PRMT5 dimethylates BCL6 at R305 to mediate the repressive activity of the BCL6 RD2 domain. (A) Mass spectroscopy of BCL6 protein following in vitro methyltransferase assay by PRMT5 identifies BCL6 methylation at R305. Representative tandem mass spectrometry spectrum of methylated R305 of BCL6 (P11482). Band corresponding to BCL6 was excised from the gel and subjected to trypsin digestion. Tryptic peptides were resolved on a reverse phase column, and high-energy collision dissociation spectra were obtained using Orbitrap Fusion Tribrid mass spectrometer. Data were searched against human protein database using Proteome Discoverer (v 1.4, ThermoScientific) by considering methylation on arginine as a potential modification. Results were filtered at 1% false discovery rate. A tandem mass spectrometry spectrum corresponding to ³⁰²EEErPSSEDEIALHFEPPNAPLNR³²⁵ of BCL6 (precursor [M+H]⁺⁴ = 698.3398 m/z. DPPM = 1.6, inset) is shown. Observed b- and y-ions are indicated. The lowercase “r” is the methylated arginine. (B) 4xBCL6 binding site luciferase reporter assays in 293T cells transfected with wild-type and R305K mutant BCL6 or control pcDNA3.1 vector in the presence or absence of cotransfected siRNAs to PRMT5. ***P < .001. Western blots in each experimental condition with the indicated antibodies. (C) Arginine dimethylation of wild-type or R305K BCL6 transfected into Raji cells. (D) Co-IP experiments of endogenous PRMT5 with wild-type or R305K BCL6 transfected into Raji and 293T cells. WT, wild-type.