Skip to main content
NCBI home page
Fundamental Research logoLink to Fundamental Research
. 2024 Jul 26;5(5):2388–2396. doi: 10.1016/j.fmre.2024.07.003

O-GlcNAcylation of YTHDF2 antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma

Jie Li a,1, Wen Zhou b,1, Jianzhi Zhang c,1, Li Ma a,1, Zhuan Lv a, Yiqun Geng d,⁎, Xing Chen b,⁎, Jing Li a,⁎
PMCID: PMC12848141  PMID: 41613430

Abstract

The intracellular O-linked N-acetylglucosamine (O-GlcNAc) glycosylation mediates many signal transduction events and regulates tumorigenesis. Previously the RNA N6-methyladenosine (m6A) reader, YTH (YT521-B homology) domain 2 (YTHDF2), has been shown to be O-GlcNAcylated on Ser-263 during Hepatitis B virus (HBV) infection and promote HBV-related hepatocellular carcinoma. Herein we mapped YTHDF2 O-GlcNAcylation at Thr-49 via electron-transfer dissociation mass spectrometry under unperturbed conditions. We show that YTHDF2 Thr-49 O-GlcNAcylation antagonizes Extracellular-signal regulated kinase (ERK)-dependent phosphorylation at Ser-39 and promotes YTHDF2 degradation. The downstream signaling pathway of YTHDF2 in lung carcinoma is thus upregulated, which leads to the downregulation of c-Myc. We further used mouse xenograft models to show that YTHDF2-T49A mutants increased lung cancer mass and size. Our work reveals a key role of YTHDF2 O-GlcNAcylation in tumorigenesis and suggests that O-GlcNAcylation exerts distinct functions under different biological stress.

Key words: O-linked N-acetylglucosamine, RNA N6-methyladenosine, c-Myc, Extracellular-signal regulated kinase, Lung carcinoma

Graphical abstract

Image, graphical abstract

Open in a new tab

1. Introduction

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification (PTM) that involves decorating the Ser/Thr residues with the O-GlcNAc moiety [1,2]. It is catalyzed by the O-GlcNAc transferase (OGT) and removed by the O-GlcNAcase (OGA). Decades of glycobiology research have witnessed emerging chemical and biological tools [3,4] that have demonstrated that O-GlcNAc occurs on about 5000 proteins and regulates tumorigenesis and immunotherapy [5], ferroptosis [6], protein aggregation and phase separation [7], stem cell biology [8], liver metabolism [9], neurodevelopment and various stress response pathways [10].

RNA N6-methyladenosine (m6A) is one of the ∼140 types of modifications in RNA, especially in mRNAs and lncRNAs [11]. It is the most abundant internal modifications that have much therapeutic potential [12,13]. The regulation of m6A is intricate, which includes its writers, readers and erasers [14]. Among the readers, the YTH (YT521-B homology) family proteins have caught much attention [15], because not only they all contain the conserved YTH domain that binds m6A, but also they are subject to many PTMs that finetune their biological functions under physiology and pathology conditions.

Our lab has been intensively studying the relationship between O-GlcNAc and m6A readers, including YT521-B homology (YTH) domain 1 (YTHDF1) and YTH domain-containing protein 1 (YTHDC1). We have shown that O-GlcNAcylation of YTHDF1 promotes its cytosolic localization and enhances downstream target expression [16]. We also demonstrated that DNA damage induces O-GlcNAcylation on YTHDC1, which promotes its binding with m6A and subsequent homologous recombination [17]. In this work we focused on YTH domain 2 (YTHDF2) and aimed to elucidate the role of O-GlcNAc in regulating YTHDF2.

YTHDF2 is a reader that regulates m6A mRNA decay [18]. Among its various roles in human diseases, it has a particular role in tumor [19,20]. In acute myeloid leukemia, YTHDF2 is often overexpressed and decreases the stability of many m6A transcripts, whose function is required for leukemic stem cell function [21]. In multiple myeloma, YTHDF2 overexpression correlates with poor prognosis, and the underlying mechanism is that YTHDF2 inhibits MAP2K2/p-ERK [22]. In lung adenocarcinoma, YTHDF2 positively regulates tumorigenesis by promoting the mRNA decay of AXIN1, which negatively regulates the Wnt/β-catenin pathway [23]. Recently YTHDF2 inhibition has also been shown to improve radiotherapy efficacy [24].

Due to its pivotal role in tumorigenesis, YTHDF2 is subject to many PTMs. YTHDF2 protein stability is dependent on cyclin dependent kinase (CDK1), although a direct phosphorylation reaction is yet to be revealed [25]. YTHDF2 is phosphorylated by the EGFR/SRC/ERK signaling pathway at Ser-39 and Thr-381 and thus stabilized and promotes invasive glioblastoma [26]. Hypoxia induces small ubiquitin-like modifier (SUMO) modification of YTHDF2 at Lys-571, which increases its binding with m6A and results in cancer [27]. Upon hepatitis B virus (HBV) infection, YTHDF2 is O-GlcNAcylated at Ser-263 to enhance its stability, and downstream target expression (including minichromosome maintenance protein 2 and 5) and HBV-related hepatocellular carcinoma (HCC) [28]. But a recent report found no O-GlcNAcylation sites on YTHDF2 [29]. Moreover, YTHDF2 is ubiquitinated by STIP1 homology and U-box-containing protein 1 (STUB1) to inhibit Sorafenib resistance in HCC [30].

