Systematic Analysis of the Impact of R-Methylation on RBPs-RNA Interactions: A Proteomic Approach.

Marianna Maniaci,Tiziana Bonaldi,Enrico Massignani,Francesca Ludovica Boffo

doi:10.3389/fmolb.2021.688973

Abstract

RNA binding proteins (RBPs) bind RNAs through specific RNA-binding domains, generating multi-molecular complexes known as ribonucleoproteins (RNPs). Various post-translational modifications (PTMs) have been described to regulate RBP structure, subcellular localization, and interactions with other proteins or RNAs. Recent proteome-wide experiments showed that RBPs are the most representative group within the class of arginine (R)-methylated proteins. Moreover, emerging evidence suggests that this modification plays a role in the regulation of RBP-RNA interactions. Nevertheless, a systematic analysis of how changes in protein-R-methylation can affect globally RBPs-RNA interactions is still missing. We describe here a quantitative proteomics approach to profile global changes of RBP-RNA interactions upon the modulation of type I and II protein arginine methyltransferases (PRMTs). By coupling the recently described Orthogonal Organic Phase Separation (OOPS) strategy with the Stable Isotope Labelling with Amino acids in Cell culture (SILAC) and pharmacological modulation of PRMTs, we profiled RNA-protein interaction dynamics in dependence of protein-R-methylation. Data are available via ProteomeXchange with identifier PXD024601.

Highlights

Arginine (R)-methylation is a widespread post-translational modification (PTM) that occurs on both histones, where it acts as an epigenetic regulator of gene expression, and non-histone proteins, where it modulates protein-protein, protein-RNA and protein-DNA interactions (Blanc and Richard, 2017), emerging as a key modulator of several cellular processes, from translation and splicing to growth factor–receptor signaling, miRNA biogenesis and DNA damage response (Guccione and Richard, 2019; Musiani et al, 2020; Spadotto et al, 2020)
Efficient inhibition of protein arginine methyltransferases (PRMTs) was achieved with a 48 h treatment with the PRMT type I inhibitor MS023 (Eram et al, 2016) and GSK591, a selective inhibitor of PRMT5 (Sachamitr et al, 2021)
Drug efficiency was confirmed by monitoring changes in arginine residues either two methyl-groups in asymmetric (ADMA) and SDMA, both globally (Supplementary Figure S1) and on the asymmetric dimethylation of Arginine 3 on histone 4 (H4R3me2a) and on the symmetric di-methylation of Arginine 3 on histone 4 (H4R3me2s), modifications known to be deposed by PRMT1 and PRMT5, respectively (Figure 1B)

Summary

Introduction

Arginine (R)-methylation is a widespread post-translational modification (PTM) that occurs on both histones, where it acts as an epigenetic regulator of gene expression, and non-histone proteins, where it modulates protein-protein, protein-RNA and protein-DNA interactions (Blanc and Richard, 2017), emerging as a key modulator of several cellular processes, from translation and splicing to growth factor–receptor signaling, miRNA biogenesis and DNA damage response (Guccione and Richard, 2019; Musiani et al, 2020; Spadotto et al, 2020). Nine enzymes have been identified and classified as type I, type II and type III protein R-methyltransferases (PRMTs), depending on their ability to transfer to the guanidino group of the arginine residues either two methyl-groups in asymmetric (ADMA) and symmetric (SDMA) manner, or one methyl-group (MMA), respectively (Blanc and Richard, 2017). PRMT1 and PRMT5 are the most active PRMTs of the type I and II families, respectively, and object of intense investigation in both basic and translational research (Zhang et al, 2019).

Methods

Results

Conclusion