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的生物学功能依赖于与蛋白质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研究活动的领域。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重要的根本问题仍未得到解答。我们将利用多维核磁共振和生化方法来研究CCCH锌指蛋白肌盲（MBNL）识别单链RNA中的特定调控位点并与致病性双链RNA发夹高亲和力结合的机制。Muscleblind含有四个CCCH锌指基元，这是其RNA结合活性所必需的。脊椎动物肌肉盲作为一种发育程序性的调节因子，调节终末肌肉分化所需的选择性前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核心作用，这些疾病与CUG和CCUG在非翻译信使RNA中重复扩增有关。扩增的RNA重复序列在核病灶中积累，异常地隔离了肌盲，从而破坏了正常的、发育调节的剪接活动。人类肌盲锌指与单链RNA靶点以及与双链CUG和CCUG重复序列的相互作用将通过核磁共振和生化方法绘制。将进行蛋白质和RNA诱变，以确定稳定单链和CUG和CCUG重复RNA复合物的相互作用，并确定结合特异性和亲和力。单链和双链RNA分子复合物的溶液结构将被确定，以阐明正常细胞发育和RNA介导的发病机制中肌盲功能的分子基础。本研究将为丰富的CCCH锌指基序与多种RNA靶标相互作用和识别的机制提供新的见解，并将为基于结构的CCCH锌指蛋白在转录后基因表达调控及其在RNA介导的发病机制中的作用的描述奠定基础。