Transcriptomic and proteomic regulation through abundant, dynamic, and independent arginine methylation by Type I and Type II PRMTs

Abstract

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.

Type I PRMTs and PRMT5 Independently Regulate Both snRNP Arginine Methylation and Post-Transcriptional Splicing

“…Additionally, by western blot these Sm proteins had reduced Rme2s (Figure 3a, middle panel) . Furthermore, consistent with the ability of different classes of PRMTs to scavenge each other’s substrates (Dhar et al 2013; Lehman et al 2020), we observed an increase in Rme2a with GSK591. We also noted an increase in Rme2s in the MS023-treated A549 cells (Figure 3a) .…”

A Semi-automated Organoid Screening Method Demonstrates Epigenetic Control of Intestinal Epithelial Differentiation

“…In agreement with the latter, we found increase of all differentiated IEC lineages after treatment with MS023 ( Figures 6A–F ). PRMT1 has a wide substrate specificity and mediates both arginine methylation of histones such as H4R3, and non-histone proteins (Huang et al, 2005 ; Lehman et al, 2020 ). Elevated PRMT1 expression is found in several cancer types and is associated with poor prognosis and chemoinsensitivity (Altan et al, 2016 ; Musiani et al, 2020 ) and pharmacological PRMT inhibitors have recently gained interest as drug candidates for cancer treatment (Guccione and Richard, 2019 ; Jarrold and Davies, 2019 ).…”

A semi-automated organoid screening method demonstrates epigenetic control of intestinal epithelial differentiation

“…6a-6f ). PRMT1 has a wide substrate specificity and mediates both arginine methylation of histones such as H4R3, and non-histone proteins 64,65 . Elevated PRMT1 expression is found in several cancer types and is associated with poor prognosis and chemoinsensitivity 66,67 and pharmacological PRMT inhibitors have recently gained interest as drug candidates for cancer treatment 43,45 .…”