Understanding microRNA Biogenesis and Function

MicroRNAs(MiRNAs)，~22nT长的单链RNAs，引导蛋白质复合体阻断 它们通过碱基配对与之结合的mRNA。第一个microRNA是在1993年发现的；第二个是在2000年发现的。 目前，已在植物、动物和病毒中鉴定出3229个microRNAs。作为一类，miRNAs可能 相互竞争的转录因子在协调基因表达变化方面的重要性。我们的目标是 了解miRNAs是如何制造、组装成功能复合体的，以及这些复合体是如何 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 这是因为miRNA途径在苍蝇和人类之间非常保守。什么 我们在苍蝇身上学习，在哺乳动物细胞提取物以及培养的人类和小鼠细胞系中进行测试。我们的目标是 以确定这些过程在苍蝇和哺乳动物中哪些地方是保守的，哪些地方是不同的，以便 了解动物miRNA途径的共同逻辑和进化出的独特特征 在哺乳动物身上。前miRNAs是miRNAs的直接前体，是-65nT的长RNA茎环结构； 前miRNA的茎是不完美的，G：U摆动对、错配和内部环会中断A 茎大约有三个螺旋转弯长。我们将使用定量生化和分子工具来 确定从前miRNAs产生miRNA所需的蛋白质和蛋白质复合体，并确定 这些蛋白质如何提高前miRNA处理的准确性和效率。迪格尔，一种酶 将前miRNAs转化为miRNAs，需要双链RNA结合蛋白伙伴来催化 MiRNA成熟。单个Dager蛋白配对是否足以满足所有前miRNA序列和结构， 或者，不同的双链RNA结合蛋白是否可以作为不同类别的Pre-Pre的Dector伙伴发挥作用？ MiRNAs？一些miRNAs位于前miRNA茎的5‘臂；另一些位于3’臂。什么顺序 而前miRNA的热力学特征确保了正确的miRNA是从正确的手臂产生的 前miRNA的茎？MiRNAs在核心含有以下成员的蛋白质-RNA复合体中发挥作用 阿加索特蛋白质家族。苍蝇有五种不同的Argavite蛋白质；人类至少有七种。 MiRNAs是如何在不同的ArgAerte蛋白之间分配的？为什么？是什么决定了 是含有相同miRNA但不同的miRNA的复合体。 ArgAerte蛋白，功能不同，每一种都专门针对不同类型的mRNA靶标？我们寻求 了解miRNAs在苍蝇和人类中的生物学功能。为什么苍蝇只受到轻微的损害 MiRNA的生产很早就死了？抵抗环境胁迫是否需要miRNAs？最后，为了 提供系统水平的miRNA途径的视图，我们将开发新的实验工具来识别 信使核糖核酸是一种微核糖核酸，通过它来调节ArgAerte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.