In this paper, we found that YTHDF2 is O-GlcNAcylated during unperturbed conditions. Using chymotrypsin for enzyme digestion followed by electron-transfer dissociation (ETD) mass spectrometry (MS), we identified Thr-49 as the O-GlcNAcylation site. Thr-49 O-GlcNAcylation antagonizes ERK-dependent phosphorylation at Ser-39, probably due to its close proximity. We show that YTHDF2 O-GlcNAcylation decreases its abundance during basal conditions. In lung carcinoma cells YTHDF2 O-GlcNAcylation downregulates c-Myc, possibly through the AXIN1/Wnt/β-catenin pathway, thus inhibiting tumor progression in mouse xenograft models. Our work reveals that O-GlcNAcylation could occur on distinct sites in different biological settings, and YTHDF2 O-GlcNAcylation provides new therapeutic potential for lung carcinoma.

2. Experimental procedures

2.1. Cell culture

HEK-293T cells were purchased from ATCC and cultured by DMEM (CellMax, CGM114.05) with 10% Fetal Bovine Serum (CellMax, SA301.02 V). H1299 and A549 cells were purchased from ATCC and cultured by RPMI 1640 Medium (Gibco, C11875500BT) with 10% Fetal Bovine Serum (CellMax, SA301.02 V) and 100 U/ml penicillin/streptomycin (Gibco, 15140122). Cell lines were validated using STR profiling and free from mycoplasma contamination for all experiments.

2.2. Immunoprecipitation (IP) and immunoblotting assays

Immunoprecipitation and immunoblotting experiments were performed as described before [31]. The following primary antibodies were used for immunoblot: anti-Myc (PTM Bio, #PTM-5390, 1:3000), anti-c-Myc (ProteinTech, #10828-1-AP, 1:1000), anti-YTHDF2 (proteinTech, #24744-1-AP, 1:1000), anti-Flag (Sigma, #F1084, 1:1000), RL2 (Abcam, #AB2739, 1:1000), anti-GST (Gene Script, #A00865, 1:1000), anti-HA (Bethyl, #A190–108A, 1:1000), anti-OGT (Santa Cruz Biotechnology, #sc-74546, 1:1000), anti-β-actin (Sigma, #A5441, 1:1000), anti-Ubiquitin (PTM Bio, #PTM-1106RM, 1:1000), anti-AXIN (ProteinTech, #16541–1-AP, 1:1000) and anti-YTHDF2-pS39 antibodies were generated using the sequence PYLpSPQAR by Dia-An Biotech, Inc. Peroxidase-conjugated secondary antibodies were from JacksonImmuno Research. The ECL detection system (Amersham) was used for immunoblotting. LAS-4000 was employed to detect signals, and signals were quantitated using Multi Gauge software (Fujifilm). All western blots were repeated at least three times.

2.3. Ubiquitination assays

HEK-293T cells were transfected with plasmids and treated with the proteasome inhibitor MG132 (10 µM) for 12 h. Transfected YTHDF2, and its binding proteins were pulled down using the Co-IP assay, and ubiquitination levels were detected using anti-ubiquitin antibodies.

2.4. IP-phosphatase assay

HEK-293T cells were transfected with HA-YTHDF2 plasmids. The anti-HA immunoprecipitates were subject to λ phosphatase (PPase) treatment or left untreated. Incubation in a water bath at 30 °C for 1 h, and then immunoblotted with the antibodies indicated. Lambda PPase was added according to the kit procedure (Lambda PP, NEB #P0753L).

2.5. Mass spectrometry

2.5.1. Sample preparation

The gel band pieces were dehydrated in acetonitrile, incubated in 10 mM DTT in 50 mM ammonium bicarbonate at 56 °C for 40 min, incubated in 55 mM iodoacetamide in 50 mM ammonium bicarbonate at ambient temperature for 1 hr in the dark, and dehydrated again. Then the gel pieces were digested in-gel with 2 ng/µL sequencing grade chymotrypsin in 50 mM ammonium bicarbonate overnight at 37 °C. The resulting peptides were extracted twice with 5% formic acid/50% acetonitrile, then vacuum-centrifuged to dryness. All samples were resuspended in 0.1% FA in water prior to LC-MS/MS analysis.

2.5.2. LC-MS/MS parameters

Peptides were separated using a loading column (100 µm × 2 cm) and a C18 separating capillary column (75 µm × 15 cm) packed in-house with Luna 3 µm C18(2) bulk packing material (Phenomenex, USA). The mobile phases (A: water with 0.1% formic acid and B: 80% acetonitrile with 0.1% formic acid) were driven and controlled by a Dionex Ultimate 3000 RPLC nano system (Thermo Fisher Scientific). The LC gradient was held at 2% for the first 8 min of the analysis, followed by an increase from 2% to 44% B in 60 min, and an increase from 44% to 99% B in 5 min.

