Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif.
Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function.
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Pedro
P
Serrano
Department of Pharmacology, University of California San Diego, La Jolla, CA 92093-0636, USA.
Brandon E
BE
Aubol
Department of Pharmacology, University of California San Diego, La Jolla, CA 92093-0636, USA.
Malik M
MM
Keshwani
Department of Pharmacology, University of California San Diego, La Jolla, CA 92093-0636, USA.
Stefano
S
Forli
Department of Pharmacology, University of California San Diego, La Jolla, CA 92093-0636, USA.
Chen-Ting
CT
Ma
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Samit K
SK
Dutta
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Michael
M
Geralt
Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA 92037, USA; Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Kurt
K
Wüthrich
Department of Pharmacology, University of California San Diego, La Jolla, CA 92093-0636, USA. Electronic address: j2adams@ucsd.edu.
Joseph A
JA
Adams
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000502:physiology*; D011485:Protein Binding / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012313:RNA / Q000378:metabolism*; D000071377:RNA Recognition Motif / Q000502:physiology*; D012326:RNA Splicing / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors / Q000378:metabolism*
D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; C000595779:SRSF1 protein, human; D000068103:Serine-Arginine Splicing Factors; D012694:Serine; D012313:RNA; D001120:Arginine; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases
NMR; RS domain; SR protein; kinetics; phosphorylation