Fig. 7

Translationally repressed transcripts associate with a PfAlba1 complex in trophozoites and are subsequently released in schizonts for efficient translation. a Schematic representation of the experimental setup. Native lysates prepared from PfAlba1-Ty1 parasites at the trophozoite (28–32 h) or schizont (42–45 h) stage were incubated with rabbit anti-Ty1 antibodies in the presence of protease and RNAse inhibitors to IP a PfAlba1-Ty1-containing ribonucleoprotein complex. IPed RNAs were extracted and analyzed by qRT-PCR. Finally, RNA association was compared with the presence of the corresponding protein in whole cell lysates. goi = gene of interest. b Lysates (20 % of the input used for immunoprecipitation) or proteins IPed with anti-Ty1 antibodies from PfAlba1-Ty1 trophozoite (T) or schizont (S) stages were separated by denaturing gel electrophoresis and analyzed by western blotting with mouse anti-Ty1 antibodies or anti-PfAlba1 antibodies. PfAldolase served as a control. c Left panel: IPed RNA from PfAlba1-Ty1 trophozoite or schizont lysates was analyzed by qRT-PCR with primers specific to the indicated genes. The y-axis of the bar graph denotes percentage enrichment, calculated as in “Materials and methods”. Data represent the means of a minimum of three independent experiments ± standard error (error bars). ** = The AMA1 transcript binds to PfAlba1 in trophozoite stages. Fold change of PfAlba1 association from trophozoites to schizonts, i.e., T/S, was calculated as in “Materials and methods”. * = The association of PfAlba1 and Clag9 mRNA does not decrease from trophozoites to schizonts. Right panel: protein lysates prepared from empty vector or PfAlba1-Ty1 trophozoites (T) and schizonts (S) were separated by denaturing gel electrophoresis and analyzed by western blotting with the indicated antibodies