For the samples analyzed by Orbitrap Fusion LUMOS Tribrid Mass Spectrometer, the precursors were ionized using an EASY-Spray ionization source (Thermo Fisher Scientific) source held at +2.0 kV compared to ground, and the inlet capillary temperature was held at 320 °C. Survey scans of peptide precursors were collected in the Orbitrap from 350 to 1800 Th with an AGC target of 400,000, a maximum injection time of 50 ms, RF lens at 30%, and a resolution of 120,000 at 200 m/z. Monoisotopic precursor selection was enabled for peptide isotopic distributions, precursors of z = 2–8 were selected for data-dependent MS/MS scans for 3 s of cycle time, and dynamic exclusion was set to 15 s with a ± 10 ppm window set around the precursor monoisotope.

In EThcD scans, an automated scan range determination was enabled. An isolation window of 2 Th was used to select precursor ions with the quadrupole. An SA collision energy of 35%, an AGC target value of 50,000, a maximum injection time of 54 ms, and a resolution of 30,000 at 200 m/z were selected.

2.5.3. Data analysis

Data processing was carried out using Thermo Proteome Discoverer 2.4 using a SwissProt homo sapiens database (https://www.expasy.org/) (TaxID = 9606 and subtaxonomy, 42253 protein sequences). Carbamidomethyl (Cys) were chosen as static modification, oxidation (Met) and HexNAc (Ser or Thr) were chosen as variable modification. Mass tolerance was 10 ppm for precursor ions and 0.02 Da for fragment ions. Maximum missed cleavages were set as 2. Peptide spectral matches (PSM) were validated using the Percolator algorithm, based on q-values at a 1% FDR at both peptide and protein level.

2.6. Colony formation assay

H1299 and A549 cells stably expressing HA-YTHDF2-WT or -T49A plasmids were seeded in 10D plates (800 cells in each plate). After being cultured for 14 days, the cells were stained by Crystal violet. The plates were photographed and the cells were counted. All experiments were repeated three times and quantified.

2.7. Cell proliferation assay

H1299 and A549 cells stably expressing HA-YTHDF2-WT or -T49A plasmids were cultured in six-well plates for proliferation assay. Cell numbers were measured every 24 h and continuously measured for 72 h. All experiments were repeated three times and quantified.

2.8. Wound healing assay

H1299 and A549 cells stably expressing HA-YTHDF2-WT or -T49A plasmids were cultured in a six-well plate and then the cells were scratched with a 200 µl pipette tip when the cells almost 100% cover the well. Wound areas were photographed at 0 h and 48 h using a microscope. All experiments were repeated three times and quantified by ImageJ (ImageJ, RRID:SCR_003070). Cellular migration rates were quantified by calculating the percentage of area reduction.

2.9. Transwell assay

H1299 and A549 cells stably expressing HA-YTHDF2-WT or -T49A plasmids were cultured in six-well plates and starved for 24 h by using serum-free DMEM (CellMax, CGM114.05). 3 × 103 cells were seeded into Transwell upper chamber (8-µm pore size; Millipore) with 50 µL serum-free DMEM, and the lower chamber was added with 200 µL DMEM with 10% FBS (CellMax, SA301.02 V). Cells on the filter side of the chamber were stained by 0.1% Crystal violet at room temperature for 10–20 min after 24 h. All experiments were repeated three times.

2.10. Mouse xenograft analysis

For xenograft assays, 2 × 105 HA-YTHDF2-WT or -T49A cells were resuspended in Matrigel (Corning) and then injected into the right abdomen of nude mice at the age of 6 weeks old. The tumor volumes and weight were measured every 3 days and continuously measured from the 7th day to the 25th day. The mice were randomly selected to be healthy, including both males and females. The mice were obtained from the Beijing SPF Biotechnology Co, Ltd (Certification NO. SCXK (Jing) 2019-0010). All animal work procedures were approved by the Animal Care Committee of Capital Normal University (Beijing, China), Certification NO. K2023-0001.

3. Results

3.1. YTHDF2 is O-GlcNAcylated at Thr-49 under unperturbed conditions

To examine if YTHDF2 is O-GlcNAcylated under basal conditions, we first tested the interaction between YTHDF2 and OGT. Cell lysates were subject to immunoprecipitation with anti-OGT antibodies, and then immunoblotted (IBed) with anti-YTHDF2 and anti-OGT antibodies (Fig. 1a). The results showed that endogenous YTHDF2 co-immunoprecipitates (coIPs) with OGT. Then cells were transfected with Myc-OGT and HA-YTHDF2 plasmids to examine the interaction between exogenously expressed proteins. Again HA-YTHDF2 coIPs with Myc-OGT (Fig. 1b). Then GST-pulldown experiments were carried out. Cells were transfected with HA-YTHDF2, and the cellular lysates were incubated with recombinant GST-OGT proteins. As shown in Fig. 1c, GST-OGT pulled-down HA-YTHDF2, suggesting the physical association between OGT and YTHDF2 without viral invasion.

Fig. 1.

Fig 1

Open in a new tab

YTHDF2 is O-GlcNAcylated at Thr-49. (a) 293T cell lysates were immunoprecipitated with anti-OGT antibodies and immunoblotted with anti-YTHDF2 and anti-OGT antibodies. (b) Cells were transfected with HA-YTHDF2 and Myc-OGT plasmids. The cell lysates were subject to immunoprecipitation and immunoblotting with the antibodies indicated. (c) Cells were transfected with HA-YTHDF2 or vector controls and the cellular lysates were incubated with recombinant GST-OGT proteins purified from E. coli. (d) Cells were treated or untreated with the OGA inhibitor Thiamet-G (TMG) and glucose (Glu) to enrich for O-GlcNAcylation. Then the cell lysates were immunoprecipitated with anti-YTHDF2 antibodies and immunoblotted with anti-O-GlcNAc RL2 antibodies. (e) Cells were transfected with HA-YTHDF2-WT or -T49A plasmids, treated or untreated with TMG and Glu to enrich for O-GlcNAcylation. Then the cell lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-O-GlcNAc RL2 antibodies. (f) Electron Transfer Dissociation (ETD) mass spectrometry identified that Thr-49 is O-GlcNAcylated. (g) Thr-49 is evolutionarily conserved among different species. All Western blots were repeated for at least three times.

