Specificity of miRNA Processing Provided by Double-Stranded RNA Binding Domains

描述（由申请人提供）：MicroRNA （miRNA）已成为基因表达的转录后调节剂，影响细胞生长和分化以及多种遗传疾病的进展。细胞中的miRNA加工涉及两种由RNase III酶和双链RNA结合域（dsRBD）伴侣组成的核糖核蛋白复合物，它们共同作用以实现两种高度特异性的内切酶切割。该建议的中心假设是，这些多蛋白复合物的miRNA加工活性固有的特异性是dsRBD相互作用的直接结果：复合物中的dsRBD对miRNA前体的特异性识别以及多个dsRBD的独特并立。至少有10个dsrbd参与miRNA的产生：2个在DGCR8中，1个在Drosha中，1个在Dicer中，3个在TRBP中，3个在PACT中。生物信息学方法极大地增加了我们对miRNA流行和功能的了解，细胞和分子生物学方法提供了miRNA生物化学和功能的一般但强大的概述。结构生物学技术已被广泛应用于单细胞生物中参与小干扰RNA （siRNA）加工的蛋白质，其结果已被推断为部分解释多细胞生物中miRNA加工的原因。然而，只有产生功能性miRNA所需的第二个内切酶介导的成熟步骤与更简单的siRNA途径共享，其功能模式在机制上也是不同的。因此，对于miRNA的成熟还有很多工作要做。该项目将采用基于整体结构生物学的方法，旨在通过miRNA加工特有的微处理器复合物以及miRNA和siRNA途径之间共享的复合物建立miRNA加工的分子机制。该项目的第一个目标是为参与miRNA加工的每个dsRBD提供原子分辨率结构，这些结构以前没有确定过，通过核磁共振波谱确定每个dsRBD的构象动力学，并量化每个dsRBD的内在RNA结合亲和力。在第二个目标中，RNase III内切酶Drosha和Dicer的dsrbd在提供其催化的裂解反应特异性方面的作用将通过结合试验和核磁共振波谱来探索。要研究的五种蛋白质中有三种含有不止一种dsRBD——由它们组成的两种核糖核蛋白复合物也是如此——因此，目的3将试图确定dsRBD之间的合作相互作用在产生与miRNA前体结合的亲和力和特异性方面的作用。本文提出的对miRNA加工过程的全面分子理解将为靶向操纵miRNA表达提供新的切入点，这可能对基础和临床科学产生广泛的影响。

DESCRIPTION (provided by applicant): MicroRNA (miRNA) has emerged as a post-transcriptional regulator of gene expression impacting - among other factors - cell growth and differentiation, and the progression of multiple genetic diseases. miRNA processing in the cell involves two ribonucleoprotein complexes composed of RNase III enzymes and double-stranded RNA binding domain (dsRBD) chaperones that work together to achieve two highly specific endonuclease cleavages. The central hypothesis of this proposal is that the specificity inherent to the miRNA processing activity of these multi-protein complexes is a direct result of dsRBD interactions: the specific recognition of the miRNA precursors by the dsRBDs in the complexes and the unique juxtaposition of the multiple dsRBDs. There are at least ten dsRBDs involved in miRNA production: two in DGCR8, one in Drosha, one in Dicer, three in TRBP, and three in PACT. Bioinformatics approaches have vastly increased our knowledge of miRNA prevalence and function, and cellular and molecular biology approaches have provided a general, but powerful, overview of miRNA biochemistry and function. Structural biology techniques have been extensively applied to the proteins involved in processing small interfering RNA (siRNA) in unicellular organisms, and the results have been extrapolated to partially explain miRNA processing in multicellular organisms. However, only the second endonuclease-mediated maturation step necessary for production of functional miRNA is shared with the simpler siRNA pathway, where the mode of function is also mechanistically distinct. Thus, much remains to be worked out for miRNA maturation. This project will take a holistic structural biology based approach, aiming to establish the molecular mechanism of miRNA processing by both the Microprocessor complex - unique to miRNA processing - and the complexes shared between the miRNA and siRNA pathways. The first aim of the project is to provide atomic resolution structures for each of the dsRBDs involved in miRNA processing, which have not previously had their structures determined, to define the conformational dynamics of each by NMR spectroscopy, and to quantify the intrinsic RNA binding affinity of each dsRBD in isolation. In the second aim, the role of the dsRBDs from the RNase III endonucleases Drosha and Dicer in providing specificity to the cleavage reactions they catalyze will be explored through binding assays and NMR spectroscopy. Three of the five proteins to be studied contain more than one dsRBD - as do both of the ribonucleoprotein complexes composed by them - and so aim 3 will seek to define the role of cooperative interactions among the dsRBDs in yielding affinity and specificity for binding to miRNA precursors. The full molecular understanding of miRNA processing proposed herein will allow novel entry points for targeted manipulation of miRNA expression, which may have broad impacts on both basic and clinical science.