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. 2019 Mar 15;30(6):778-793.
doi: 10.1091/mbc.E18-05-0330. Epub 2019 Jan 30.

PRMT7 Methylates Eukaryotic Translation Initiation Factor 2α and Regulates Its Role in Stress Granule Formation

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Free PMC article

PRMT7 Methylates Eukaryotic Translation Initiation Factor 2α and Regulates Its Role in Stress Granule Formation

Nasim Haghandish et al. Mol Biol Cell. .
Free PMC article

Erratum in

  • Correction.
    Mol Biol Cell. 2019 Dec 1;30(25):3073. doi: 10.1091/mbc.E18-05-0330-corr. Mol Biol Cell. 2019. PMID: 31778347 Free PMC article. No abstract available.

Abstract

Protein arginine methyltransferases (PRMTs) are a family of enzymes that modify proteins by methylating the guanidino nitrogen atoms of arginine residues to regulate cellular processes such as chromatin remodeling, pre-mRNA splicing, and signal transduction. PRMT7 is the single type III PRMT solely capable of arginine monomethylation. To date, other than histone proteins, there are very few identified substrates of PRMT7. We therefore performed quantitative mass spectrometry experiments to identify PRMT7's interactome and potential substrates to better characterize the enzyme's biological function(s) in cells. These experiments revealed that PRMT7 interacts with and can methylate eukaryotic translation initiation factor 2 alpha (eIF2α), in vitro and in breast cancer cells. Furthermore, we uncovered a potential regulatory interplay between eIF2α arginine methylation by PRMT7 and stress-induced phosphorylation status of eIF2α at serine 51. Finally, we demonstrated that PRMT7 is required for eIF2α-dependent stress granule formation in the face of various cellular stresses. Altogether, our findings implicate PRMT7 as a novel mediator of eIF2α-dependent cellular stress response pathways.