We then assessed YTHDF2 O-GlcNAcylation. Cells were treated with Thiamet-G (TMG, OGA inhibitor) plus glucose to enrich for O-GlcNAcylation, as previously described [32]. As shown in Fig. 1d, endogenous YTHDF2 showed robust O-GlcNAc signaling, indicated by RL2 (O-GlcNAc antibody) staining. We also evaluated O-GlcNAcylation of over-produced YTHDF2 (Fig. 1e), and exogenous YTHDF2 is also O-GlcNAcylated. The immunoprecipitates were then subject to electron-transfer dissociation (ETD) mass spectrometry (MS) analysis, and a single O-GlcNAc site (Thr-49) was revealed (Fig. 1f), which was quite distinct from the previously identified Ser-263 under HBV infection. We generated T49A mutants accordingly, and the mutant almost abolished O-GlcNAc signals (Fig. 1e). We further tested YTHDF2 O-GlcNAcylation in H1299 lung cancer cells (Supplementary Fig. S1a), and the results showed that YTHDF2 is O-GlcNAcylated at Thr-49. Thr-49 is also conserved among different species (Fig. 1g). These results suggest that YTHDF2 is O-GlcNAcylated under basal conditions.

3.2. YTHDF2 O-GlcNAcylation antagonizes ERK-dependent phosphorylation

As YTHDF2 has been shown to be phosphorylated by ERK at Ser-39 [26], which is quite close to Thr-49, we wonder whether there is crosstalk between these two PTMs. Thus, we manufactured a phospho-specific antibody targeting p-S39. We first tested the specificity and efficiency of the phospho-antibody (Fig. 2a-b). Cells were transfected with YTHDF2-wild type (WT) or -S39A plasmids, and the p-S39 antibody did not show any signal in the YTHDF2-S39A extracts (Fig. 2a). Then YTHDF2-WT lysates were treated with phosphatase, which again abolished the p-S39 signal (Fig. 2b), suggesting that the antibody is specific.

Fig. 2.

Fig 2

Open in a new tab

O-GlcNAcylation of YTHDF2 at Thr-49 antagonizes ERK-dependent phosphorylation at Ser-39. (a-b) A rabbit anti-pS39 antibody was generated and its specificity was detected. 293T cells were transfected with HA-YTHDF2-WT or HA-YTHDF2-S39A, and the lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-pS39 antibodies (a). Cells were transfected with HA-YTHDF2 plasmids. The anti-HA immunoprecipitates were subject to λ phosphatase treatment or left untreated. They were then subjected to immunoblotting with anti-pS39 antibodies (b). (c-f) Cells were transfected with HA-YTHDF2-WT or HA-YTHDF2-S39A (c-d), or -T49A (e-f) plasmids, and then the lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with indicated antibodies. (g-h) Cells were transfected with HA-YTHDF2-WT plasmids, treated or untreated with TMG and Glu to enrich for O-GlcNAcylation. Then the cell lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-pS39 antibodies. (i-l) Cells were transfected with Myc-OGT together with HA-YTHDF2-WT or -S39A plasmids (i-j), or Flag-Erk together with HA-YTHDF2-WT and -T49A plasmids (k-l), and then the lysates were immunoprecipitated and immunoblotted with the antibodies indicated. Statistics analysis was carried out with Student's t-test. * indicates P < 0.05; ** indicates P < 0.01. All Western blots were repeated for at least three times.

We then assessed the potential crosstalk between Thr-49 O-GlcNAcylation and Ser-39 phosphorylation of YTHDF2. Cells were transfected with YTHDF2-WT or -S39A, and the S39A mutant showed a drastic increase of O-GlcNAc signals (Fig. 2c-d). Cells were also transfected with YTHDF2-WT or T49A, and the p-S39 antibody was used for IB. The T49A mutant increased the p-S39 signals markedly (Fig. 2e-f). On the contrary, the p-S39 signals were decreased when the cells were treated with TMG plus glucose (Fig. 2g-h). We further investigated whether the changes in modification levels were due to different binding affinity with the catalyzing enzymes. HA-YTHDF2-S39A increased binding with Myc-OGT (Fig. 2i-j) and HA-YTHDF2-T49A increased binding with ERK (Fig. 2k-l). These results suggest that O-GlcNAcylation and phosphorylation counteract each other.

As these results reveal a mechanistic antagonism between phosphorylation and O-GlcNAcylation, we also validated it in H1299 cells (Supplementary Fig. S1b-i). We found that in H1299 cells, the phospho-deficient S39A mutant also increased O-GlcNAcylation (Supplementary Fig. S1b-c), and the O-GlcNAc mutant T49A upregulated pS39 (Supplementary Fig. S1d-e). Mechanistically, S39A elevated binding with OGT (Supplementary Fig. S1f-g), and T49A shows more robust association with Erk (Supplementary Fig. S1h-i), suggesting the molecular mechanism also applies in lung cancer cells.

