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Assembly and Dynamics of Uber Complex RNPs

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The majority of protein-coding genes in multicellular organisms contain introns, noncoding regions that must be excised from newly made transcripts (pre-mRNAs) to generate messenger RNAs (mRNAs). Intron excision is mediated by the spliceosome, a highly dynamic macromolecular machine containing five stable small nuclear RNAs (snRNAs) and scores of proteins. By altering the splice sites it utilizes, the spliceosome can produce many different mRNAs from a single gene. Such alternative splicing greatly enhances the number of different proteins that can be encoded within a single genome and is crucial for development, maintenance, and function of diverse tissue types.

A central goal of our research is to elucidate the basic mechanisms by which spliceosomes accurately identify pre-mRNA splice sites and then catalyze intron excision. Recently the lab has developed methodologies for fluorescently labeling individual proteins in crude cell lysates. In collaboration with Jeff Gelles (Brandeis University), we use these lysates to monitor spliceosome assembly and intron excision on individual pre-mRNA molecules. The use of a multiwavelength fluorescence method, colocalization of single-molecule spectroscopy (CoSMoS), allows us to analyze the dynamic characteristics of individual spliceosomes in real time. This opens exciting new windows onto previously unaddressable questions regarding spliceosome assembly and internal its structural transitions.

This talk is part of the MRC LMB Seminar Series series.

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