Fig. 5. miR-3191-5p inhibits the translational initiation of CACNA1A IRES–driven α1ACT by eIF4AII and eIF4GII. miR-3191-5p inhibits the translational initiation.

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Fig. 5. miR-3191-5p inhibits the translational initiation of CACNA1A IRES–driven α1ACT by eIF4AII and eIF4GII. miR-3191-5p inhibits the translational initiation of CACNA1A IRES–driven α1ACT by eIF4AII and eIF4GII. (A to D) Relative dual-luciferase CACNA1A IRES reporter activities in HEK293 cells treated by overexpression of either eIF4AI (AI) (A), eIF4AII (AII) (B), eIF4GI (GI) (C), eIF4GII (GII) (D), or a control vector (C) in the presence of miR-3191-5p or an miRNA NC (n = 6). The ratio of firefly luciferase to Renilla luciferase activities of a bicistronic CACNA1A IRES reporter vector was normalized to that of a bicistronic control reporter vector. (E and F) Western blot and densitometric analyses showing α1A-Q33-FLAG and α1ACT-Q33-FLAG expression in HEK293 cells treated by overexpression of either AII (E), GII (F), or C in the presence of miR-3191-5p or NC (n = 6). (G and H) Relative CACNA1A-Q33 mRNA expression in HEK293 cells treated by overexpression of either AII (G), GII (H), or C in the presence of miR-3191-5p or NC (n = 6). (I and J) Amount of IRES-α1ACT-Q33 mRNA binding to AII (I), GII (J), or a control IgG in the presence of vectors expressing miR-3191-5p (miR-3191) or an miRNA control (miR-C) that was immunoprecipitated using antibodies against AII, GII, or a control IgG (n = 6). (K) Immunoprecipitation (IP) from HEK293 cells transfected with AII- and GII-expressing vectors using an antibody against AII, GII, or a control IgG. Recovered proteins were analyzed using Western blot. Input, 10%. (L) Relative dual-luciferase CACNA1A IRES reporter activities in HEK293 cells treated with si-AII, si-GII, or a scrambled si-C (n = 6). The ratio of firefly luciferase to Renilla luciferase activities of a bicistronic CACNA1A IRES reporter vector was normalized to that of a bicistronic control reporter vector. All data represent means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. Student’s t test in (A) to (F), (I), (J), and (L). One-way ANOVA in (G) and (H). Yu Miyazaki et al., Sci Transl Med 2016;8:347ra94 Copyright © 2016, American Association for the Advancement of Science