Fig. 7: Effect of TgCet depletion on mRNA capping in Toxoplasma.

A Experimental workflow showing steps involved to enrich capped mRNA. Total RNA was isolated from TgCet-mAID-3HA parasites grown with IAA or vehicle. RNA was treated with the indicated enzymes and subsequently used for library construction and RNA sequencing. B Agarose gel image showing rRNA levels in ±RppH+Xrn1 treatment in TgCet-mAID-3HA parasites grown with IAA or vehicle (n = 3). C Immuno-dot blot showing m⁷G RNA levels in ±RppH treatment (Thermopol buffer 2) in TgCet-mAID-3HA parasites grown with IAA or vehicle (n = 3). Methylene blue stain serves as a loading control. D Immuno-dot blot showing m⁷G RNA levels in ±RppH treatment (NEBuffer 2) in TgCet-mAID-3HA parasites grown with IAA or vehicle (n = 3). Methylene blue stain serves as a loading control. E Volcano plot showing differentially expressed genes in TgCet-mAID-3HA parasites with IAA or vehicle. Genes with a log2 fold change ≥1 and p ≤ 0.05 are presented (n = 2, Benjamini–Hochberg test, p value in DESeq2). F Validation of altered gene expression in TgCet parasites (−IAA/ + IAA) in a subset of genes, including H3, H2A1, H2Bb, H2Ba, PYK, PGK, ENR, PGD, RPS12, and RPS27 RT-qPCR. Gene expression was normalized to 18 s rRNA levels (n = 2 replicates). G RT-qPCR of H3, H2A1, H2Bb, H2Ba, PYK, PGK, ENR, PGD, RPS12, and RPS27 genes in TgCet parasites (−Actinomycin D/+ Actinomycin D). 18 s rRNA levels were used for normalization (n = 2 replicates). Gene ontology analysis of downregulated (H), and upregulated (I) expressed genes with respect to cellular components and biological processes. Genes with a log2 fold change ≥1 and p ≤ 0.05 are presented (n = 2, Benjamini–Hochberg test, p value in DESeq2). Source data are provided as a source data file.