Fig. 7 | Nature Communications

Fig. 7

From: Pentatricopeptide repeat poly(A) binding protein KPAF4 stabilizes mitochondrial mRNAs in Trypanosoma brucei

Fig. 7

KPAF4-bound adenylated RNA is partially resistant to uridylation and degradation in vitro. a Western blotting of affinity purified KPAF4-WT and KPAF4-Mut samples. Protein samples were purified from mitochondrial fraction by rapid affinity pulldown with IgG-coated magnetic beads. KPAF4 polypeptides were detected with an antibody against the calmodulin-binding peptide. Single experiment performed. b Electrophoretic mobility shift assay with KPAF4-WT. Increasing amounts of affinity-purified KPAF4 were incubated with 5′ radiolabeled RNAs and separated on 3–12% native PAGE. Representative of six experiments is shown. c Electrophoretic mobility shift assay with KPAF4-Mut was performed as in (b). Representative of two experiments is shown. d RNA adenylation and uridylation. KPAP1, RET1, or in combination, were incubated with 5′ radiolabeled RNA and ATP, UTP, or ATP/UTP mix, respectively, in the absence or presence of KPAF4. Recombinant enzymes were purified from bacteria as described8,48. Reactions were terminated at indicated time intervals and products were resolved on 10% polyacrylamide/ 8 M urea gel. Representative of seven experiments are shown. e RNA degradation. The same RNA substrates as in (d) were incubated with increasing concentrations of KPAF4, and the reactions were initiated by adding buffer or the MPsome for a fixed period of time (DSS1, left panels). RNAs were incubated with a fixed concentration of KPAF4 for 20 min, and reactions were initiated by adding the MPsome (right panel). Reactions were terminated at indicated time intervals and products were resolved on a 10% polyacrylamide/8 M urea gel. Input RNA and final degradation products of 4–5 nt (FP) are shown. Representative of two experiments are shown and quantified in Supplementary Fig. 4

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