3.3. O-GlcNAcylation decreases YTHDF2 stability

As YTHDF2 Ser-39 phosphorylation plays a role in stabilizing YTHDF2 [26], we wondered whether Thr-49 O-GlcNAcylation would destabilize YTHDF2. Thus, we tested the ubiquitination levels of YTHDF2-WT and -T49A. As shown in Fig. 3a-b, T49A reduced the ubiquitination levels of YTHDF2. To exclude the possibility that this is caused by the mutation itself, we used the OGA plasmid to suppress cellular O-GlcNAcylation (Fig. 3c-d). Consistently, OGA overexpression decreased YTHDF2 ubiquitination levels. Cycloheximide (CHX) pulse-chase experiments were performed, and T49A again increased YTHDF2 stability (Fig. 3e-f). Taken together, O-GlcNAcylation at T49 destabilizes YTHDF2.

Fig. 3.

Fig 3

Open in a new tab

YTHDF2 O-GlcNAcylation promotes ubiquitination. (a) 293T cells were transfected with HA-YTHDF2-WT or -T49A plasmids and the lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-Ubiquitin antibodies. (b) Quantitation of (a). (c) 293T cells were transfected with HA-YTHDF2-WT together with vector or Flag-OGA plasmids and the lysates were immunoprecipitated with anti-HA antibodies and immunoblotted with anti-Ubiquitin antibodies. (d) Quantitation of (c). (e-f) cycloheximide (CHX) pulse-chase assays. HEK-293T cells were transfected with HA-YTHDF2-WT or -T49A plasmids, then treated with CHX for different durations (e). The quantitation is in (f). Student's t-test was used for statistical analysis in (b) and (d), two-way Anova was used for (f). * indicates P < 0.05. All Western blots were repeated for at least three times.

We further studied YTHDF2 stability in lung cancer cells (Fig. S1j-o). H1299 cells were used to study YTHDF2 ubiquitination and subsequent proteasome-mediated degradation. We found that T49A significantly decreased YTHDF2 ubiquitination levels (Fig. S1j-k). Consistently, OGA overproduction reduced YTHDF2 ubiquitination (Fig. S1l-m). CHX pulse-chase experiments correlated with the ubiquitination studies and showed that T49A is more stable (Fig. S1n-o). These data indicate that O-GlcNAc reduces YTHDF2 abundance in lung cancer cells.

3.4. YTHDF2 O-GlcNAcylation decreases c-Myc via the β-catenin pathway in lung carcinoma cells

As YTHDF2 has been demonstrated to exert its function in lung adenocarcinoma through the AXIN/Wnt/β-catenin pathway [23], we examined whether Thr-49 O-GlcNAcylation plays a role. We used both A549 and H1299 lung adenocarcinoma cells, and found that overexpression of T49A markedly increased c-Myc abundance, concomitantly with increased β-catenin levels and reduced AXIN1 protein abundance (Fig. 4a-d), suggesting that Thr-49 O-GlcNAcylation attenuates c-Myc in lung carcinoma.

Fig. 4.

Fig 4

Open in a new tab

O-GlcNAcylation of YTHDF2 downregulates c-Myc in H1299 and A549 lung cancer cells. (a) A549 cells were transfected with HA-YTHDF2-WT or -T49A plasmids and the lysates were immunoblotted with anti-β-catenin, anti-c-Myc and anti-AXIN1 antibodies. (b) Quantitation of (a). (c) A549 cells were transfected with HA-YTHDF2-WT or -T49A plasmids and the lysates were immunoblotted with anti-β-catenin, anti-c-Myc and anti-AXIN1 antibodies. (d) Quantitation of (d). Statistics analysis were carried out with one-way Anova. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. All Western blots were repeated for at least three times.

3.5. YTHDF2 O-GlcNAcylation downregulates invasion and migration of lung carcinoma cells

To test the effect of YTHDF2 O-GlcNAcylation in cells, we constructed stable transfectants of YTHDF2-WT and -T49A in both A549 and H1299 cells (Fig. 5a). Colony formation assays were carried out and T49A enhanced cellular growth and proliferation (Fig. 5b-d). H1299 and A549 cells stably expressing HA-YTHDF2-WT or -T49A plasmids were seeded. After being cultured for 14 days, the cells were stained by Crystal violet. Trypan blue live cell count assays were performed, and again T49A increased cell growth and proliferation (Fig. 5e-f). We also measured the metastatic capacity by Transwell invasion (Fig. 5g-i). In both cell lines, T49A upregulated cell migration capabilities. In wound healing assays (Fig. 5j-k), H1299 and A549 cells stably expressing HA-YTHDF2-WT or -T49A plasmids were cultured in a six-well plate and then the cells were scratched with a pipette tip when the cells almost 100% covered the well. The results (Fig. 5j-k) suggested that Thr-49 O-GlcNAcylation attenuates lung carcinoma metastasis.

Fig. 5.

