title: Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA.
abstract: Splicing factor proline- and glutamine-rich (SFPQ) also commonly known as polypyrimidine tract-binding protein-associated-splicing factor (PSF) and its binding partner non-POU domain-containing octamer-binding protein (NONO/p54nrb), are highly abundant, multifunctional nuclear proteins. However, the exact role of this complex is yet to be determined. Following purification of the endogeneous SFPQ/NONO complex, mass spectrometry analysis identified a wide range of interacting proteins, including those involved in RNA processing, RNA splicing, and transcriptional regulation, consistent with a multifunctional role for SFPQ/NONO. In addition, we have identified several sites of arginine methylation in SFPQ/PSF using mass spectrometry and found that several arginines in the N-terminal domain of SFPQ/PSF are asymmetrically dimethylated. Furthermore, we find that the protein arginine N-methyltransferase, PRMT1, catalyzes this methylation in vitro and that this is antagonized by citrullination of SFPQ. Arginine methylation and citrullination of SFPQ/PSF does not affect complex formation with NONO. However, arginine methylation was shown to increase the association with mRNA in mRNP complexes in mammalian cells. Finally we show that the biochemical properties of the endogenous complex from cell lysates are significantly influenced by the ionic strength during purification. At low ionic strength, the SFPQ/NONO complex forms large heterogeneous protein assemblies or aggregates, preventing the purification of the SFPQ/NONO complex. The ability of the SFPQ/NONO complex to form varying protein assemblies, in conjunction with the effect of post-translational modifications of SFPQ modulating mRNA binding, suggests key roles affecting mRNP dynamics within the cell.
authors.affiliation: ChELSI Institute, Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom.
authors.forename: Ambrosius P
authors.initials: AP
authors.lastname: Snijders
authors.affiliation: Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield S10 2HQ, United Kingdom.
authors.forename: Guillaume M
authors.initials: GM
authors.lastname: Hautbergue
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Alex
authors.initials: A
authors.lastname: Bloom
authors.affiliation: ChELSI Institute, Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom.
authors.forename: James C
authors.initials: JC
authors.lastname: Williamson
authors.affiliation: ChELSI Institute, Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Minshull
authors.affiliation: ChELSI Institute, Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom.
authors.forename: Helen L
authors.initials: HL
authors.lastname: Phillips
authors.affiliation: Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Sheffield S10 2HQ, United Kingdom.
authors.forename: Simeon R
authors.initials: SR
authors.lastname: Mihaylov
authors.affiliation: PhyNexus Inc., San Jose, California 95136, USA.
authors.forename: Douglas T
authors.initials: DT
authors.lastname: Gjerde
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: David P
authors.initials: DP
authors.lastname: Hornby
authors.affiliation: Department of Molecular Biology and Biotechnology, Krebs Institute, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Stuart A
authors.initials: SA
authors.lastname: Wilson
authors.affiliation: School of Biological and Chemical Sciences, Queen Mary University of London, London E1 4NS, United Kingdom.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Hurd
authors.affiliation: ChELSI Institute, Chemical and Biological Engineering, University of Sheffield, Sheffield S1 3JD, United Kingdom m.dickman@sheffield.ac.uk.
authors.forename: Mark J
authors.initials: MJ
authors.lastname: Dickman
mesh_terms: D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008745:Methylation; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics; D034521:Nuclear Matrix-Associated Proteins / Q000737:chemistry / Q000235:genetics*; D050810:Octamer Transcription Factors / Q000737:chemistry / Q000235:genetics*; D000072670:PTB-Associated Splicing Factor; D011499:Protein Processing, Post-Translational; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics; D012261:Ribonucleoproteins / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D050810:Octamer Transcription Factors; D000072670:PTB-Associated Splicing Factor; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D012261:Ribonucleoproteins; C026452:messenger ribonucleoprotein; D001120:Arginine; C099988:PRMT1 protein, human; D011484:Protein-Arginine N-Methyltransferases
keywords: arginine methylation; citrullination; mRNP binding; mass spectrometry; non-POU domain-containing octamer-binding protein; polypyrimidine tract-binding protein-associated-splicing factor; splicing factor proline- and glutamine-rich