Stabilization of ERK-Phosphorylated METTL3 by USP5 Increases m6A Methylation

Abstract

N⁶-methyladenosine (m⁶A) is the most abundant mRNA modification and is installed by the METTL3-METTL14-WTAP methyltransferase complex. Although the importance of m⁶A methylation in mRNA metabolism has been well documented recently, regulation of the m⁶A machinery remains obscure. Through a genome-wide CRISPR screen, we identify the ERK pathway and USP5 as positive regulators of the m⁶A deposition. We find that ERK phosphorylates METTL3 at S43/S50/S525 and WTAP at S306/S341, followed by deubiquitination by USP5, resulting in stabilization of the m⁶A methyltransferase complex. Lack of METTL3/WTAP phosphorylation reduces decay of m⁶A-labeled pluripotent factor transcripts and traps mouse embryonic stem cells in the pluripotent state. The same phosphorylation can also be found in ERK-activated human cancer cells and contribute to tumorigenesis. Our study reveals an unrecognized function of ERK in regulating m⁶A methylation.

Keywords: ERK; METTL3 phosphorylation; USP5; m(6)A methylation; stem cell differentiation.

Figure 1.. ERK Activation Promotes mRNA m⁶A Methylation

(A) Schematic diagram of a circular RNA (circRNA) translation reporter consisting of a single exon and two introns with complementary sequences. The exon containing GGACU can be back-spliced to generate circRNAs that drive GFP translation. (B) Overview of the CRISPR screen. Cas9 KO libraries are packaged into lentivirus and then transduced into HeLa cells contain circRNA GFP reporters. Cells with the top and bottom 5% GFP expression were collected by flow cytometry. The sgRNA were amplified from genomic DNA and then sequenced, followed by statistical analyses to identify candidate genes. (C) Positive regulators for the m⁶A pathway identified in the screen using circular GGACU-GFP reporters. (D) Pathway analysis of sgRNA enriched in the bottom 5% GFP cells with circRNA GFP reporters. See also Figure S1, Table S1, and Table S2.

Figure 2.. ERK Interacts and Phosphorylates METTL3 and WTAP

(A) A375 cells were treated with/without 0.1 μM trametinib for 1 hr. Lysates were analyzed by IP and IB as indicated. (B) Sequence alignment of the conserved D-domain on METTL3 and WTAP predicted by the ELM website. The D domain possesses a consensus binding sequence of (Lys/Arg)0–2-(X)1–6-Φ-X-Φ; where Φ is a hydrophobic residue such as Leu, Ile, Val, Phe, and X is any amino acid. (C) Interaction between wild-type (WT) or mutant METTL3, WTAP, and ERK2 in lysates from BRAF-expressing 293T cells transfected as indicated was examined by co-IP. EE, R415E/R416E METTL3 or R71E/R72E WTAP. (D) Sequence alignment of the conserved serine residues on METTL3 that are phosphorylated by ERK. (E) Mass spectrometry detected S43, S50 and S525 phosphorylation in METTL3 in 293T cells co-transfected with BRAF V600E. (F) Phos-tag SDS-PAGE showing the phosphorylation status of WT or non-phosphorylatable alanine mutants of METTL3 in 293T cells co-transfected with BRAF. 2A, T43A/S50A; 3A, S43A/S50A/S525A. (G) Sequence alignment of the serine/threonine-proline (S/T-P) motif on WTAP. (H) Phos-tag SDS-PAGE showing the phosphorylation status of WT or non-phosphorylatable alanine mutants of human WTAP in 293T cells co-transfected with BRAF. 2A, S306A/S341A. See also Figure S2.

Figure 3.. USP5 is Required for ERK-Mediated METTL3 Stabilization

(A) Comparison of METTL3 and WTAP protein levels in mESCs and A375 stable transfectants by immunoblotting (IB). (B) 293T cells transfected as indicated were treated with MG-132 (10 μM, 8hr) followed by IP/IB. (C) 293T cells transfected as indicated for 48 hr, followed by cycloheximide (CHX) 10 μg/ml for 0–12 h. Lysates were used for IB to measure the protein levels of METTL3. Density of METTL3 expression was quantified by ImageJ and the relative fold compared to the untreated WT was indicated and plotted at the right panel. (D) BRAF expression promotes METTL3-USP5 interaction. Lysates of 293T cells transfected as indicated for 48 hr were subjected to IP with anti-myc antibody followed by IB. (E) USP5 decreases ubiquitination of METTL3. Lysates of 293T cells transfected as indicated for 48 hr were subjected to IP with anti-myc antibody followed by IB. (F) Knockdown of SPOP, TRIM28, and ANAPC1 attenuated USP5 inhibition-induced degradation of METTL3. A375 cells were transfected with siRNA for 72 hr or treated with 10 μM EOAI3402143 (EOAI) for 8 hr before IB analysis. See also Figure S3 and Figure S4.

