TY - JOUR
T1 - Proteomics profiling of arginine methylation defines PRMT5 substrate specificity
AU - Musiani, Daniele
AU - Bok, Jabez
AU - Massignani, Enrico
AU - Wu, Liling
AU - Tabaglio, Tommaso
AU - Ippolito, Marica Rosaria
AU - Cuomo, Alessandro
AU - Ozbek, Umut
AU - Zorgati, Habiba
AU - Ghoshdastider, Umesh
AU - Robinson, Robert C.
AU - Guccione, Ernesto
AU - Bonaldi, Tiziana
N1 - Funding Information:
We thank the SGC (Toronto, Canada) for providing the PRMT5 small-molecule inhibitor GSK591 and the SGC20969 inactive compound. We also thank the Kaldis Lab for providing the pMX-IRES-blasticidin overexpression retroviral vector (Cell Biolabs, #RTV-016). Research activity in the T.B. laboratory was supported by grants from the Italian Association for Cancer Research (AIRC [15741]), the Italian Ministry of Health (GR-2011-02347880), and CNR-EPIGEN flagship project. D.M. was supported by the CNR-EPIGEN flagship project, the Fondazione Umberto Veronesi (FUV) fellowship, and the Fondazione Istituto Europeo di Oncologia (FIEO) fellowship. L.W. and T.T. were supported by the A*STAR SINGA fellowship. J.B. was supported by the A*STAR returning scholar funds. E.G. acknowledged funding support from the NRF2016-CRP001-103 CRP award and from the RNA Biology Center at the Cancer Science Institute (CSI) of Singapore, National University of Singapore, and funding by the Singapore Ministry of Education’s Tier 3 grants (grant number MOE2014-T3-1-006).
Publisher Copyright:
Copyright © 2019 The Authors.
PY - 2019/4/2
Y1 - 2019/4/2
N2 - 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.
AB - 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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U2 - 10.1126/scisignal.aat8388
DO - 10.1126/scisignal.aat8388
M3 - Article
C2 - 30940768
AN - SCOPUS:85064163981
VL - 12
JO - Science's STKE : signal transduction knowledge environment
JF - Science's STKE : signal transduction knowledge environment
SN - 1937-9145
IS - 575
M1 - eaat8388
ER -