Figures

FIGURE 1:
FIGURE 1:
Preparation and confirmation of immunoprecipitation for SILAC-based quantitative mass spectrometry. (A) Representative fluorescent images of MCF7 cells infected with lentiviruses expressing either mGFP control or PRMT7-mGFP showing diffused localization of PRMT7 at 40× magnification (scale bar: 50 µm). (B) Confirmation of affinity immunodepletion of PRMT7-mGFP fusion protein and mGFP using GFP-Trap beads (ChromoTek). (C) Coomassie-stained SDS–PAGE gel of the eluate (1:1 mixture of PRMT7-mGFP/mGFP) from affinity purification using GFP-Trap beads. The gel was sliced into five fragments to be sent for mass spectrometric analysis of coimmunoprecipitated proteins.
FIGURE 2:
FIGURE 2:
Analysis of SILAC-based mass spectrometry results. (A) Comparison of the normalized SILAC H:L ratio of identified proteins from the two independent mass spectrometry experiments (L:H ratio for experiment 2). Thresholds for putative protein interactors are indicated by red-dashed lines. (B) Scatter plot of two independent mass spectrometry experiments comparing shared interactor proteins represented as Log2 ratios of the heavy-labeled protein to light-labeled protein. PRMT7 interactors are highlighted in purple within the graph (above red median lines).
FIGURE 3:
FIGURE 3:
Identified PRMT7 interactors. (A) Strong interactors (high enrichment) of experiments 1 and 2 are represented in a table displaying their relative enrichment and number of identified peptides. (B) Interaction between exogenous PRMT7 and endogenous eIF2α, eIF2β, and eIF2γ was confirmed in MCF7 cells infected with either pLenti-C-MycDDK or pLenti-C-MycDDK-PRMT7 lentiviruses, using affinity pull downs with Myc-Trap beads.
FIGURE 4:
FIGURE 4:
Presence of PRMT7 within the translational machinery. (A) Representative polyribosome profile of parental HEK293T cells. Relative absorbance of RNA was read at 254 nm. (B) Polyribosome fractions were run on an SDS–PAGE gel including an input of total cell lysate. Endogenous PRMT7 cofractionates with eIF2α. FMRP was used as a positive control polysome fraction. (C) Pretranslational fractions were pooled from MCF7 cells transiently expressing either mGFP or PRMT7-mGFP. (D) GFP-Trap beads were used to immunoprecipitate control mGFP and PRMT7-mGFP from pooled samples. Inputs were run alongside immunoprecipitates. PRMT7-mGFP interacts with eIF2α in pretranslational fractions.
FIGURE 5:
FIGURE 5:
Confirmation that PRMT7 methylates eIF2α in vitro. (A) A motif recognized by PRMT7, RXRXR motif, exists adjacent to the regulatory Ser51 residue of eIF2α. Using site-directed mutagenesis, the arginine residues were mutated into lysine to create six mutants within the RXRXR motif. (B) In vitro methylation assays using 3HSAM as methyl donor, PRMT7 as the enzyme, and eIF2α as a substrate. Experiments revealed that R52R53R54I55R56 sequence motif is methylated by PRMT7—specifically, R54 is critical for its methylation. Methylation of histones was used as a positive control and GST as a negative control. Automethylation of PRMT7 was observed. Methylation assays are revealed through fluorography.
FIGURE 6:
FIGURE 6:
eIF2α is a PRMT7-specific substrate. (A) In vitro methylation assay using 3HSAM as a methyl donor, purified PRMT 1, 3, 4, 5, 6, 7, 8, and 9 as the enzyme, and purified GST-eIF2α as a substrate (both wild type and RKK mutant). Experiments revealed that eIF2α is a PRMT7-specific substrate (asterisks). Automethylation of PRMTs 4, 6, 7, and 8 was observed. (B) Methylation of histones was used as a positive control. Automethylation was also observed for PRMTs 4, 6, 7, and 8. Methylation assays are revealed through fluorography.
FIGURE 7:
FIGURE 7:
PRMT7 methylates eIF2α in vivo. (A) In vivo methylation assay using 3H methionine in MDA-MB-231 cell lines transiently expressing wild-type eIF2α-mGFP (in the presence of control or PRMT7-targeting shRNAs), KRR, or RRK mutant alleles. (B) Representative 35S metabolic labeling demonstrating that no incorporation of isotopic amino acid is observed following treatment with translation inhibitors. (C) Quantification of the relative methylation status of eIF2α normalized to the wild-type eIF2α-mGFP mock conditions, as well as to the amount of eIF2-mGFP detected on the Coomassie stain. A significant decrease in methylation of eIF2α was observed upon PRMT7 knockdown and for the RRK mutant. No change was observed for the KRR mutant; data are presented as mean ± SEM for n = 5; **, p = 0.002; ***, p = 0.001 (ANOVA). (D) Representative Western blot depicting the efficiency of the knockdown in cells infected with the PRMT7-targeting shRNA.
FIGURE 8:
FIGURE 8:
Interplay between eIF2α methylation and phosphorylation. Phosphorylation of eIF2α decreased upon transient knockdown of PRMT7 in MDA-MB-231 cells as shown in the (A) Western blot and (B) quantification (data are presented as mean ± SEM for n = 5; **, p = 0.01, ANOVA). This effect is not seen when experiments were performed in the presence of cycloheximide (CHX). (C) Representative Western blot depicts an increase in eIF2α phosphorylation upon transient overexpression of PRMT7-Myc in MDA-MB-231 cells and (D) its quantification (data are presented as mean ± SEM for n = 5; *, p = 0.05; two-tailed t test). (E) Western blots depicting phosphorylation of transiently expressed wild-type and KRR mutant eIF2α upon treatment with sodium arsenite for 30 min at 500 µM in MDA-MB-231 cells. Phosphorylation is absent in the RRK mutant.
FIGURE 9:
FIGURE 9:
PRMT7 methylates eIF2α under stressed conditions. Transiently expressed eIF2α-mGFP and GFP (negative control) were immunoprecipitated from untreated and AsNaO2-treated (500 µM for 30 min) HEK293T cells, followed by immunoblotting using a monomethylation-specific antibody. Western blot depicts a significant increase in eIF2α monomethylation within AsNaO2-treated cells. Furthermore, immunoblotting of the same membrane for PRMT7 reveals a drastic reduction in the interaction of PRMT7 with eIF2α-mGFP under stress conditions.
FIGURE 10:
FIGURE 10:
PRMT7 activity toward eIF2α is increased under stressed conditions. (A) PRMT7-Myc was transiently expressed in MDA-MB-231 cells and then affinity-purified using Myc-Trap beads. Affinity-purified PRMT7-Myc, from Mock- or AsNaO2-treated (500 µM for 30 min) was then used as a source of enzyme to perform in vitro methylation assays using 3HSAM as methyl-donor wild-type eIF2α-GST as a substrate. Cells transfected with a Myc empty vector were used as a negative control. As a positive control, purified PRMT7 with either GST or wild-type eIF2α-GST was used. (B) Quantification of the methylation status of eIF2α was calculated for the AsNaO2-treated condition and normalized to the amount of PRMT7-Myc detected in the Coomassie stain. A significant increase in methylation of eIF2α was observed in AsNaO2-treated cells; data are presented as mean ± SEM for n = 5; *, p = 0.02; two-tailed t test. (C) Western blot depicting unchanged endogenous PRMT7 expression AsNaO2 treatment (500 µM for the indicated times) in parental MDA-MB-231 cells.
FIGURE 11:
FIGURE 11:
Stress granule formation is abrogated upon PRMT7 knockdown. (A) Representative immunofluorescent images of MDA-MB-231 cells depicting decreased stress granule formation upon transient knockdown of PRMT7 when exposed to AsNaO2 (500 µM for 30 min) and thapsigargin (10 µM for 2 h). No change was observed when cells were treated with rocaglamide A (2 µM for 2 h). FMRP (green) was used as a stress granule marker (scale bar: 20 µm). (B) Quantitation of “percentage of cells with at least five stress granules” for n = 5 independent experiments in triplicate when treated with AsNaO2; data are presented as mean ± SEM; *, p = 0.04; **, p = 0.005 (ANOVA). (C) Similar quantitation was performed when treating with thapsigargin; ***, p = 0.0001 (ANOVA), and (D) rocaglamide A (not significant).
FIGURE 12:
FIGURE 12:
Working model depicting how PRMT7 participates in the eIF2α-dependent cellular response to stress. Upon exposure to stress, such as oxidative or ER stress, PRMT7 activity is stimulated, which leads to increased methylation of eIF2α within the RXR motif. Methylation of eIF2α by PRMT7 then promotes stable phosphorylation levels of eIF2α at Ser51, ultimately inducing stress granule formation.

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References

    1. Agolini E, Dentici ML, Bellacchio E, Alesi V, Radio FC, Torella A, Musacchia F, Tartaglia M, Dallapiccola B, Nigro V, et al. (2018). Expanding the clinical and molecular spectrum of PRMT7 mutations: 3 additional patients and review. Clin Genet , 675–681. - PubMed
    1. Anderson P, Kedersha N, Ivanov P. (2015). Stress granules, P-bodies and cancer. Biochim Biophys Acta , 861–870. - PMC - PubMed
    1. Aulas A, Fay MM, Lyons SM, Achorn CA, Kedersha N, Anderson P, Ivanov P. (2017). Methods to classify cytoplasmic foci as mammalian stress granules. J Vis Exp , 10.3791/55656 - DOI - PMC - PubMed
    1. Baguet A, Degot S, Cougot N, Bertrand E, Chenard M-P, Wendling C, Kessler P, Le Hir H, Rio MC, Tomasetto C. (2007). The exon-junction-­complex-component metastatic lymph node 51 functions in stress-­granule assembly. J Cell Sci , 2774. - PubMed
    1. Balagopal V, Parker R. (2009). Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr Opin Cell Biol , 403–408. - PMC - PubMed

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