Project Summary/Abstract: Splicing converts a single gene into multiple unique mRNA fragments to expand the size of the proteome and regulate cell function. While splicing is integral for normal function in complex organisms, mistakes in splice-selection can lead to disease. In fact, splicing errors are associated with numerous human disorders including muscular dystrophy, Alzheimer's disease, parkinsonism, cardiovascular disease, ataxias and cancers. Splicing occurs at the spliceosome, a macromolecular complex that includes both RNA and protein. In the latter group, SR proteins are essential splicing factors that control where the spliceosome assembles on precursor mRNA. SR proteins contain C-terminal domains rich in arginine-serine repeats whose polyphosphorylation controls splice-site selection. The SRPK family of protein kinases phosphorylates these RS domains directing SR proteins into the nucleus for splicing activity. While SRPKs are normally localized to the cytoplasm for this function, they can enter the nucleus under certain conditions but their function in this cellular compartment is not well understood. We will now investigate the functions of SRPKs in both the cytoplasm and nucleus using genome-wide splicing assays, cell imaging, fast-mixing kinetics, protease footprinting and structural techniques. We will determine how the unique SRPK phosphorylation mechanism governs transport of SR proteins from the cytoplasm to the nucleus. We will explore factors that regulate SRPK nuclear import through a novel kinase- kinase complex. Finally, we will investigate how this complex affects splicing reactions through the mobilization of SR proteins in the nucleus. Overall, the studies outlined in this proposal will greatly expand our knowledge of SRPK-induced phosphorylation of SR proteins and their splicing function.

Narrative/Public Health Relevance Statement The splicing of genes is essential for normal cell function but can lead to many human diseases when performed incorrectly. To better understand the connection between disease and gene processing, we are studying the role of SRPKs, a critical enzyme family that regulates splicing activity in the cell.