Arginine methylation in subunits of mammalian pre-mRNA cleavage factor I

Only arginines within the GGRGRGRF motif are methylated in CF I_m68. Site directed point mutations within the GAR motif in the GST-68_GR construct and fragments of CF I_m68 were subjected to methylation in vitro by the recombinant PRMT5/WD45 complex. GST-68_GR_3-mut is GST-68_GR with all three arginines (202, 204, and 206) in the GAR motif mutated to alanines. (A) One microgram of the recombinant substrate protein was incubated with 100 ng of PRMT5/WD45 and [³H]SAM. (B) The same as A except that 1 μg of PRMT5/WD45 was used. (C) The same as B except that the reaction contained cold SAM and the blot was probed with the sym10 polyclonal antibody against the SDM arginines. (D) Blot from C stained with Ponceau-S.