Arginine methylation and citrullination of splicing factor proline- and glutamine-rich (SFPQ/PSF) regulates its association with mRNA

Citrullination blocks arginine methylation of SFPQ/PSF in vitro. (A) CID MS/MS fragmentation spectra of the in vitro citrullinated peptide GGGGGCitGGLHDFR [M+2H]²⁺ from SFPQ. The prominent y, b ions and the characteristic neutral losses associated with isocyanic acid are highlighted. (B) MS spectra highlighting the isotopic distribution due to the incorporation of H₂¹⁸O during the enzymatic citrullination of SFPQ using PADI4. (C) Methylation assays were performed in conjunction with the addition of tritiated S-adenosyl-L-methionine ([³H] AdoMet) to detect methylation of recombinant SFPQ using PRMT before and after citrullination using PADI4. Left panel shows the autorad, right panel shows the Coomassie stained SDS-PAGE. (D) Citrullination of SFPQ affects mRNA binding. Poly(A)⁺ RNA from 293T cells +/− AdOx transfected with FLAG, FLAG-SFPQ, FLAG-SFPQ + HA PADI4/Ca²⁺ ionophore was purified on oligo(dT) in denaturing conditions after UV cross-linking (+) or not (−). Total extract (1% of input) and eluted proteins were analyzed by Western blotting (WB) with α-FLAG antibody, α-ALYREF, or Chtop antibody. (E) Co-IP of FLAG control, FLAG-SFPQ in conjunction with PADI4 in the presence of Ca²⁺ ionophore. Total extract (1% of input) and eluted proteins were analyzed by Western immunoblotting using α-FLAG, α-NONO, α-Chtop, or α-Tubulin antibodies. (F) PADI4/Ca²⁺ increases SFPQ/PSF abundance in mammalian cells. 293T cells were cotransfected with either a FLAG control, FLAG-SFPQ in conjunction with PADI4 in the presence of Ca²⁺ ionophore and 5 mM CaCl₂. Of note, 100 mM cycloheximide was added for 6 h when indicated (+) in order to block de novo protein synthesis. Twelve percent SDS-PAGE was analyzed using Western immunoblotting using α-FLAG, α-ALYREF, α-Chtop, or α-Tubulin antibodies.