Recognition of Regulatory and Pathogenic RNA by Muscleblind Zinc Fingers

描述(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非常活跃的研究领域。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根本重要的问题仍然没有得到回答。多维核磁共振和生化方法将被用来研究CCCH锌指蛋白肌肉盲(MBNL)识别单链RNA中的特定调控位点并与致病双链RNA发夹具有高亲和力的机制。粘虫含有四个CCCH锌指基序，是其RNA结合活性所必需的。脊椎动物肌肉盲目的功能是作为一种发育程序化的调节器，调节末端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。肌肉盲性肌营养不良在1型和2型强直性肌营养不良中起核心作用，这两种疾病与非翻译信使RNA中的CUG和CCUG重复扩张有关。扩展的RNA重复序列聚集在核内，异常地隔离肌肉，从而扰乱其正常的、发育调节的剪接活动。人类肌肉盲锌指与单链RNA靶标以及双链CUG和CCUG重复序列的相互作用将使用核磁共振和生物化学方法进行绘制。将进行蛋白质和RNA突变，以确定稳定单链以及CUG和CCUG重复RNA复合体的相互作用，并确定结合特异性和亲和力。将测定单链和双链RNA分子的复合体的溶液结构，以阐明在正常细胞发育和RNA介导的发病机制中肌肉功能的分子基础。这项研究将为高度丰富的CCCH锌指基序与不同的RNA靶标相互作用和识别不同的RNA靶标提供新的见解，并将为基于结构的描述CCCH锌指蛋白在基因表达的转录后调控中的作用及其在RNA介导的致病机制中的作用奠定基础。

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.