Figure 1

Translational enhancement of p53 mRNA by pentatricopeptide repeat (PPR)-eIF4G fusion protein. (A) p53 mRNA 5′ UTR and target position of designed PPR-eIF4G fusion gene (pP1-4G to pP17-4G). Number of Hela cells was determined 2 d after transfection with designed PPR-eIF4G fusion genes. Mock indicates transfection with empty vector. Unpaired two-tailed Student’s t-test, N = 3, **p < 0.01. Error bars indicate standard deviation. (B) Structure of PPR-eIF4G fusion gene plasmid. The PPR protein gene with 18 PPR motifs (i.e., 18 nt recognition) was fused with 3 × FLAG and truncated eIF4G (607–1600 aa), and integrated in expression plasmid under the CMV promoter. (C) p53 protein level analyzed by ELISA with or without pP10-4G transfection. (D) RT-PCR analysis for p53 mRNA with or without pP10-4G transfection. β-actin was used as an internal control. (E) Target RNA sequence for PPR-eIF4G fusion gene (pP3-4G, pP10-4G, and pP15-4G). (F) Western blot analysis to examine p53 protein levels in absence (mock) or in presence of pP3-4G, pP10-4G, and pP15-4G. (G) RNA–protein co-immunoprecipitation and RT-qPCR (RIP-PCR) analysis of interaction between PPR-eIF4G fusion protein and targeted p53 mRNA in cultured cells. The RNA from total lysate (input) or coprecipitated RNA (IP) with PPR-eIF4G fusion protein (pP3-4G, pP10-4G, and pP15-4G) was used for RT-PCR analysis. GAPDH was used as an internal control.