Proteomic analysis of interactors for yeast protein arginine methyltransferase Hmt1 reveals novel substrate and insights into additional biological roles

Abstract

Protein arginine methylation is a PTM catalyzed by an evolutionarily conserved family of enzymes called protein arginine methyltransferases (PRMTs), with PRMT1 being the most conserved member of this enzyme family. This modification has emerged to be an important regulator of protein functions. To better understand the role of PRMTs in cellular pathways and functions, we have carried out a proteomic profiling experiment to comprehensively identify the physical interactors of Hmt1, the budding yeast homolog for human PRMT1. Using a dual-enzymatic digestion linear trap quadrupole/Orbitrap proteomic strategy, we identified a total of 108 proteins that specifically copurify with Hmt1 by tandem affinity purification. A reverse coimmunoprecipitation experiment was used to confirm Hmt1's physical association with Bre5, Mtr4, Snf2, Sum1, and Ssd1, five proteins that were identified as Hmt1-specific interactors in multiple biological replicates. To determine whether the identified Hmt1-interactors had the potential to act as an Hmt1 substrate, we used published bioinformatics algorithms that predict the presence and location of potential methylarginines for each identified interactor. One of the top hits from this analysis, Snf2, was experimentally confirmed as a robust substrate of Hmt1 in vitro. Overall, our data provide a feasible proteomic approach that aid in the better understanding of PRMT1's roles within a cell.

Analysis of the Physiological Activities of Scd6 through Its Interaction with Hmt1

“…Hmt1 was previously shown to catalyze arginine methylation of mRNP components with RGG motifs such as Npl3, Sbp1, and Ded1, which are reportedly localized in RNA granules [ 21 ]. In addition, the P-body component Ebs1 [ 35 ] was recovered in our experiments, confirming the specificity of our screening methods, and Ebs1 was also recovered in proteomic analysis of Hmt1-TAP associating proteins [ 36 ]. These observations suggest the presence of a functional link between Hmt1 and components of P-bodies [ 37 ].…”

Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation

“…The ribosomal protein Rps2 is arginine methylated in yeast and humans, albeit by different PRMTs and possibly with different impacts in the two systems [50–52]. The Mtr4 RNA helicase was recently shown to interact with the major yeast type I PRMT in vivo , suggesting it might be methylated like its T. brucei homologue [53]. These findings suggest that some functions of arginine methylation exhibit extraordinary evolutionary conservation.…”

“…Regarding DNA repair, we identified the DNA excision repair protein, TbSnf2, as an arginine methylated protein. In yeast, Snf2 is associated in vivo with and methylated in vitro by the major type I PRMT, suggesting that modulation of Snf2 function by arginine methylation may be evolutionarily conserved [53]. We detected methylarginine residues in the T. brucei homologues of the PI3K-like kinases, ATM and ATR, which are master regulators of the DNA damage response and repair pathways in other eukaryotes [65].…”

Arginine methylation in yeast proteins during stationary-phase growth and heat shock

“…Ras2p regulates the nitrogen starvation response (Broek et al 1985;Parrini et al 1996) and has been shown to associate with Rmt1p (Jackson et al 2012). The recycling of amino acids is also a component of stationary growth (Herman 2002), and some proteins involved in this process are arginine methylated.…”