The removal of introns by RNA splicing is an essential process in the expression of almost all genes in human cells. This process is regulated in some cell types, the same gene sequence is expressed as two distinct proteins by variation in the combination of coding sequence spliced together. A better understanding of the splicing process is essential for studies in development and oncogenesis as well as for an appreciation of the evolutionary origin of genes. It is thought that the process of splicing of nuclear precursors to mRNA is RNA catalyzed, thus reflecting chemical processes which might have evolved before many protein catalyzed processes. The intron structure of genes could reflect early evolutionary events in biological systems. Many proteins must recognize specific sequences in the precursor RNA in selection of splice sites for reaction. Assays of photo-crosslinking of RNA to protein and shifts in the electrophoretic mobility of RNA-protein complexes will be used to identify and purify protein which specifically binds to sequences in splicing substrates. Correlation of the effects of mutation on the binding affinity of the protein and splicing efficiency in vitro will be used to initially establish the role of the protein in splicing. Subsequent mechanistic studies will establish the steps in splicing dependent upon the protein-RNA interaction. Sequence-specific RNA-binding factors, which regulate the differential splicing of the calcitonin/calcitonin-related protein gene in brain and thyroid cells will be identified and studied. The role of snRNA in splicing of nuclear precursors to mRNAs will be investigated. Methods for reconstitution of active snRNPs from purified snRNA, will be incorporated into particles and tested for activity in formation of multi-snRNP complexes and spliceosomes. Analysis of nucleosides with photo-activatable azido groups will be incorporated into splicing substrates and snRNAs. These will be used to locate snRNA-precursor contact, protein-precursor contacts and snRNA-snRNA contracts.
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