Figure 4.

Netrin-1 mRNA is translated through IRES-dependent translation. HepaRG cells were transfected with bicistronic vectors carrying the netrin-1 5’UTR or the HCV IRES between the Rluc and Fluc coding regions. Three days after transfection, cells were treated for 4 hours with increasing amounts of DTT or left untreated. (A) The netrin-1 5’UTR allows Fluc translation in a bicistronic construct upon UPR. (B) Renilla luciferase activity (RLU) is decreased after DTT treatment. (C) Firefly luciferase activity (RLU) is differentially modulated after DTT treatment. (D) Northern blot showing the presence of a unique mRNA population of the expected size being synthesized. Equal loading was confirmed by 18S and 28S intensities. (E) FLuc/RLuc mRNA ratio is unchanged after DTT treatment. (F) The interaction between the netrin-1 5’UTR and the 40S ribosomal subunit is concentration-dependent. Internally ³²P-labeled netrin-1 5’UTR transcript was incubated with purified 40S particles in binding buffer. Complexes were detected by filter retention (mean ± SD, n = 3). (G) The interaction between the netrin-1 5’UTR and 40S subunit is displaced by the HCV IRES. The internally ³²P-labeled netrin-1 5’UTR RNA was incubated with the purified 40S particle in the presence of increasing amounts of the unlabeled transcripts (mean ± SD, n = 3).