Figure 4

Increased 5′ UTR and 3′ UTR coverage in the polysomal mRNA. (A) Comparison of genome-wide 5′ UTR coverage at the 18 h time point between steady-state mRNA (red), polysomal mRNA (blue) and the subset of apicoplast-encoded genes in polysomal mRNA (green). (B) Increased 5′ UTR coverage in a subset of genes in polysomal mRNA. (C) Gel electrophoresis of steady-state RNA (S) and polysome-associated RNA (P) (left) from trophozoite-stage parasites, and northern blots of the same RNA fractions using a probe designed to detect sense transcripts containing the 5′ UTR of gene PF3D7_0823800 (middle), or gene PF3D7_1216200 (right). Smaller transcript fragments that are observed in the polysomal fraction but not in the steady-state fraction are marked with an asterisk. (D) Heatmap showing the percentage of genes with high 5′ UTR coverage out of the total number of genes for each combination of steady-state mRNA and polysomal mRNA profiles. Combinations where transcription and translation peak at the same time point are boxed. For clarity, expression profiles were grouped based on the time point of highest expression (for example, expression profiles 18 h and 18 h to (36 h) were grouped as 18 h). (E) Distribution of the longest upstream open reading frame (uORF) in genes that are expressed at the 18 h stage and that show high 5′ UTR coverage and translational delay, in comparison to genes that have low 5′ UTR coverage and no translational delay. The difference in distribution was tested for statistical significance using the Kolmogorov-Smirnov test. (F) Comparison of genome-wide 3′ UTR coverage at the 18 h time point between steady-state mRNA (red) and polysomal mRNA (blue). (G) Increased 3′ UTR coverage in polysomal mRNA for genes with a large ORF directly downstream of the stop codon (encoding a >65 amino acid (aa) peptide) as compared to genes with short (<25 amino acids) downstream ORFs, suggestive of stop codon readthrough.