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.

Public Health Relevance

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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067969-08
Application #
8204680
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Bender, Michael T
Project Start
2004-02-01
Project End
2013-03-31
Budget Start
2011-12-01
Budget End
2013-03-31
Support Year
8
Fiscal Year
2012
Total Cost
$292,975
Indirect Cost
$96,955
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
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
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 63:218-28
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 2β1. 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
Barkho, Sulyman; Pierce, Levi C T; McGlone, Maria L et al. (2013) Distal loop flexibility of a regulatory domain modulates dynamics and activity of C-terminal SRC kinase (csk). PLoS Comput Biol 9:e1003188

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