Comparisons of the genomes from humans and lower organisms reveal that the complexity in humans is achieved not by a dramatic increase in the number of genes but by alternative splicing events that stitch together different portions of genes to generate diverse proteins. Correct splicing allows normal healthy function, however, incorrect splicing is linked to many human diseases. For example, muscular dystrophy, ataxias, parkinsonism, neurofibromatosis, psychiatric disorders and cancer have their origins in splicing errors. Splicing reactions are catalyzed by a large macromolecular machine known as the spliceosome. Composed of both RNA and protein, the spliceosome can accurately select the proper splice sites from a considerably large precursor mRNA in healthy cells. The assembly of the spliceosome, the identification of the correct 5'and 3'splice sites and the chemical splicing reaction itself is regulated by a large class of splicing factors known as SR proteins. SR proteins contain one or two RNA recognition motifs and a long C-terminal domain rich in numerous arginine-serine dipeptide repeats. The phosphorylation of the RS domain serves many RNA processing functions including splice-site selection, import of SR proteins into the nucleus and export of mature mRNA to the cytoplasm. This project will investigate how two principal families of splicing enzymes uniquely impact SR protein function through regiospecific, multi-site phosphorylation of the RS domains. Using engineered footprinting methods, the directionality of the splicing enzymes will be defined and shown to control which serines in the RS domain are modified. The effects of these selective phosphorylation reactions on SR protein structure and interaction/function within the spliceosome will then be evaluated using kinetic, structural, splicing and cellular assays. The goal is to identify how splicing kinases recognize and phosphorylate specific regions of the RS domains and determine how these chemical modifications impact splicing componentry.
Health Relevance Statement Comparisons of the genomes from humans and lower organisms reveal that the complexity in humans is achieved not by a dramatic increase in the number genes but by splicing events that stitch together different portions of genes to generate diverse proteins. Correct splicing allows normal healthy function, however, incorrect splicing is linked to many human neurodegenerative diseases and cancer. We are investigating how the newly identified drug targets for diverse diseases known as splicing enzymes (named SR-kinases) regulate important splicing factors (a specific family of proteins known as SR proteins) which cooperate in the control of alternative splicing reactions important in both health and disease.
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