Fig 5

Open in a new tab

O-GlcNAcylation of YTHDF2 regulates the metastatic capacity of H1299 and A549 lung cancer cells. (a) Stable cell lines were generated expressing HA-YTHDF2-WT or -T49A in H1299 and A549 cells. (b) Colony formation assays of cells in (a). (c-d) Quantitation of (b). (e-f) Cell growth assays of H1299 (e) and A549 (f) cells described in (a). (g) Transwell assays of H1299 and A549 cells described in (a). (h-i) Quantitation of (g). (j) Wound healing assay of H1299 and A549 cells described in (a). (k) Quantitation of (j). Statistics analysis were carried out with two-way Anova for (e-f), and one-way Anova for (c-d), (h-i) and (k). * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

3.6. YTHDF2 O-GlcNAcylation downregulates lung carcinoma in xenograft mouse models

We then used mouse xenograft models to study Thr-49 O-GlcNAcylation in vivo. Stable transfectants were injected into nude mice. After sacrificing the animals, we isolated the tumors (Fig. 6a-c), and T49A increased tumor volume and weight more than YTHDF2-WT. Taken together, these results suggest a model where O-GlcNAcylation at Thr-49 destabilizes YTHDF2, and suppresses lung cancer progression by downregulating c-Myc (Fig. 6d).

Fig. 6.

Fig 6

Open in a new tab

YTHDF2 O-GlcNAcylation inhibits lung cancer. (a-c) Xenografts in nude mice. HA-YTHDF2-WT and -T49A cells were injected into nude mice. Tumors were photographed after 25 days. Tumor images are in (a), tumor volumes are quantitated in (b) and tumor weights are quantitated in (c). Statistics analysis were carried out with two-way Anova for (b), and one-way Anova for (c). * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001. (d) Model depicting the role O-GlcNAcylation of YTHDF2: it antagonizes ERK-dependent phosphorylation and inhibits lung carcinoma.

4. Discussion and conclusion

In this work we found that YTHDF2 is O-GlcNAcylated at Thr-49 under basal conditions. Thr-49 O-GlcNAcylation antagonizes Ser-39 phosphorylation by ERK and promotes YTHDF2 ubiquitination and proteasome-mediated degradation. By downregulating c-Myc expression, YTHDF2 O-GlcNAcylation inhibits lung adenocarcinoma (Fig. 6d).

Our work is distinct from previous reports, where O-GlcNAcylation was found to stabilize YTHDF2 upon HBV infection and promote HBV-related HCC [28]. In our experience, O-GlcNAcylation usually carries out different functions under basal conditions versus viral invasion. For example, under unperturbed conditions UNC51-like kinase-1 (ULK1) is O-GlcNAcylated at Thr-635/Thr-754, which facilitates AMP-activated protein kinase (AMPK)-mediated ULK1 phosphorylation at Ser-555 and Ser-638, and promotes autophagy [33]. But upon Human papillomavirus (HPV) infection, ULK1 is O-GlcNAcylated at Ser-409/Ser-410, which antagonizes PKCα-mediated phosphorylation at Ser423 and stabilizes ULK1 by counteracting chaperone-mediated autophagy [34]. As a nutrient sensor that links stress response with metabolism, perhaps O-GlcNAc responds to virion particles by changing the modified sites and subsequent biological pathways.

Of the three m6A readers we investigated (YTHDF1, YTHDC1 and YTHDF2), O-GlcNAc plays distinct roles. YTHDF1 is a nuclear-cytoplasmic shuttling protein, whose nuclear import is inhibited by O-GlcNAcylation. By promoting downstream targets of YTHDF1, such as c-Myc, O-GlcNAc promotes colorectal cancer progression [16]. YTHDC1 O-GlcNAcylation is induced by DNA damage. Occurring right on the YTH domain, O-GlcNAc promotes YTHDC1-m6A binding and subsequent DNA damage repair by homologous recombination [17]. Here we show that YTHDF2 is also O-GlcNAcylated. By promoting YTHDF2 degradation, O-GlcNAcylated YTHDF2 inhibits lung carcinoma. It is interesting that YTHDF1 and YTHDF2 O-GlcNAcylation occur in their low-complexity domains, as often found for many PTMs. It is recently proposed that YTHDF1 and YTHDF2 behave differently in condensate formation [35], and YTHDF2 is not O-GlcNAcylated like YTHDF1 and YTHDF3 [29]. Our work identified that YTHDF2 is O-GlcNAcylated and suggests that different enzyme digest may render interesting biological results.

As small noncoding RNAs have recently been shown to be modified by sialylated glycans [36], more tools are being developed to enable the discovery of glycoRNAs [37]. We think that glycosylation is bound to be found in many RNA metabolism pathways.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [38] partner repository with the dataset identifier PXD045137 and 10.6019/PXD045137.

Declaration of competing interest

The authors declare that they have no conflicts of interest in this work.

Acknowledgements

Jing L. is supported by the National Natural Science Foundation of China (32271285) and R & D Program of Beijing Municipal Education Commission (KZ202210028043). Y. G. is supported by Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences (2021-RC350-002), CAMS Innovation Fund for Medical Sciences (2021-I2M-1-026).

Biographies

Jie Li is a doctoral student in the College of Life Sciences, Capital Normal University. During her doctoral studies, she focuses on the mechanistic studies of protein post-translational modifications. She was awarded the first-prize academic scholarship by Capital Normal University in 2020 and third-prize academic scholarship in 2021 and 2022, and the National Scholarship in 2023. She has published four papers as the first author.

