m6A RNA methylation facilitates pre-mRNA 3’-end formation and is essential for viability of Toxoplasma gondii
Fig 6
m6A deficiency impairs 3’-end formation.
A) Western blot of YTH1HA-AID parasites treated with 500 μM IAA for the indicated times. B-D) Differential gene expression analysis after 4 h IAA treatment for METTL3HA-AID (B), WTAPHA-AID (C) and YTH1HA-AID (D) parasites. E-F) Venn diagram showing the relationship of upregulated (E) and downregulated (F) differentially expressed genes between the indicated parasite lines after 4 h IAA treatment. G) De novo transcriptome assembly shows that IAA treatment produces run-on transcripts. ToxoDB gene models are shown at the top (uncolored) for reference. m6A peaks detected from tachyzoite and bradyzoite-induced samples by MeRIPseq are denoted by black bars. Arrowhead shows the m6A-associated motif. Predicted transcripts are shown for untreated (cyan), after 4 h IAA (yellow), and after 16 h IAA (magenta) treatment. RNAseq mapping density for each experiment (black heatmaps) are shown immediately below predicted transcripts for reference. H) Violin plot demonstrating that estimated transcript length increases upon IAA treatment. Mean transcript size (in kb) is stated for each treatment. Statistical significance was assessed by Student’s t-test assuming unequal variances. ** represents p ≤ 10−50.