Proteomics profiling of arginine methylation defines PRMT5 substrate specificity

Abstract

Protein arginine methyltransferases (PRMTs) catalyze arginine methylation on both chromatin-bound and cytoplasmic proteins. Accumulating evidence supports the involvement of PRMT5, the major type II PRMT, in cell survival and differentiation pathways that are important during development and in tumorigenesis. PRMT5 is an attractive drug target in various cancers, and inhibitors are currently in oncological clinical trials. Nonetheless, given the complex biology of PRMT5 and its multiple nonhistone substrates, it is paramount to fully characterize these dynamic changes in methylation and to link them to the observed anticancer effects to fully understand the functions of PRMT5 and the consequences of its inhibition. Here, we used a newly established pipeline coupling stable isotope labeling with amino acids in cell culture (SILAC) with immunoenriched methyl peptides to globally profile arginine monomethylation and symmetric dimethylation after PRMT5 inhibition by a selective inhibitor. We adopted heavy methyl SILAC as an orthogonal validation method to reduce the false discovery rate. Through in vitro methylation assays, we validated a set of PRMT5 targets identified by mass spectrometry and provided previously unknown mechanistic insights into the preference of the enzyme to methylate arginine sandwiched between two neighboring glycines (a Gly-Arg-Gly, or “GRG,” sequence). Our analysis led to the identification of previously unknown PRMT5 substrates, thus both providing insight into the global effects of PRMT5 and its inhibition in live cells, beyond chromatin, and refining our knowledge of its substrate specificity.

Deep protein methylation profiling by combined chemical and immunoaffinity approaches reveals novel PRMT1 targets

“…The modifications SDMA and ADMA were added to MaxQuant's library with the added mass of dimethyl on arginine and the corresponding neutral loss masses of 31.042 for SDMA and 45.058 for ADMA assigned in the "Neutral Loss" table in Configuration (26). The missed cleavage rate was set to 5 and all other settings were kept unchanged.…”

“…We next sought to investigate the effect of PRMT1 knockdown on protein arginine dimethylation (DMA). Distinguishing the isobaric ADMA and SDMA PTMs should be possible based on characteristic neutral losses from both dimethylarginine forms (26,32,(50)(51)(52). For ADMA, the neutral loss of dimethyamine causes mass loss of 45.058 Da (Fig.…”

“…Enrichment of methyl peptides is more difficult than many other Deep methylation profiling reveals novel PRMT1 targets 5 PTMs because methylation does not add significant steric bulk or change the charge of the amino acids (20). Despite this difficulty, advances in methyl peptide enrichment for mass spectrometry have been made using immunoaffinity enrichment (IAP) with antibodies that recognize various forms of protein methylation (21)(22)(23)(24)(25)(26). By combining IAP against MMA with sample fractionation, as many as 8,000 MMA sites have been identified in human cells (24).…”

Middle-down proteomics reveals dense sites of methylation and phosphorylation in arginine-rich RNA-binding proteins

“…alternative splicing [81,85]. Another type II enzyme, PRMT9, regulates alternative splicing by methylation of spliceosome-associated protein 145 (SAP145) at R508, priming SAP145 for interaction with the protein SMN [86].…”

“…examination with alternative proteolytic enzymes, advanced search engines and high resolution Orbitrap MS/MS strategies will greatly aid in the advancement of unreported combinatorial PTM identification and quantification.A vital discovery achieved by our analysis is the combinatorial nature of PTMs within Argrich domains, highlighted by a shared RSRS motif between arginine dimethylation and serine phosphorylation in nucleoplasm proteins[73,79]. Serine-arginine protein kinases (SRPKs) and protein arginine methyltransferase (PRMT) enzymes modify arginine-rich domains in RNAbinding proteins[12,68,80,81]. Due to the proximal relationship of methylation and phosphorylation PTMs, cross-talk between adjacent arginine and serine residues within RSRS motifs is likely.…”