Global profiling of arginine dimethylation in regulating protein phase separation by a steric effect–based chemical-enrichment method

Abstract

Protein arginine methylation plays an important role in regulating protein functions in different cellular processes, and its dysregulation may lead to a variety of human diseases. Recently, arginine methylation was found to be involved in modulating protein liquid–liquid phase separation (LLPS), which drives the formation of different membraneless organelles (MLOs). Here, we developed a steric effect–based chemical-enrichment method (SECEM) coupled with liquid chromatography–tandem mass spectrometry to analyze arginine dimethylation (DMA) at the proteome level. We revealed by SECEM that, in mammalian cells, the DMA sites occurring in the RG/RGG motifs are preferentially enriched within the proteins identified in different MLOs, especially stress granules (SGs). Notably, global decrease of protein arginine methylation severely impairs the dynamic assembly and disassembly of SGs. By further profiling the dynamic change of DMA upon SG formation by SECEM, we identified that the most dramatic change of DMA occurs at multiple sites of RG/RGG–rich regions from several key SG-contained proteins, including G3BP1, FUS, hnRNPA1, and KHDRBS1. Moreover, both in vitro arginine methylation and mutation of the identified DMA sites significantly impair LLPS capability of the four different RG/RGG–rich regions. Overall, we provide a global profiling of the dynamic changes of protein DMA in the mammalian cells under different stress conditions by SECEM and reveal the important role of DMA in regulating protein LLPS and SG dynamics.

Investigating the effects of arginine methylation inhibitors on microdissected brain tumour biopsies maintained in a miniaturised perfusion system

“…101 In summary, the switch to sDMA in FUS could contribute to the mechanisms underlying the effects of PRMT inhibitors on GBM cell death. This hypothesis is supported by recent observations of changes in the dimethylation patterns of FUS in cells under stress (oxidative stress, heat stress), 102 however, it is not consistent with the lack of synergistic effects between type I and type II PRMT inhibitors in causing cell death (Fig. 3B).…”

“…101 In summary, the switch to sDMA in FUS could contribute to the mechanisms underlying the effects of PRMT inhibitors on GBM cell death. This hypothesis is supported by recent observations of changes in the dimethylation patterns of FUS in cells under stress (oxidative stress, heat stress), 102 however, it is not consistent with the lack of synergistic effects between type I and type II PRMT inhibitors in causing cell Table 2 Identification of FUS peptides labelled with 13 CD 3 while GBM samples were maintained in the perfusion device. Pool 1: lysates from samples treated with light Met and MS023, pooled with lysates from control samples treated with heavy Met.…”

ETD-Based Proteomic Profiling Improves Arginine Methylation Identification and Reveals Novel PRMT5 Substrates

“…Considering the low complexity of the RGG/RG motif, it is reasonable to speculate that SERBP1 is not the only case where two types of dimethylation coexist in the same region . Unfortunately, accurately distinguishing between aDMA and sDMA still poses a significant challenge to MS analysis on these multiple methylated peptides . Future method development is needed to facilitate our understanding of arginine methylation functions and the interplay among various PRMTs.…”

“…70 Unfortunately, accurately distinguishing between aDMA and sDMA still poses a significant challenge to MS analysis on these multiple methylated peptides. 71 Future method development is needed to facilitate our understanding of arginine methylation functions and the interplay among various PRMTs.…”

Lipidation alters the phase-separation of resilin-like polypeptides

“…21,22 Due to the well-recognized role of electrostatic interactions in modulating protein phase-separation, the significance of 'chargealtering' PTMs is under intense investigation and is beginning to come into a sharper focus. These include but are not limited to, serine phosphorylation, 23,24 arginine-methylation, 25 and lysine acetylation, 26 which modify multivalent electrostatic interactions by altering coulombic charges or their distributions. However, more than 300 types of PTMs have been identified, and many do not alter the electrostatic charge significantly.…”