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

双链 RNA 结合域提供的 miRNA 加工特异性

• 批准号: 8537952
• 负责人: Scott A Showalter
• 金额: $ 28.75万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8537952
• 关键词: Affinity; Autoimmune Diseases; Back; Basic Science; Binding; Biochemistry; Bioinformatics; Biological Assay; C-terminal; Cardiovascular Diseases; Cell Differentiation process; Cells; Characteristics; Clinical Sciences; Communicable Diseases; Complex; Data; Development; Diabetes Mellitus; Double-Stranded RNA; Double-Stranded RNA Binding Domain; Enzymes; Gene Expression; Health; Hereditary Disease; Human; Individual; Insulin; Intervention; Knowledge; Link; Literature; Mediating; Mediator of activation protein; Messenger RNA; MicroRNAs; Microprocessor; Molecular; Molecular Abnormality; Molecular Chaperones; Molecular and Cellular Biology; NMR Spectroscopy; Neurodegenerative Disorders; Organism; Outcome; Pathway interactions; Positioning Attribute; Prevalence; Process; Production; Proteins; RNA; RNA Binding; RNA Precursors; RNA Processing; RNA-Protein Interaction; Reaction; Recombinants; Recruitment Activity; Regulator Genes; Research; Resolution; Ribonuclease III; Ribonucleoproteins; Role; Small Interfering RNA; Small RNA; Specificity; Stimulus; Structure; Techniques; Tertiary Protein Structure; Testing; Therapeutic; Translating; Work; base; cell growth; endonuclease; heart metabolism; human DICER1 protein; novel; programs; protein complex; protein protein interaction; research study; response; scaffold; simulation; structural biology; tumor progression

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.

描述（由申请人提供）：MicroRNA（miRNA）已成为基因表达的转录后调节因子，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加工涉及由RNase III酶和双链RNA结合结构域（dsRBD）分子伴侣组成的两种核糖核蛋白复合物，其共同作用以实现两种高度特异性的核酸内切酶切割。该提议的中心假设是，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的独特并置。至少有10个dsRBD参与miRNA的产生：DGCR 8中有2个，Drosha中有1个，Dicer中有1个，TRBP中有3个，PACT中有3个。生物信息学方法极大地增加了我们对miRNA的流行和功能的了解，细胞和分子生物学方法提供了一个一般的，但强大的，概述了miRNA的生物化学和功能。结构生物学技术已被广泛应用于处理小干扰RNA（siRNA）在单细胞生物体中的蛋白质，其结果已被外推到部分解释在多细胞生物体中的miRNA加工。然而，只有产生功能性miRNA所必需的第二个核酸内切酶介导的成熟步骤与更简单的siRNA途径共享，其中功能模式也在机制上不同。因此，对于miRNA的成熟仍有许多工作要做。该项目将采取基于整体结构生物学的方法，旨在通过微处理器复合物（miRNA加工所特有的）以及miRNA和siRNA途径之间共享的复合物来建立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dsRBD以前没有确定其结构，通过NMR光谱确定每个dsRBD的构象动力学，并量化每个dsRBD的固有RNA结合亲和力。在第二个目标中，将通过结合试验和NMR光谱来探索来自RNase III内切核酸酶Drosha和Dicer的dsRBD在为它们催化的裂解反应提供特异性方面的作用。待研究的五种蛋白质中有三种含有多于一个的dsRBD --它们组成的两种核糖核蛋白复合物也是如此--因此目标3将试图确定dsRBD之间的协同相互作用在产生与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加工的完整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影响。