Jing Li(BRID: 09606.00.06595) is a professor in the College of Life Sciences, Capital Normal University, Beijing, China. Dr. Li earned her B.S. degree from Peking University and Ph.D. from Yale University. Her research investigates the interlinked relationship among glycosylation, phosphorylation and ubiquitination in signal transduction. She has served as a guest editor for PLOS Genetics, and was awarded a third prize by the Fok Ying Tung Education Foundation. With more than 20 papers such as Nat. Comm., JCI Insight and JBC, she currently serves on the Glycobiology Committee of the Biophysical Society of China.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.fmre.2024.07.003.

Contributor Information

Yiqun Geng, Email: gengyiqun@imm.ac.cn.

Xing Chen, Email: xingchen@pku.edu.cn.

Jing Li, Email: jing_li@mail.cnu.edu.cn.

Appendix. Supplementary materials

mmc1.pdf (115.5KB, pdf)
mmc2.pdf (149.7KB, pdf)

References

  • 1.Hart G.W., Slawson C., Ramirez-Correa G., et al. Cross talk between O-GlcNAcylation and phosphorylation: Roles in signaling, transcription, and chronic disease. Annu. Rev. Biochem. 2011;80:825–858. doi: 10.1146/annurev-biochem-060608-102511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Yang X., Qian K. Protein O-GlcNAcylation: Emerging mechanisms and functions. Nat. Rev. Mol. Cell Biol. 2017;18:452–465. doi: 10.1038/nrm.2017.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Alteen M.G., Tan H.Y., Vocadlo D.J. Monitoring and modulating O-GlcNAcylation: Assays and inhibitors of O-GlcNAc processing enzymes. Curr. Opin. Struct. Biol. 2021;68:157–165. doi: 10.1016/j.sbi.2020.12.008. [DOI] [PubMed] [Google Scholar]
  • 4.Ma J., Wu C., Hart G.W. Analytical and biochemical perspectives of protein O-GlcNAcylation. Chem. Rev. 2021;121:1513–1581. doi: 10.1021/acs.chemrev.0c00884. [DOI] [PubMed] [Google Scholar]
  • 5.He X.F., Hu X., Wen G.J., et al. O-GlcNAcylation in cancer development and immunotherapy. Cancer Lett. 2023;566:216258. doi: 10.1016/j.canlet.2023.216258. [DOI] [PubMed] [Google Scholar]
  • 6.Zhang H., Zhang J., Dong H., et al. Emerging field: O-GlcNAcylation in ferroptosis. Front. Mol. Biosci. 2023;10:1203269. doi: 10.3389/fmolb.2023.1203269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Li X., Pinou L., Du Y., et al. Emerging roles of O-glycosylation in regulating protein aggregation, phase separation, and functions. Curr. Opin. Chem. Biol. 2023;75:102314. doi: 10.1016/j.cbpa.2023.102314. [DOI] [PubMed] [Google Scholar]
  • 8.Minh G.Le, Reginato M.J. Role of O-GlcNAcylation on cancer stem cells: Connecting nutrient sensing to cell plasticity. Adv. Cancer Res. 2023;157:195–228. doi: 10.1016/bs.acr.2022.06.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Xie Z., Xie T., Liu J., et al. Emerging role of protein O-GlcNAcylation in liver metabolism: Implications for diabetes and NAFLD. Int. J. Mol. Sci. 2023;24:2142. doi: 10.3390/ijms24032142. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Wenzel D.M., Olivier-Van Stichelen S. The O-GlcNAc cycling in neurodevelopment and associated diseases. Biochem. Soc. Trans. 2022;50:1693–1702. doi: 10.1042/BST20220539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Wei J., He C. Chromatin and transcriptional regulation by reversible RNA methylation. Curr. Opin. Cell Biol. 2021;70:109–115. doi: 10.1016/j.ceb.2020.11.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.He P.C., He C. m(6) A RNA methylation: From mechanisms to therapeutic potential. EMBO J. 2021;40 doi: 10.15252/embj.2020105977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Pomaville M.M., He C. Advances in targeting RNA modifications for anticancer therapy. Trends Cancer. 2023;9:528–542. doi: 10.1016/j.trecan.2023.04.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Shi H., Wei J., He C. Where, when, and how: Context-dependent functions of RNA methylation writers, readers, and erasers. Mol. Cell. 2019;74:640–650. doi: 10.1016/j.molcel.2019.04.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Sikorski V., Selberg S., Lalowski M., et al. The structure and function of YTHDF epitranscriptomic m(6)A readers. Trends Pharmacol. Sci. 2023;44:335–353. doi: 10.1016/j.tips.2023.03.004. [DOI] [PubMed] [Google Scholar]
  • 16.Li J., Ahmad M., Sang L., et al. O-GlcNAcylation promotes the cytosolic localization of the m(6)A reader YTHDF1 and colorectal cancer tumorigenesis. J. Biol. Chem. 2023;299 doi: 10.1016/j.jbc.2023.104738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Li M., Li J., Wang Y., et al. DNA damage-induced YTHDC1 O-GlcNAcylation promotes homologous recombination by enhancing m(6)A binding. Fundam. Res. 2023;5(2):868–879. doi: 10.1016/j.fmre.2023.04.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Wang X., Lu Z., Gomez A., et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505:117–120. doi: 10.1038/nature12730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Wang J.Y., Lu A.Q. The biological function of m6A reader YTHDF2 and its role in human disease. Cancer Cell Int. 2021;21:109. doi: 10.1186/s12935-021-01807-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Chen X., Zhou X., Wang X. m(6)A binding protein YTHDF2 in cancer. Exp. Hematol. Oncol. 