RT Journal Article
SR Electronic
T1 Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.06.23.167601
DO 10.1101/2020.06.23.167601
A1 Stephanie M. Lehman
A1 Hongshan Chen
A1 Emmanuel S. Burgos
A1 Maxim Maron
A1 Sitaram Gayatri
A1 Edward Nieves
A1 Dina L. Bai
A1 Simone Sidoli
A1 Varun Gupta
A1 Matthew R. Marunde
A1 James R. Bone
A1 Zu-Wen Sun
A1 Mark T. Bedford
A1 Jeffrey Shabanowitz
A1 Donald F. Hunt
A1 David Shechter
YR 2020
UL http://biorxiv.org/content/early/2020/06/24/2020.06.23.167601.abstract
AB Arginine methylation is essential for both cellular viability and development and is also dysregulated in cancer. PRMTs catalyze the post translational monomethylation (Rme1/MMA, catalyzed by Type I-III), asymmetric (Rme2a/ADMA, Type I enzymes)-, or symmetric (Rme2s/SDMA, Type II enzymes) dimethylation of arginine. Despite many studies, a thorough integration of PRMT enzyme substrate determination and proteomic and transcriptomic consequences of inhibiting Type I and II PRMTs is lacking. To characterize cellular substrates for Type I (Rme2a) and Type II (Rme2s) PRMTs, human A549 lung adenocarcinoma cells were treated with either Type I (MS023) or Type II (GSK591) inhibitors. Using total proteome hydrolysis, we developed a new mass spectrometry approach to analyze total arginine and lysine content. We showed that Rme1 was a minor population (∼0.1% of total arginine), Rme2a was highly abundant (∼1.1%), and Rme2s was intermediate (∼0.4%). While Rme2s was mostly eliminated by GSK591 treatment, total Rme1 and Rme2a were more resistant to perturbation. To quantitatively characterize substrate preferences of the major enzymes PRMT1, PRMT4(CARM1), and PRMT5, we used oriented peptide array libraries (OPAL) in methyltransferase assays. We demonstrated that while PRMT5 tolerates aspartic acid residues in the substrate, PRMT1 does not. Importantly, PRMT4 methylated previously uncharacterized hydrophobic motifs. To integrate our studies, we performed PTMScan on PRMT-inhibited A549 cells and enriched for methylated arginine containing tryptic peptides. For detection of highly charged peptides, a method to analyze the samples using electron transfer dissociation was developed. Proteomic analysis revealed distinct methylated species enriched in nuclear function, RNA-binding, intrinsically disordered domains, and liquid-liquid phase separation. Parallel studies with proteomics and RNA-Seq revealed distinct, but ontologically overlapping, consequences to PRMT inhibition. Overall, we demonstrate a wider PRMT substrate diversity and methylarginine functional consequence than previously shown.Competing Interest StatementCo-author M.T.B. is a co-founder of EpiCypher, Inc.