FIGURE 3.

YTHDF2 depletion affects transcripts required for neural development. (A) Volcano plot generated from sequencing data showing the adjusted P-value (y-axis) plotted against the fold change (x-axis) for individual genes. Differentially expressed genes are shown in green (differential expression: read counts >5, Benjamini-Hochberg adjusted P-value <0.05, and log₂-fold change >1.0, represented by gray dotted lines). Neural-specific transcripts evaluated in subsequent figures are labeled. (B–E) Venn diagrams showing the overlap between transcripts up-regulated (B,D) or down-regulated (C,E) following YTHDF2 depletion and transcripts previously shown to be m⁶A methylated in hESCs (B,D) (Batista et al. 2014) or bound by YTHDF2 (C,E) (Wang et al. 2014a). P-values were determined using a hypergeometric test. (F) Boxplot of the log₂ fold change in expression (YTHDF2 KD/Control), taken from results generated by DESeq2, vs. number of m⁶A sites. Transcripts were binned based on the number of m⁶A sites identified in Batista et al. (2014). The central line of the box plot represents the median log₂ fold change expression value. n denotes the number of genes in each bin. Binned groups were compared using an unpaired two-sided Student's t-test ([****] P-value <0.0001). (G–I) Functional annotation clustering of biological process (G), cellular component (H), and tissue expression (I) performed by DAVID on genes up-regulated following YTHDF2 depletion. Annotation clusters with the highest enrichment according to FDR P-value are listed. The number of genes in each cluster is shown next to the respective bar and fold enrichment over background is given in parentheses. (J) RT-dPCR analysis of mRNA abundances in control and YTHDF2 depleted samples normalized to GAPDH. Data are reported as fold change (YTHDF2 KD/Control). Asterisks indicate significant difference in mean abundance between control and YTHDF2 KD samples ([*] P-value <0.05, [**] P-value <0.005).