Non-canonical miRNA biogenesis mechanisms in Drosophila and mammals

microRNAs（miRNAs）是一类丰富的小分子RNA，它们来源于不同的 发夹前体转录物。它们的生物起源通常涉及Drosha的分裂， Dicer RNase III酶和所得的成熟miRNA介导了广泛的网络， 转录后调节。在分析果蝇中的miRNA通路的过程中， 发现了来自短发夹内含子的非典型miRNA，我们称之为mirtron。他们的 生物发生用Drosha RNase III酶代替了一个看似专性的加工过程 用拼接机我们描述了mirtron的生物起源和进化范围 在四个动物物种中，使用生物化学，分子，遗传和计算方法。 这项工作导致了一系列意想不到的途径，进一步扩大了非- 典型的miRNAs，包括一个杂交途径，它将剪接与RNA外泌体结合， 产生作为切粒机底物的发夹，以及产生 在脊椎动物中的Dicer独立机制中的miRNA。此外，这项工作还作为 这是我们对miRNA功能的生物学研究的基础。 对于我们的更新申请，我们选择了了解酶的几个方向 以及介导mirtron和Dicer非依赖性miRNAs生物发生的机制。我们 为我们提出的目标提供了大量未发表的数据，包括发现了数百个 哺乳动物mirtron，包括一种新的3'尾亚型在哺乳动物中，第一次证明 Dicer非依赖性miRNA在果蝇中的功能，并建立体外检测Dicer非依赖性miRNA的方法。 不依赖切丁酶的miRNA产生。这些初步数据是个体基因的基础 研究，以了解详细的机制，我们将扩展与全基因组的观点。 此外，我们希望将这些发现与其对其他小的 RNA（即典型的miRNA）和新底物的发现。最后，我们关注 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，并利用我们的机制知识， 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 We present extensive unpublished data for our proposed Aims, including the discovery of hundreds of mammalian mirtrons, including a novel 3' tailed subtype in mammals, the first demonstration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.