Translation repression in parasites ectopically expressing TcUBP1.

<p>(A) Wt parasites were incubated with CHX (50 μg/ml) or vehicle and then with puromycin (10 μg/ml) or vehicle for 30 minutes. Puromycin incorporation was determined by Immunoblot (IB) using an anti-puromycin antibody. Protein loading was monitored using an anti-tubulin antibody. Image is representative of 3 independent experiments. Proteins unspecifically detected by the anti-puromycin antibody in the absence of puromycin, are pointed with arrowheads. (B) GFP or TcUBP1-GFP induced or GFP uninduced parasites were cultured for 5 and 10 days. 3 x 10⁷ parasites were incubated with puromycin (10 μg/ml) or vehicle for 30 minutes. Puromycin incorporation was determined by immunoblot using an anti-puromycin antibody. Protein loading was monitored using an anti-tubulin antibody. The ratio between Puromycin fluorescence/Tubulin fluorescence (Puro/Tub) is shown in arbitrary units (AU). Images are representative of 4 independent experiments. Unspecific bands are pointed with arrowheads. (C) Left panel, polysome profiles of induced (green line) and uninduced TcUBP1-GFP (black line), and from induced GFP parasites (filled light blue profile) after 5 days of induction, in the presence of CHX. Right panel, a control polysome profile from wt parasites using CHX is shown (black line), together with the disruption of 80S ribosomes into 60S and 40S ribosomal subunits with EDTA as a specificity control (red line). (D) Percentage of parasites displaying mRNA granules under optimal starvation conditions. (E) Percentage of parasites displaying mRNA granules under suboptimal starvation conditions. (F) Microscopic images of epimastigotes expressing TcUBP1-GFP, or GFP, under suboptimal starvation conditions, together with poly(A) mRNA detection by FISH and staining of DNA with DAPI, shown in cyan. Images are representative of 5 different experiments. (G) Analysis of granule area. (H) Analysis of the number of mRNA granules per parasite. Values represent mean ± SD of 5 independent replicates. *P< 0.05 by Anova-Dunnett test. Scale bar, 5 μm.