microRNA-122 Dependent Binding of Ago2 Protein to Hepatitis C Virus RNA Is Associated with Enhanced RNA Stability and Translation Stimulation
Figure 6
Influence of insertions between miR-122 target sites and IRES on miR-122-mediated stimulation of HCV translation.
(A) In the HCV reporter RNA, 5 or 10 nucleotides (nt) were inserted between the second miR-122 binding site and the base of stem-loop II. (B) An insert between the second miR-122 target site and the stem-loop II was designed to mask the two miR-122 target sites in an additional stem (st). (C) Relative translation activities of the wild-type (wt) and mutant HCV reporter RNAs in HuH-7 cells. Additional ectopic miR-122 (or miR-124 as a control) was added as indicated. A capped and polyadenylated Renilla-Luciferase (Rluc) was co-transfected. The Rluc readouts were used for normalization of the Fluc readouts from the HCV reporter RNA. The expression of the HCV reporter RNA in the presence of the control miR-124 was set 100%. The p-value of the statistical difference is indicated for the stem insertion construct compared with the wt HCV reporter RNA (* = p<0.05). (D–F) Immunoprecipitation of Ago2-HCV 5′-UTR RNA complexes with the constructs shown in A and B. The experiments were performed essentially as in Fig. 2 B–D. (D) Examination of the radioactive HCV RNA re-extracted from the cell lysate prior to immunoprecipitation (as in Fig. 2B). A quantification of the RNA amounts in lanes 2–7 is shown in Fig. S1D. (E) Radiolabelled HCV RNA recovered by anti-Ago2 immunoprecipitation. In lanes 2–5, the HCV RNA with the additional stem (st) was used, in lanes 6–7, the wt HCV 5′-UTR RNA was used. In lane 1, the input RNA with the stem insert, in lane 8 the input wt HCV 5′-UTR RNA is shown (each in 1∶100 dilution). In lane 2, an anti-eIF3 antibody was used as a positive control. In lane 3, an anti-Flag antibody was used as a negative control. (F) Anti-Ago2 immunoblot control of the samples analyzed in (E).
