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.
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