Fig 2: YTHDF2 downregulates LXRA and HIVEP2 through m6A-dependent mRNA decay.a Western blotting of LXRa and HIVEP2 in GSC11 cells stably transfected with control shRNA or two YTHDF2 shRNAs. b Western blotting of LXRa and HIVEP2 in LN229/EGFRvIII tet-on cells cultured with vehicle or Doxycycline and transfected with control siRNA or two individual YTHDF2 siRNAs. c, d MeRIP-seq of GSC11 cells shows m6A peaks at individual mRNAs of LXRA and HIVEP2. The y-axis shows normalized reads distribution for input (blue) and m6A IP (red) along the transcripts. The schematic representation of mRNA of LXRA or HIVEP2 (top panel). e, f Lifetime of LXRA (e) and HIVEP2 (f) mRNA in GSC11 cells expressing shYTHDF2 (shDF2), or shControl (shCtrl). Transcription was inhibited by actinomycin D. g CLIP-qPCR showing the association of LXRA and HIVEP2 transcripts with wild-type (WT) or m6A recognition defective (MUT) Flag-YTHDF2 in LN229 cells. Data are mean ± S.E.M., n = 4 biologically independent experiments. h mRNA levels of YTHDF2, LXRA, and HIVEP2 in LN229 cells transfected with vector, wild type (WT) or m6A recognition defective (MUT) YTHDF2. Data are mean ± S.E.M., n = 3 biologically independent experiments. i Western blotting of YTHDF2, LXRa, and HIVEP2 in LN229 cells transfected with vector, wild-type (WT) or recognition defective (MUT) YTHDF2. j, k Lifetime of LXRA (j) or HIVEP2 (k) mRNA in LN229 cells expressing wild-type (WT) or m6A recognition defective YTHDF2 (W432A/W486A/W491A, MUT). Transcription was inhibited by actinomycin D. l mRNA levels of LXRA and HIVEP2 in GSC11 cells treated with inhibitor of EGFR (Gefitinib), SRC (SU6656), or ERK1/2 (SCH772984). Data are mean ± S.E.M., n = 5 biologically independent experiments. m mRNA levels of LXRA and HIVEP2 in GSC11 cells with EGF intervention transfected with control or two individual YTHDF2 siRNAs. Data are mean ± S.E.M., n = 6 biologically independent experiments. n mRNA levels of LXRA and HIVEP2 in LN229/EGFRvIII tet-on cells cultured with or without Dox, and transfected with control or two individual YTHDF2 siRNAs. Data are mean ± S.E.M., n = 6 biologically independent experiments. For a, b, and i, representative blots of three independent experiments are shown. For g, h, and l–n, data were analyzed by one-way ANOVA Tukey’s post hoc test. Source data are provided as a Source data file.

Fig 3: Identification of YTHDF2 targets by high-throughput RNA-seq and RIP-seq.a Bland-Altman plot of YTHDF2 RIP-seq in GSC11 cells. For each transcript, the average signal (measured as log2 normalized reads counts) against the RIP-seq log2 fold enrichment (RIP versus INPUT) was plotted. Significantly enriched targets are highlighted in red. b Volcano plot of RNA-seq results in siControl and siYTHDF2 GSC11 cells. Red dots indicate fold change >1.5 and p value <0.05. c Heatmap of expression and enrichment of 19 overlapped genes in RNA-seq, RIP-seq, and TCGA datasets results. d Schematic workflow of YTHDF2 downstream targets analysis. e, f Plots showing YTHDF2 binding to individual mRNAs of LXRA (e) and HIVEP2 (f) genes in GSC11 cells, as measured by RIP-seq. The normalized reads distribution for input (blue) and YTHDF2 RIP along mRNAs (red). g CLIP-qPCR showing the association of LXRA and HIVEP2 transcripts with YTHDF2 in GSC11 cells. Data are mean ± S.E.M., n = 4 biologically independent experiments (unpaired two-sided t test). h Analysis of mRNA levels of YTHDF2, LXRA, HIVEP2, and LXRB in GSC11 cells stably expressing control or YTHDF2 shRNA, using qPCR. Data are mean ± S.E.M., n = 3 biologically independent experiments (unpaired two-sided t test). i Analysis of mRNA levels of LXRA, HIVEP2, and LXRB in LN229 cells transfected with vector or YTHDF2 expression constructs, using qPCR. Data are mean ± S.E.M., n = 5 biologically independent experiments (unpaired two-sided t test). j Kaplan–Meier overall survival plot showing survival rates for lower-grade glioma and GBM patients having both low LXRA expression (HIVEP2 expression (LXRA expression (>median) and high HIVEP2 expression (>median) (red) in the TCGA dataset (two-sided log-rank test). k Percentage of low LXRA/HIVEP2 expression and high LXRA/HIVEP2 expression in YTHDF2 low expression and high expression group. Chi-squared test, ?2 = 69.63, p < 0.0001. Source data are provided as a Source data file.