Recognition of Regulatory and Pathogenic RNA by Muscleblind Zinc Fingers

肌盲锌指识别调节性和致病性 RNA

• 批准号: 7924930
• 负责人: PETER Edwin WRIGHT
• 金额: $ 7.77万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7924930
• 关键词: 5' Splice Site; Adipocytes; Affinity; Area; Binding; Biochemical; Biological Process; Blood Vessels; Cardiac; Complex; DNA; Development; Disease; Double-Stranded RNA; Exons; Foundations; Gene Expression; Genetic Recombination; Genetic Transcription; Genetic Translation; Goals; Human; Indium; Integrins; Lead; Maps; Mediating; Messenger RNA; Methods; Molecular; Muscle; Mutagenesis; Myotonic Dystrophy; Neurons; Nuclear; Nucleic Acids; Pathogenesis; Photoreceptors; Physiological; Play; Post-Transcriptional Regulation; Process; Proteins; RNA; RNA Binding; RNA Sequences; RNA Splicing; RNA-Protein Interaction; Regulation; Reporting; Research; Research Activity; Role; Site; Solutions; Specificity; Structure; Therapeutic; Translations; Troponin T; Untranslated RNA; Work; Zinc Fingers; base; cellular development; insight; mRNA Precursor; novel; programs; research study; stem; stoichiometry; three dimensional structure; treatment strategy

DESCRIPTION (provided by applicant): The biological functions of DNA and RNA are dependent upon highly specific and complex interactions with proteins. Elucidation of the mechanisms by which proteins recognize and manipulate nucleic acids in such fundamental processes as transcription, translation, mRNA splicing, replication, and recombination remains an area of intense research activity. Despite recent progress, the enormous diversity of protein-nucleic acid interactions means that many questions of fundamental importance remain unanswered. Multi-dimensional NMR and biochemical methods will be used to investigate the mechanisms by which the CCCH zinc finger protein muscleblind (MBNL) recognizes specific regulatory sites in single stranded RNA and also binds with high affinity to pathogenic double stranded RNA hairpins. Muscleblind contains four CCCH zinc finger motifs that are absolutely required for its RNA binding activity. Vertebrate muscleblind functions as a developmentally programmed regulator of alternative pre-mRNA splicing required for regulation of terminal muscle differentiation; it is also involved in differentiation of photoreceptors, neurons, adipocytes, and blood vessels. Muscleblind plays a central role in type 1 and type 2 myotonic dystrophy, diseases that are associated with CUG and CCUG repeat expansions in untranslated messenger RNA. The expanded RNA repeats accumulate in nuclear foci that aberrantly sequester muscleblind and thereby disrupt its normal, developmentally regulated splicing activities. The interactions of the human muscleblind zinc fingers with single stranded RNA targets and with double stranded CUG and CCUG repeats will be mapped using NMR and biochemical methods. Protein and RNA mutagenesis will be performed to identify the interactions that stabilize the single stranded and CUG and CCUG repeat RNA complexes and determine binding specificity and affinity. Solution structures of complexes with single stranded and duplex RNA molecules will be determined to elucidate the molecular basis for muscleblind function in normal cellular development and in RNA-mediated pathogenesis. This research will provide novel insights into the mechanisms by which the highly abundant CCCH zinc finger motif interacts with and recognizes diverse RNA targets, and will form the foundations for a structure-based description of the role of CCCH zinc finger proteins in post-transcriptional regulation of gene expression and their role in RNA-mediated pathogenesis.

描述（由申请人提供）：DNA和RNA的生物学功能取决于与蛋白质的高度特异性和复杂的相互作用。阐明蛋白质在转录、翻译、mRNA剪接、复制和重组等基本过程中识别和操纵核酸的机制仍然是一个激烈的研究活动领域。尽管最近取得了进展，但蛋白质-核酸相互作用的巨大多样性意味着许多具有根本重要性的问题仍然没有答案。多维NMR和生物化学方法将被用来研究CCCH锌指蛋白肌盲（MBNL）识别单链RNA中的特定调控位点，并以高亲和力结合致病性双链RNA发夹的机制。Muscleblind含有四个CCCH锌指基序，这是其RNA结合活性所必需的。脊椎动物muscleblind的功能是作为一种发育程序的调节器，调节终末肌肉分化所需的替代性前mRNA剪接;它还参与光感受器、神经元、脂肪细胞和血管的分化。肌盲在1型和2型肌强直性营养不良中起着核心作用，这些疾病与非翻译信使RNA中的CUG和CCUG重复扩增相关。扩增的RNA重复序列在核灶中积累，异常地隔离肌盲，从而破坏其正常的发育调节的剪接活动。将使用NMR和生物化学方法绘制人肌盲锌指与单链RNA靶标以及与双链CUG和CCUG重复序列的相互作用。将进行蛋白质和RNA诱变，以鉴定稳定单链和CUG和CCUG重复RNA复合物的相互作用，并测定结合特异性和亲和力。解决方案与单链和双链RNA分子的复合物的结构将被确定，以阐明在正常细胞发育和RNA介导的发病机制中肌盲功能的分子基础。这项研究将提供新的见解的机制，其中高度丰富的CCCH锌指基序相互作用，并识别不同的RNA靶点，并将形成基于结构的描述CCCH锌指蛋白的作用在转录后调控基因表达和RNA介导的发病机制中的作用的基础。