Fig 1: YTHDC2 suppressed cystine uptake dependent on SLC7A11. (A, B) SLC7A11 protein and mRNA expression in H1299 control cells, YTHDC2WT and YTHDC2ΔYTH overexpression cells, as determined by IB and RT-qPCR assay. (C, D) IHC and RT-qPCR assay showed Slc7a11 expression in KPE, KPYWT and KPYΔYTH tumor tissues (n = 30 tumors from 8 mice per group), scar bar 50 μm. (E–G) SLC7A11 protein expression, mRNA level and cystine uptake were measured in H1299 control cells, YTHDC2WT overexpression cells with or without simultaneously overexpressed SLC7A11, as detected by IB, RT-qPCR and L-14C-cystine (0.2 μCi/mL). (H–J) SLC7A11 protein expression, mRNA level and cystine uptake were measured in H1299 control cells, ATF4 overexpression cells with or without simultaneously overexpressed YTHDC2WT, as detected by IB, RT-qPCR and L-14C-cystine (0.2 μCi/mL). (K–M) Representative IHC images of YTHDC2 and SLC7A11 expression from two paired LUAD cases in corhort#1 TMA (K), scar bar 200 μm. Proteins were quantified by IHC scores (L). And the correlation between YTHDC2 protein and SLC7A11 protein level in cohort#1 (pearson analysis, p = 0.0002) (M). Statistical analysis was performed using one-way ANOVA (B, D, F, G, I, J) Chi-squared test (L). Data are means ± SEMs, **p < 0.01, N·S.: no significant.
Fig 2: YTHDC2 preferred to interact and destabilize m6A methylated SLC7A11 mRNA. (A) Predominant consensus GGAC motif was showed in H1975 cells with or without METTL3 knocked down from MeRIP-seq. (B) Density distribution of m6A peaks across mRNA transcripts, the percentages of m6A peaks were determined. (C) MeRIP-qPCR analysis of m6A levels at the indicated sites within SLC7A11 mRNA in control cells and H1975 cells with METTL3 knockdown, and H1299 cells with METTL3 overexpressed. (D) PAR-CLIP assay of RNA pulled down by HA-tagged YTHDC2WT or YTHDC2ΔYTH in H1299 cells with or without overexpressing METTL3. RNA labeled with biotin at 3′ end was visualized by the chemiluminescent nucleic acid detection module. SLC7A11 mRNA levels in the pulled down products were also verified by RT-qPCR. (E) In vitro partial SLC7A11 3′UTR probe pulldown assay in H1299 control cells, HA tagged YTHDC2WT and YTHDC2ΔYTH overexpression cells. GGYC (Y = m6A or A), CCYG (Y = m6A or A). (F, G) RIP-qPCR analysis showing the binding between YTHDC2 and SLC7A11 mRNA in H1975 and H1299 cells under indicated treatment. (H–J) Schematic generation strategy for the pmir-Glo luciferase reporters containing WT and Mut (GGAC to GGCC) SLC7A11 3′-UTR. Luciferase activities from the indicated pmir-Glo vector were measured in H1299 and H1975 cells with or without YTHDC2 overexpression or knockout. (K) Schematic presentation of the primer sets designed to detect WT or Mut F-Luc-SLC7A11 fusion mRNA. (L, M) The decay curve of exogenous F-Luc-SLC7A11 fusion mRNA in indicated groups. The F-Luc-SLC7A11 fusion mRNA level were normalized to that of internal Renilla luciferase mRNA. Statistical analysis was performed using Student's t tests (C, D, F, G), one-way ANOVA (I, J) and two-way ANOVA (L, M). Data are means ± SEMs, **p < 0.01, N·S.: no significant.
Fig 3: Model of the study. The diagram reveals an m6A-dependent YTHDC2 regulation of SLC7A11 mRNA to modulate cystine uptake in LUAD cells. In our model, YTHDC2 destabilizes SLC7A11 mRNA via its m6A-reading YTH domain. METTL3-mediated m6A methylation of SLC7A11 mRNA at its 3′UTR is prerequisite for this process. YTHDC2 is frequently suppressed in LUAD; thereby its anti-tumor activity to impair cystine uptake and downstream antioxidant program is blocked. This model may explain a possible mechanism underlying tumorigenesis in LUAD.
Fig 4: YTHDC2 suppressed cystine uptake and downstream antioxidant program in LUAD. (A, B) Statistics of top 20 enriched KEGG pathways, and alterations in metabolites involved in GSH metabolism, as measured by metabolomics in YTHDC2low (n = 20) and YTHDC2high (n = 20) LUAD tissues. (C) Correlation between intracellular cystine level and YTHDC2 protein in LUAD tissues from cohort #1 (n = 100, pearson analysis, p = 0.001). (D–F) Cystine uptake in xenografts (n = 8 per group) generated by H1299 cells with YTHDC2WT or YTHDC2ΔYTH overexpression, H1975 cells with or without YTHDC2 knockout and reconstitution, and tumors from KPE/KPYWT/KPYΔYTH mice (n = 30 tumors from 8 mice per group), as detected by L-14C-cystine (0.2 μCi/mL). (G) Schematic presentation of antioxidant program from cystine uptake to lipid peroxidation. (H–J) Schematic illustration of drug treatment strategy. Representative H&E staining of lungs bearing tumors (black arrows indicate tumors), quantification of tumors and relative GSH/GSSG ratio in KPE and KPYWT mice (n = 8 per group) administrated with vehicle, NAC (100 mg/kg/day) or GSH (100 mg/kg/day) for 4 weeks. (K, L) 4-HNE, MDA (n = 30 tumors from 8 mice per group), and survival curves in KPYWT mice (n = 8 per group) administrated with vehicle, NAC (100 mg/kg/day) or GSH (100 mg/kg/day), as determined by IHC, lipid peroxidation assays, and the Kaplan-Meier method (log-rank tests, vehicle versus NAC: p = 0.0027, vehicle versus GSH: p = 0.009), scar bar 50 μm. Statistical analysis was performed using one-way ANOVA (D-F, I-K). Data are means ± SEMs, **p < 0.01, N·S.: no significant.
Fig 5: RBM46 interacts with RNA close to YTHDC2-binding sites.(A) RBM46 and YTHDC2 exhibit a similar distribution pattern along the length of mRNA. (B) RBM46 eCLIP mRNA targets substantially overlapped with YTHDC2 mRNA targets by CLIP-seq in P10 testis. In total, 53 and 52% of P10 YTHDC2–binding sites were co-occupied by RBM46 within a 100- and 70-nt window, respectively. (C) Top motif at best-scored YTHDC-binding sites from P10 and adult YTHDC2 CLIP-seq using HOMER. Sequences within ±100 nt relative to YTHDC2-binding sites were used for de novo motif analysis. (D) RBM46-binding motif AAUCAUGU is the top motif within a 100-nt window centered on YTHDC2 U-rich motifs in both P10 and adult testis. Motif analysis by HOMER was performed with best-scored P10 and adult YTHDC2 CLIP targets. (E) Position and distance between RBM46 and YTHDC2 binding on their shared targets. (F) Schematic illustrations for the function of RBM46-MEIOC-YTHDC2 complex during spermatogenesis. RNA binding protein RBM46 forms an ancient posttranscriptional network with MEIOC and YTHDC2 to recognize and destabilize mitotic transcripts for a successful meiotic entry.
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