Understanding microRNA Biogenesis and Function

MicroRNA（miRNAs），约22 nt长的单链RNA，引导蛋白复合物阻断 通过碱基配对结合的mRNA。第一个microRNA是在1993年发现的，第二个是在2000年。 目前，已经在植物、动物和病毒中发现了3,229种microRNA。作为一类，miRNAs可能 竞争转录因子在协调基因表达变化中的重要性。我们的目标是 了解miRNAs是如何制造的，组装成功能性复合物，以及这些复合物是如何形成的。 调节mRNA表达。我们使用果蝇作为模型系统，因为它提供了强大的遗传和 这是因为微RNA通路在果蝇和人类之间是非常保守的。什么 我们在苍蝇身上学习，我们在哺乳动物细胞提取物和培养的人类和小鼠细胞系中测试。我们的目标是 以确定这些过程在哪里是保守的，以及它们在苍蝇和哺乳动物之间的分歧， 了解动物中miRNA通路的共同逻辑和进化的独特特征， 在哺乳动物中。pre-miRNAs是miRNAs的直接前体，是~ 65 nt长的RNA茎环结构; pre-miRNA的茎是不完美的，有G：U摆动对，错配和内部环中断了一个完整的序列。 茎大约三个螺旋圈长。我们将使用定量生物化学和分子工具， 鉴定从pre-miRNA产生miRNA所需的蛋白质和蛋白质复合物，并确定 这些蛋白质如何提高pre-miRNA加工的准确性和效率。切丁酶， 将pre-miRNAs转化为miRNAs，需要双链RNA结合蛋白伴侣催化 miRNA成熟。单个Dicer蛋白伴侣是否足以用于所有前体miRNA序列和结构， 或者不同的双链RNA结合蛋白作为不同种类的前体的Dicer伴侣起作用， miRNAs？一些miRNA位于前体miRNA茎的5'臂，另一些位于3'臂。 前体miRNA的热力学特征确保正确的miRNA从正确的臂产生 前体miRNA茎的位置miRNAs在蛋白质-RNA复合物中发挥作用，其核心含有 Argonaute家族的蛋白质。苍蝇有五种不同的Argonaute蛋白;人类至少有七种。 miRNAs是如何以及为什么在不同的Argonaute蛋白中分配的？是什么决定了 Argonaute蛋白a miRNA相关物？是含有相同的miRNA，但不同的 Argonaute蛋白，功能不同，每一种都专门针对不同类型的mRNA靶点？我们寻求 了解miRNAs在苍蝇和人类中的生物学功能。为什么苍蝇只会轻微受损， miRNA的产生会夭折？miRNAs是抵抗环境胁迫所必需的吗？最后为 为了提供miRNA途径的系统级视图，我们将开发新的实验工具来识别 mRNA的种类是一种miRNA，通过它调节Argonaute蛋白。

MicroRNAs (miRNAs), ~22 nt long, single-stranded RNAs, guide protein complexes to block expression of mRNAs to which they bind by base pairing. The first microRNA was discovered in 1993; the second, in 2000. Currently, 3,229 microRNAs have been identified in plants, animals and viruses. As a class, miRNAs may rival transcription factors for their importance in orchestrating changes in gene expression. Our goal is to understand how miRNAs are made, assembled into functional complexes, and how these complexes regulate mRNA expression. We use Drosophila as a model system, because it offers powerful genetic and biochemical tools and because the miRNA pathway is closely conserved between flies and humans. What we learn in flies, we test in mammalian cell extracts and in cultured human and mouse cell lines. Our goal is to identify where these processes are conserved and where they diverge between flies and mammals, so as to understand the common logic of the miRNA pathway in animals and the unique features that have evolved in mammals. Pre-miRNAs, the immediate precursors of miRNAs, are -65 nt long RNA stem loop structures; the stems of pre-miRNA are imperfect, with G:U wobble pairs, mismatches, and internal loops interrupting a stem approximately three helical turns long. We will use quantitative biochemical and molecular tools to identify the proteins and protein complexes required to produce miRNA from pre-miRNAs, and to determine how these proteins enhance the accuracy and efficiency of pre-miRNA processing. Dicer, the enzyme that converts pre-miRNAs to miRNAs, requires a double-stranded RNA-binding protein partner to catalyze miRNA maturation. Does a single Dicer protein partner suffice for all pre-miRNA sequences and structures, or do different double-stranded RNA-binding proteins function as Dicer partners for distinct classes of pre- miRNAs? Some miRNAs reside in the 5' arm of the pre-miRNA stem; others, in the 3' arm. What sequence and thermodynamic features of the pre-miRNA ensure that the right miRNA is produced from the correct arm of the pre-miRNA stem? miRNAs function in protein-RNA complexes containing at their core a member of the Argonaute family of proteins. Flies have five different Argonaute proteins; humans have at least seven. How¿and why¿are miRNAs partitioned among different Argonaute proteins? What determines with which Argonaute protein a miRNA associates?Are complexes containing the same miRNA, but a different Argonaute protein, functionally distinct, each specialized for a different type of mRNA target? We seek to understand the biological functions of miRNAs in flies and humans. Why do flies only modestly impaired in miRNA production die young? Are miRNAs required for resistance to environmental stress? Finally, to provide a systems-level view of the miRNA pathway, we will develop new experimental tools to identify the mRNA species a miRNA regulates and through which Argonaute protein.