Fig. 5. Identification of YTHDF2 targets via m⁶A MeRIP-seq, eCLIP-seq, and RNA-seq in LFS astrocytes.

a m⁶A MeRIP-seq indicates distribution of m⁶A peaks in different regions (5’UTR, first exon, other exon, and 3’UTR) of transcripts. Pie chart shows the percentage of m⁶A peaks within distinct regions of transcripts in LFS astrocytes. b Violin plot demonstrates significant elevation of highly m⁶A-marked transcripts upon YTHDF2 depletion. c Examination of YTHDF2 IP enrichment by eCLIP-seq. d Metagene plot of YTHDF2 eCLIP-seq indicates enrichment of YTHDF2-interacting mRNA peaks in the 3’UTR clustered around stop codons. e Motif analysis demonstrates that YTHDF2 binding motifs are similar to the consensus m⁶A motif RRACH. f Venn diagram identifying 84 YTHDF2-targeted m⁶A transcripts validated by a combination of m⁶A MeRIP-seq, YTHDF2 eCLIP-seq, and RNA-seq in LFS astrocytes. These transcripts include CDKN2B and SPOCK2 mRNAs. g IGV track views of m⁶A peaks located on CDKN2B and SPOCK2 transcripts in LFS astrocytes. h RT-qPCR indicates decreased expression of YTHDF2-targeted CDKN2B and SPOCK2 transcripts in LFS astrocytes (n = 3 biologically independent samples). i Low expression of YTHDF2-targeted CDKN2B and SPOCK2 is correlated with poor overall survival of LGG/GBM patients. Log-rank (Mantel–Cox) test is performed to compute significance. j Immunostaining demonstrates lower CDKN2B in engrafted LFS cerebral organoids (upper panel) and increased CDKN2B in YTHDF2-depleted engrafted LFS cerebral organoids (lower panel), Scale bar, 100 µm. Anti-CDKN2B antibodies only recognize human but not mouse CDKN2B proteins. k mRNA stability assay demonstrates that YTHDF2 knockdown leads to an increased half-life of CDKN2B and SPOCK2 mRNAs. shCtrl-LFS and shYTHDF2-LFS astrocytes are treated with actinomycin D and total RNAs are isolated at 0, 30, and 60 min. (n = 3 biologically independent samples). l RT-qPCR demonstrates elevated expression of YTHDF2 targets CDKN2B and SPOCK2 upon depletion of p53, YTHDF2, or SVIL as well as inhibition of MLL1 function by OICR-9429 or MI-2-2 in LFS astrocytes (n = 3 biologically independent samples). The results are representative of at least three independent experiments (c, j). The data are presented as the mean ± SEM); two-way ANOVA with Bonferroni’s multiple comparison test (l); unpaired two-tailed Student’s t test (h); multiple t test (k); **P < 0.01, ***P < 0.001. Source data and exact P values are provided in the Source Data file.