Research ResourceBiochemistry

Proteomics profiling of arginine methylation defines PRMT5 substrate specificity

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Science Signaling  02 Apr 2019:
Vol. 12, Issue 575, eaat8388
DOI: 10.1126/scisignal.aat8388

Profiling PRMT5

The enzyme PRMT5 methylates arginine residues in target proteins to modify their function. PRMT5 functions in various cellular processes and is implicated in cancer. Musiani et al. used advanced and modified proteomics to explore the substrate specificity of PRMT5 and identified a preferred target site (a glycine-sandwiched arginine), as well as previously unknown substrates of the enzyme. This resource therefore offers clues to the function of PRMT5 in normal physiology and potential ways to target it in disease.


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.

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