Figure 4.. Phosphorylation of METTL3/WTAP by ERK Facilitates Resolution of Pluripotency

(A) LC-MS/MS quantification of the m⁶A/A ratio in mRNA of mESCs stable transfectants. *p < 0.05, **p < 0.01, ***p < 0.001. (B) Cell growth of R-WT and R-3A2A mESCs were measured by sulforhodamine B dye (SRB assay). Data are presented as relative to Day 1. ***p < 0.001. (C) MeRIP-qPCR of pluripotency transcripts in mESCs stable transfectants. *p < 0.05, **p < 0.01. (D) qPCR analysis of pluripotency genes in mESCs stable transfectants. *p < 0.05, **p < 0.01. (E) qPCR analysis for pluripotency and differentiation markers expression after 8 days of embryonic body induction. *p < 0.05, **p < 0.01, ***p < 0.001. Unless otherwise indicated, all data in this figure contain n = 3 replicates per group, and represent mean ± SEM. All p-values were also calculated by Student’s t test. See also Figure S5.

Figure 5.. Transcripts Affected by Phosphorylation of Methyltransferase Complex in mESCs

(A) Cumulative distribution function of log2 peak intensity of m⁶A-modified sites in R-WT and R-3A2A mESCs. (B) Volcano plot for peaks with differential m⁶A intensity between R-WT and R-3A2A mESCs. Fold change (FC) is the ratio of IP over Input for R-WT and R-3A2A. (C) Coverage plots of the m⁶A peaks of Nanog, Lefty1, and Zfp219 comparing R-WT and R-3A2A mESCs. Plotted coverages are the medians of three replicates. (D) Gene enrichment analysis with WikiPathway terms of differentially m⁶A methylated peaks in R-WT and R-3A2A mESCs for molecular functions. (E) Gene enrichment analysis with WikiPathway terms of differentially expressed genes (p < 0.05). (F) A histogram showing relative m⁶A peak enrichment of R-3A2A compared to R-WT mESCs, indicating higher m⁶A methylation in pluripotency genes (PluriNetwork) for R-WT mESCs. See also Figure S6, Table S3, Table S4, and Table S5.

Figure 6.. Phosphorylation of the m⁶A Methyltransferase Complex May Affect Tumorigenesis

(A) Lysates of A375 stable transfectants harvested at different time points after treatment with cycloheximide (CHX) 10 μg/ml were analyzed by IB. (B) LC-MS/MS quantification of the m⁶A/A ratio in mRNA of A375 stable transfectants. **p < 0.01. (C) After 8 hr treatment with 10 μM PD0325901 or 0.1 μM trametinib, lysates from A375 cells were analyzed by IB. (D) LC-MS/MS quantification of the m⁶A/A ratio in mRNA of A375 cells treated with 10 μM PD0325901 or 0.1 μM trametinib for 48 hr. **p < 0.01, ***p < 0.001. (E) After 8 hr treatment with 10 μM EOAI3402143 (EOAI) or 30 μM vialinin A, cell lysates from A375 cells were analyzed by immunoblot. (F) A375 stable transfectants as indicated were treated with 3 μM EOAI3402143 (EOAI) or 10 μM vialinin A before measuring cell viability by SRB assay. Data are presented as relative to the R-WT cells without drug treatment. *p < 0.05, **p < 0.01, ***p < 0.001. (G) Immunofluorescence analysis of METTL3 (green) in SKBR3 cells treated with 1 μM tucatinib and 1 μM lapatinib for 8 hr. DAPI (blue) was used to mark the nucleus. Scale bars, 10 μm. (H) LC-MS/MS quantification of the m⁶A/A ratio in mRNA of SKBR3 and BT474 cells treated with 1 μM tucatinib and 1 μM lapatinib for 48 hr. *p < 0.05, **p < 0.01, ***p < 0.001. Unless otherwise indicated, all data with error bars in this figure represent n = 3 replicates per group, and represent mean ± SEM. All p-values were also calculated by Student’s t test. See also Figure S7.

Figure 7.

A Schematic Model of the Role of the m⁶A Methyltransferase Phosphorylation by ERK.