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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067969-14
Application #
9419305
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Bender, Michael T
Project Start
2004-02-01
Project End
2021-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
14
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Pharmacology
Type
Schools of Medicine
DUNS #
804355790
City
La Jolla
State
CA
Country
United States
Zip Code
92093
Aubol, Brandon E; Hailey, Kendra L; Fattet, Laurent et al. (2017) Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform. J Mol Biol 429:2178-2191
Aubol, Brandon E; Wu, Guowei; Keshwani, Malik M et al. (2016) Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing. Mol Cell 63:218-228
Serrano, Pedro; Aubol, Brandon E; Keshwani, Malik M et al. (2016) Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif. J Mol Biol 428:2430-45
Keshwani, Malik M; Aubol, Brandon E; Fattet, Laurent et al. (2015) Conserved proline-directed phosphorylation regulates SR protein conformation and splicing function. Biochem J 466:311-22
Jamros, Michael A; Aubol, Brandon E; Keshwani, Malik M et al. (2015) Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2?1. J Biol Chem 290:17269-81
Barkho, Sulyman; Pierce, Levi C T; Li, Sheng et al. (2015) Theoretical Insights Reveal Novel Motions in Csk's SH3 Domain That Control Kinase Activation. PLoS One 10:e0127724
Aubol, Brandon E; Adams, Joseph A (2014) Recruiting a silent partner for activation of the protein kinase SRPK1. Biochemistry 53:4625-34
Sumida, Kyohei; Kawana, Makiko; Kouno, Emi et al. (2013) Importance of UDP-glucuronosyltransferase 1A1 expression in skin and its induction by UVB in neonatal hyperbilirubinemia. Mol Pharmacol 84:679-86
Chen, Shujuan; Yueh, Mei-Fei; Bigo, Cyril et al. (2013) Intestinal glucuronidation protects against chemotherapy-induced toxicity by irinotecan (CPT-11). Proc Natl Acad Sci U S A 110:19143-8
Aubol, Brandon E; Plocinik, Ryan M; Hagopian, Jonathan C et al. (2013) Partitioning RS domain phosphorylation in an SR protein through the CLK and SRPK protein kinases. J Mol Biol 425:2894-909

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