Understanding microRNA Biogenesis and Function

描述（由申请人提供）：微小RNA（miRNAs），约22 nt长的单链RNA，引导蛋白复合物通过碱基配对阻断与其结合的mRNA的表达。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，miRNA可能与转录因子竞争，因为它们在协调基因表达变化中的重要性。我们的目标是了解miRNAs是如何产生、组装成功能性复合物的，以及这些复合物如何调节mRNA的表达。我们使用果蝇作为模型系统，因为它提供了强大的遗传和生物化学工具，因为miRNA途径在果蝇和人类之间非常保守。我们在苍蝇身上学到的东西，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进行测试。我们的目标是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，从而了解动物中miRNA通路的共同逻辑和哺乳动物中进化的独特特征。pre-miRNA是miRNA的直接前体，是约65 nt长的RNA茎环结构; pre-miRNA的茎是不完美的，具有G：U摆动对、错配和中断茎约三个螺旋转角长的内环。我们将使用定量生物化学和分子工具来鉴定从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。Dicer是将pre-miRNA转化为miRNA的酶，需要双链RNA结合蛋白伴侣来催化miRNA成熟。单个Dicer蛋白伴侣是否足以满足所有pre-miRNA序列和结构，或者不同的双链RNA结合蛋白是否作为不同类别的pre-miRNA的Dicer伴侣起作用？一些miRNA位于pre-miRNA茎的5'臂;另一些位于3'臂。pre-miRNA的哪些序列和热力学特征确保正确的miRNA从pre-miRNA茎的正确臂产生？miRNA在蛋白质-RNA复合物中起作用，其核心含有Argonaute蛋白质家族的成员。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miRNA与哪种Argonaute蛋白相关联？含有相同miRNA但不同Argonaute蛋白质的复合物是否功能不同，各自专门针对不同类型的mRNA靶标？我们试图了解miRNAs在苍蝇和人类中的生物学功能。为什么果蝇在产生miRNA时只受到轻微损害，就会过早死亡？miRNAs是抵抗环境胁迫所必需的吗？最后，为了提供miRNA通路的系统水平视图，我们将开发新的实验工具来识别miRNA调节的mRNA种类以及Argonaute蛋白通过哪些mRNA种类。

DESCRIPTION (provided by applicant): 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.