Figure 5.

CD46-CYT2 regulates IRES-dependent translation via hnRNPA1

(A) EJ-1 cells were transiently transfected with StrepII-GST-CYT1/2 and/or FLAG-hnRNPA1. The cell lysates were precipitated with StrepII-Tactin and immunoblotted with an anti-FLAG antibody. (B) Co-immunoprecipitation experiments were conducted with an CD46 antibody in CD46-KO cells stably expressing CD46-CYT1 or CD46-CYT2, respectively. (C) Lysates from 293T cells transfected with FLAG-hnRNPA1 were incubated with either purified protein GST-CYT1 or GST-CYT2. Bound FLAG-hnRNPA1 proteins were immunoblotted by anti-FLAG. (D) EJ-1 cells expressing sh-LacZ or sh-hnRNPA1 were transfected with indicated IRES-dependent reporters, respectively. The firefly and Renilla luciferase activities were measured. (E and F) EJ-1 cells expressing sh-LacZ or sh-hnRNPA1 were transfected with CD46-CYT2 or pHAGE-negative control (NC) (control/empty vector) and the indicated IRES-dependent reporter plasmids. The firefly and Renilla luciferase activities were measured. (G) Relative luciferase activity of wild-type (WT) or CD46-KO EJ-1 cells transfected with Lenti-NC (control) or FLAG-hnRNPA1 with the indicated tethering reporter. (H) CD46-KO cells stably expressing CD46-CYT2, MS2-GST together with sh-LacZ, or sh-hnRNPA1 were transfected with the 3′ MS2 stem-loop-tagged IRES sequence of HIF1a or c-Myc. After 48 h of culture, cells were lysed and incubated overnight with glutathione Sepharose beads. Precipitates were subjected to western blotting with anti-hnRNPA1 or anti-CD46 antibodies. Levels of hnRNPA1 and CD46 are normalized against input and expressed as fold change relative to base expression determined using control sh-LacZ. (I) Cell lysates from WT or CD46-KO cells were incubated with IgG or anti-hnRNPA1 antibody and immunoprecipitated with protein A/G-conjugated beads. Bound RNAs were then eluted, purified, and subjected to qPCR for CCND1 and c-Myc mRNAs.