short hairpins that generate miRNAs in a Dicer-independent mechanism in vertebrates. In addition, this work served as a foundation of our biological studies of miRNA functions. For our renewal application, we selected several directions on understanding enzymes and mechanisms that mediate the biogenesis of mirtrons and Dicer-independent miRNAs. We present extensive unpublished data for our proposed Aims, including the discovery of hundreds of mammalian mirtrons, including a novel 3' tailed subtype in mammals, the first demonstration of Dicer-independent miRNA function in Drosophila, and establishment of in vitro assays for Dicer-independent miRNA production. These preliminary data are the basis of individual gene studies to understand detailed mechanisms, which we will extend with genomewide perspectives. In addition, we wish to relate these findings to their broader implications with respect to other small RNAs (i.e. canonical miRNAs) and discovery of novel substrates. Finally, we are concerned with relating our research to larger evolutionary questions, including the evolutionary emergence of miRNAs and of RNAi itself, and with exploiting our mechanistic knowledge to advance RNA silencing as a technological tool.