Fig 1.
Toxoplasma RNA is modified with the m6A mark.
A) Immunofluorescence assays show m6A-specific marks enriched in the cytoplasm of i) extracellular tachyzoites compared to ii) a control without primary antibody. The m6A mark is also present in the cytoplasm of iii) intracellular tachyzoites and iv) stress-induced bradyzoites (cyst wall is stained with DBA). Scale bars represent 5 μm. B) Toxoplasma genomic DNA and differing amounts of RNA, as indicated, were immunoblotted for the m6A mark. SYBR Gold staining serves as a loading control.
Table 1.
m6A writer components in Toxoplasma.
Fig 2.
Components of Toxoplasma m6A writer complex.
A) Immunofluorescence assay of endogenously HA-tagged WTAP, METTL3, and METTL14 subunits of the writer complex. Each protein is localized to the parasites’ nuclei. The outlines of parasites are shown in the merge panel for reference. B) Western blot of endogenously HA-tagged writer complex members. C) Relative quantification of writer complex member expression as assessed by Western blot densitometry. Data are presented as average ± standard deviation normalized to WTAPHA signal. * represents p ≤ 0.05 as assessed by Student’s t-test assuming unequal variances. D) Schematic summarizing the Toxoplasma writer complex membership as identified by co-immunoprecipitation experiments outlined in Table 2.
Fig 3.
The m6A mark is enriched near mRNA 3’-boundaries.
A) Volcano plot displaying gene expression changes upon 24 h treatment with alkaline stress. The 295 upregulated genes, displayed in purple, include classic bradyzoite markers and the 19 downregulated genes are displayed in green. B) Venn diagram showing the overlap between m6A-modified genes from tachyzoites (Tz) and parasites after 24 h stress treatment (Bz). C) Motif identification of m6A-enriched sequencing. Significance (e value) is displayed in a bar chart for sequencing data from tachyzoites (Tz), bradyzoite-induced sample (Bz) and a combination of all m6A-enriched sequences (All). The same motif was independently identified from all annotated 3’-UTRs in ToxoDB. D) Metagene plot showing average distribution of m6A density along a normalized transcript from a single tachyzoite and bradyzoite-induced replicate. The m6A mark is most abundant in annotated 3’-UTRs. All three replicates from each condition displayed similar distributions. E) Distribution of m6A-enriched MeRIPseq (red) and input RNAseq (blue) reads from tachyzoite (Tz) and alkaline stressed (Bz) samples. m6A-containing peaks are displayed as black bars for both conditions. The m6A motif is indicated by the white arrowhead and the putative 3’-boundary is shown by the black arrowhead. All data are shown to scale. F) m6A-like motifs are enriched near mRNA 3’-boundaries (3’-B) in Toxoplasma transcripts. Each motif variant detected in this study is displayed. An enrichment of AU-rich elements was also seen downstream of the 3’-boundary. Statistical significance of the localized enrichment (Bonferroni-corrected one-tailed binomial test) is shown on the bottom right of the panel for each individual motif.
Table 2.
Interactors of the m6A writer complex.
The m6A writer complex was identified from immunoprecipitation of HA-tagged METTL3, METTL14, and WTAP. Peptide counts from two independent experiments are shown.
Fig 4.
Effect of METTL3 and WTAP depletion in Toxoplasma.
A) Western blot of METTL3HA-AID and WTAPHA-AID parasites treated with 500 μM IAA for the indicated times. Rapid protein depletion is seen upon addition of IAA. A decreased signal in the SAG1 loading control is also apparent upon prolonged IAA exposure. B) Quantitation of m6A levels after treatment of parasites with 500 μM IAA for 4 h. C) Replication assay upon treatment with 500 μM IAA or DMSO vehicle for 16 h. Data are presented as the average of three replicates ± standard deviation. E) Plaque assay and schematic representation of IAA dosing regimens. Parasites were allowed to grow for 24 h before undergoing one of three treatment regimens. Parasites were treated with i) DMSO vehicle for 24 h followed by 4 days in fresh media with DMSO; ii) 500 μM IAA for 24 h followed by 4 days in fresh media with DMSO; or iii) 500 μM IAA followed by 4 days in fresh media with IAA. Representative images of each treatment regimen are displayed. F) Quantitation of the plaque assay described in (E). Values are presented as the average of 4 replicates ± standard deviation. Statistical significance was assessed by Student’s t-test assuming unequal variances. * represents p ≤ 0.05, ** represents p ≤ 0.001. All experiments were repeated at least twice with similar results.
Fig 5.
Localization of YTH domain-containing m6A reader proteins.
A-B) Western blot of endogenously HA-tagged A) YTH1 and B) YTH2 reader proteins. Both proteins display reproducible additional bands that are not present in the RHΔΔ parental strain. C) Immunofluorescence assay of each HA-tagged YTH reader proteins. Outlines of the parasites are shown in the merge panel for reference. Scale bars represent 5 μm.
Table 3.
Proteins interacting with the YTH-domain m6A reader proteins.
Interacting proteins were identified from immunoprecipitation of HA-tagged YTH1 and YTH2. Peptide counts from two biological replicates are shown.
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.