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

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.

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“…4 C ). This observation was corroborated by a very recent study published by our group showing that R methylation is involved in the assembly of membrane-less organelles through the modulation of RBP–RNA interaction ( 17 ).…”

“…While methylation of histones and its implication in transcriptional regulation has been extensively studied in several model systems and functional states ( 6 ), the concept that R methylation is widespread on non-histone proteins is supported by more recent studies ( 7 , 8 , 9 , 10 , 11 ). PRMT1, PRMT3, PRMT4, PRMT5, and PRMT7 have all been reported to catalyze methylation on RNA-binding proteins (RBPs), modulating their interactions with RNAs and thus impacting on biological processes, such as mRNA splicing, miRNA maturation, translation, and ribonucleoprotein (RNP) granules assembly ( 8 , 12 , 13 , 14 , 15 , 16 , 17 ). Importantly, PRMTs are often aberrantly expressed in cancer ( 18 , 19 ) and in neurodegenerative and metabolic disorders ( 20 , 21 ).…”

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“…This region is on the border of the DBD and the hinge domain, and R260 is within this region. RBPs are a main group of arginine-methylated proteins, and the arginine methylation of RBPs affects RBP-RNA interactions [ 86 ]. Although it is unclear how the methylation status of the ERα R260 (and R264 in mouse ERα) influences ERα’s function as an RBP, the substitution of four amino acids including R260 to unmethylated alanine disrupts the ERα’s RBP function, leading to changes in an alternative splicing and translation of the target genes without affecting the DNA-binding ability [ 85 ].…”

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“…Indeed, even if previous studies from her group have demonstrated that cisplatin treatment could change PRMT subcellular localization, her results suggest a novel layer of complexity. In response to the cisplatin treatment the R-hypomethylation on RBPs, the consequent modulation of RBP-RNA binding and their subcellular localization can occur ( Maniaci et al, 2021 ).…”