Assaying epigenome functions of PRMTs and their substrates

Rakow, Sinja; Pullamsetti, Soni Savai; Bauer, Uta‐Maria; Bouchard, Caroline

doi:10.1016/j.ymeth.2019.09.014

Cited by 18 publications

(12 citation statements)

References 198 publications

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“…Determining the molecular basis of substrate recognition by PRMTs is crucial for understanding their roles in the cell [4,7,10]. Here, we have systematically and comprehensively investigated the substrate specificity of PRMT6, in the context of the histone H3 tail, and show that it has a broad substrate specificity, with a preference for basic and bulky amino acids in positions around the target arginine.…”

Section: Discussionmentioning

confidence: 99%

“…In human, there are nine PRMTs that catalyse Abbreviations AdoMet, S-adenosyl L-methionine; ETD, electron transfer dissociation; LC, liquid chromatography; MS, mass spectrometry; MT-MAMS, methyltransferase motif analysis by mass spectrometry; PRM, parallel reaction monitoring; PRMT, protein arginine methyltransferase. methylation on a wide variety of cellular substrates [2][3][4]. Phenotypes of mice PRMT knockouts range from mild defects to embryonic lethality [5].…”

Section: Introductionmentioning

confidence: 99%

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Systematic investigation of PRMT6 substrate recognition reveals broad specificity with a preference for an RG motif or basic and bulky residues

et al. 2021

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Protein arginine methyltransferase 6 (PRMT6) catalyses the asymmetric dimethylation of arginines on numerous substrate proteins within the human cell. In particular, PRMT6 methylates histone H3 arginine 2 (H3R2) which affects both gene repression and activation. However, the substrate specificity of PRMT6 has not been comprehensively analysed. Here, we systematically characterise the substrate recognition motif of PRMT6, finding that it has broad specificity and recognises the RG motif. Working with a H3 tail peptide as a template, on which we made 204 amino acid substitutions, we use targeted mass spectrometry to measure their effect on PRMT6 in vitro activity. We first show that PRMT6 methylates R2 and R8 in the H3 peptide, although H3R8 is methylated with lower efficiency and is not an in vivo PRMT6 substrate. We then quantify the effect of 194 of these amino acid substitutions on methylation at both H3R2 and H3R8. In both cases, we find that PRMT6 tolerates essentially any amino acid substitution in the H3 peptide, but that positively charged and bulky residues are preferred near the target arginine. We show that PRMT6 also has preference for glycine, but only in the position immediately following the target arginine. This indicates that PRMT6 recognises the RG motif rather than the RGG motif. We further confirm this preference for the RG motif on another PRMT6 substrate, histone H4R3. This broad specificity and recognition of RG rather than RGG are distinctive among the PRMT family and has implications for the development of drugs to selectively target PRMT6.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Systematic investigation of PRMT6 substrate recognition reveals broad specificity with a preference for an RG motif or basic and bulky residues

et al. 2021

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“…Because there is a correlation between the expression level of some PRMTs and cancer (Yang and Bedford, 2013), investigation in both academic laboratories and the pharmaceutical industry has been leading to the discovery of several small molecules targeting different PRMTs that at micromolar and submicromolar concentrations display anti-tumour effects in cellular assays (Hu et al ., 2016; Zhang and Zheng, 2016; Guccione and Richard, 2019). In fact, three specific inhibitors for PRMT5 (GSK3325695, JNJ-64619178 and EPZ015938) and one for type I PRMTs (GSK3368715) have been analysed in phase I clinical trials (Rakow et al ., 2020). As PRMTs of protozoan parasites play important roles in their cell growth, virulence, conversion stages and response to stress conditions (Table 1), anti-PRMT drugs could also be effective for the treatment of diseases produced by those pathogenic microorganisms.…”

Section: Discussionmentioning

confidence: 99%

“…In general, PRMTs are found in the nucleus and cytoplasm, but PRMT3 is located principally in the cytoplasm, PRMT6 is concentrated mainly in the nucleus and PRMT8 is linked to the plasma membrane (Frankel and Clarke, 2000; Frankel et al ., 2002; Lee et al ., 2005). Arginine methylation on histones is involved in the co-activation or co-repression of gene transcription and the effects on transcription of specific residues modified by different PRMTs have been determined (reviewed by Rakow et al ., 2020). PRMT1 and PRMT4/CARM1 are the main co-activators (Bedford and Clarke, 2009), PRMT2 and PRMT7 function as co-repressors (Ganesh et al ., 2006; Karkhanis et al ., 2012), whereas PRMT5 and PRMT6 have been reported to have both functions (Zhao et al ., 2009; Migliori et al ., 2012; Casadio et al ., 2013; Cheng et al ., 2020).…”

Section: Protein Arginine Methyltransferases (Prmts)mentioning

confidence: 99%

Protein arginine methyltransferases in protozoan parasites

Rodríguez

2021

Parasitology

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Arginine methylation is a post-translational modification involved in gene transcription, signalling pathways, DNA repair, RNA metabolism and splicing, among others, mechanisms that in protozoa parasites may be involved in pathogenicity-related events. This modification is performed by protein arginine methyltransferases (PRMTs), which according to their products are divided into three main types: type I yields monomethylarginine (MMA) and asymmetric dimethylarginine; type II produces MMA and symmetric dimethylarginine; whereas type III catalyses MMA only. Nine PRMTs (PRMT1 to PRMT9) have been characterized in humans, whereas in protozoa parasites, except for Giardia intestinalis, three to eight PRMTs have been identified, where in each group there are at least two enzymes belonging to type I, the majority with higher similarity to human PRMT1, and one of type II, related to human PRMT5. However, the information on the role of most of these enzymes in the parasites biology is limited so far. Here, current knowledge of PRMTs in protozoan parasites is reviewed; these enzymes participate in the cell growth, stress response, stage transitions and virulence of these microorganisms. Thus, PRMTs are attractive targets for developing new therapeutic strategies against these pathogens.

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“…We further validated the screening assay by analysis of the histone modifications by mass spectrometry (MS). This technique measures directly the abundance of histone proteins without use of antibodies that can present problems of specificity 42 . U2OS cells were incubated with compound 4 or EPZ-5676 at 3.2µM for 8 days or with DMSO as negative control.…”

Section: Validation By Mass Spectrometrymentioning

confidence: 99%