2022;11:21. doi: 10.1186/s40164-022-00269-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Paris J., Morgan M., Campos J., et al. Targeting the RNA m(6)A reader YTHDF2 selectively compromises cancer stem cells in acute myeloid leukemia. Cell Stem Cell. 2019;25:137–148. doi: 10.1016/j.stem.2019.03.021. e136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Hua Z., Wei R., Guo M., et al. YTHDF2 promotes multiple myeloma cell proliferation via STAT5A/MAP2K2/p-ERK axis. Oncogene. 2022;41:1482–1491. doi: 10.1038/s41388-022-02191-3. [DOI] [PubMed] [Google Scholar]
  • 23.Li Y., Sheng H., Ma F., et al. RNA m(6)A reader YTHDF2 facilitates lung adenocarcinoma cell proliferation and metastasis by targeting the AXIN1/Wnt/beta-catenin signaling. Cell Death. Dis. 2021;12:479. doi: 10.1038/s41419-021-03763-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Wang L., Dou X., Chen S., et al. YTHDF2 inhibition potentiates radiotherapy antitumor efficacy. Cancer Cell. 2023;41:1294–1308. doi: 10.1016/j.ccell.2023.04.019. e1298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Fei Q., Zou Z., Roundtree I.A., et al. YTHDF2 promotes mitotic entry and is regulated by cell cycle mediators. PLoS Biol. 2020;18 doi: 10.1371/journal.pbio.3000664. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Fang R., Chen X., Zhang S., et al. EGFR/SRC/ERK-stabilized YTHDF2 promotes cholesterol dysregulation and invasive growth of glioblastoma. Nat. Commun. 2021;12:177. doi: 10.1038/s41467-020-20379-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Hou G., Zhao X., Li L., et al. SUMOylation of YTHDF2 promotes mRNA degradation and cancer progression by increasing its binding affinity with m6A-modified mRNAs. Nucleic Acids Res. 2021;49:2859–2877. doi: 10.1093/nar/gkab065. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Yang Y., Yan Y., Yin J., et al. O-GlcNAcylation of YTHDF2 promotes HBV-related hepatocellular carcinoma progression in an N(6)-methyladenosine-dependent manner. Signal Transduct. Target. Ther. 2023;8:63. doi: 10.1038/s41392-023-01316-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chen Y., Wan R., Zou Z., et al. O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins. Nat. Cell Biol. 2023;25:1676–1690. doi: 10.1038/s41556-023-01258-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Liao Y., Liu Y., Yu C., et al. HSP90beta impedes STUB1-induced ubiquitination of YTHDF2 to drive sorafenib resistance in hepatocellular carcinoma. Adv. Sci. 2023 doi: 10.1002/advs.202302025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Li J., Wang J., Hou W., et al. Phosphorylation of Ataxin-10 by polo-like kinase 1 is required for cytokinesis. Cell Cycle. 2011;10:2946–2958. doi: 10.4161/cc.10.17.15922. [DOI] [PubMed] [Google Scholar]
  • 32.Ramakrishnan P., Clark P.M., Mason D.E., et al. Activation of the transcriptional function of the NF-kappaB protein c-Rel by O-GlcNAc glycosylation. Sci. Signal. 2013;6:ra75. doi: 10.1126/scisignal.2004097. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ruan H.B., Ma Y., Torres S., et al. Calcium-dependent O-GlcNAc signaling drives liver autophagy in adaptation to starvation. Genes Dev. 2017;31:1655–1665. doi: 10.1101/gad.305441.117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Shi Y., Yan S., Shao G.C., et al. O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection. J. Biol. Chem. 2022;298 doi: 10.1016/j.jbc.2022.102341. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Zou Z., Sepich-Poore C., Zhou X., et al. The mechanism underlying redundant functions of the YTHDF proteins. Genome Biol. 2023;24:17. doi: 10.1186/s13059-023-02862-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Flynn R.A., Pedram K., Malaker S.A., et al. Small RNAs are modified with N-glycans and displayed on the surface of living cells. Cell. 2021;184:3109–3124. doi: 10.1016/j.cell.2021.04.023. e3122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Li J., Yue S., Gao Z., et al. Novel approach to enriching glycosylated RNAs: Specific capture of GlycoRNAs via solid-phase chemistry. Anal. Chem. 2023;95:11969–11977. doi: 10.1021/acs.analchem.3c01630. [DOI] [PubMed] [Google Scholar]
  • 38.Perez-Riverol Y., Bai J., Bandla C., et al. The PRIDE database resources in 2022: A hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 2022;50:D543–D552. doi: 10.1093/nar/gkab1038. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.pdf (115.5KB, pdf)
mmc2.pdf (149.7KB, pdf)

Data Availability Statement

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [38] partner repository with the dataset identifier PXD045137 and 10.6019/PXD045137.

Articles from Fundamental Research are provided here courtesy of The Science Foundation of China Publication Department, The National Natural Science Foundation of China

RESOURCES