Sequestration of miR-122 in Huh-7 hepatoma cells decreases HCV translation. (A) The reporter RNA with the HCV 5′-UTR, partial core, ubiquitin (not shown) and firefly luciferase (Fluc) sequences and the HCV 3′-UTR with an authentic 3′-end (). miR-122 and its target sequences (grey boxes) are indicated. The core–ubiquitin–Fluc fusion reading frame is drawn interrupted to adjust image scale. (B) Detection of miR-122 in Huh-7 cells, HeLa cells and nuclease-treated rabbit reticulocyte lysate (RRL) by northern blot. U6 snRNA was detected as a control. The indicated amounts of synthetic miR-122 were applied for comparison. (C) Fluc activity after translation of HCV reporter RNA in Huh-7 cells together with 2′-O-methylated antisense oligonucleotides. (D) RNA stability controls by northern blot with antisense RNA probes for HCV Fluc reporter RNA (Fluc 3′-region) and for glyceraldehyde-3-phosphate dehydrogenase (GDH) mRNA. (E) RNA 5′-end integrity controls by RNase protection assay for HCV reporter RNA (using an HCV 5′-UTR 5′-region antisense probe) and for GDH mRNA. In, input RNA; P, RNA probe; C, untransfected cells. (F) Capped and polyadenylated Rluc reporter RNA with the miR-122 target sequence in the 3′-UTR. (G) Translation efficiency of Rluc reporter RNA (F) in Huh-7 cells. (H) Northern blot controls for (G).

The miR-122 target sites reside in the HCV 5′-UTR. (A) Substitution mutations (white boxes) of the miR-122 target sequences (grey) in the HCV 5′- and 3′-UTR (see also ) and the miR-122 mu with the compensatory seed mutation. (B) Translation of the 5′- and 3′-UTR mutant HCV reporter RNAs in Huh-7 cells in the presence of anti-miR-122 or anti-miR-124 oligonucleotides. Lower panels, RNA stability controls by northern blot. (C) Translation of the 5′-UTR mutant HCV reporter RNAs in Huh-7 cells in the presence of ectopically added duplex miR-122, miR-122 mu or miR-124, respectively.

miR-122 stimulates HCV full-length genome translation. (A) The full-length HCV RNA. The polyprotein reading frame is drawn interrupted to adjust image scale. (B) Translation of the full-length HCV RNA genome in Huh-7 cells after co-transfection of ectopically added miR-122 or miR-124. (C) Translation as in (B) but of a replication-deficient full-length HCV RNA genome with an NS5B polymerase GND mutation. (D) Translation of the full-length HCV GND mutant genome in the presence of anti-miR-122. All values represent core antigen amounts in lysates 4 h after transfection. Lower panels: northern blot controls.

miR-122 stimulates HCV translation in HeLa cells and reticulocyte lysate. (A) Translation of the wild-type HCV reporter RNA (see ) in HeLa cells in the presence of miR-122 duplex and sequestration control with anti-miR-122. Duplex miR-124 and anti-miR-124 were used as controls. (B) Influence of increasing amounts of mature single-stranded miR-122 on translation of the HCV Fluc reporter RNA (see ) in rabbit reticulocyte lysate (RRL). (C) HCV reporter RNA translation efficiency in RRL in the presence of mature single-stranded or duplex miR-122 and miR-124. (D) Sequestration of added single-stranded miR-122 by antisense anti-miR-122 in RRL. Lower panels, northern blot controls.

miR-122 stimulates HCV translation at the stage of ribosomal 48S initiation complex formation. (A) The short RNA used for initiation complex formation contains the HCV IRES fused to an artificial 96 nt ORF that includes partial core coding sequences, followed by the HCV 3′-UTR with a precise 3′-end. (B) Sucrose gradient analysis of initiation complexes formed after 10 min incubation in RRL with the radiolabelled short HCV RNA shown in (A) in the presence or absence of miR-122. Reactions were performed in the presence of the elongation inhibitor anisomycin to ‘freeze' reactions after assembly of complete 80S ribosomes. The gradients were run for 5.5 h to resolve 48S and 80S initiation complexes. (C) Initiation complexes formed in the presence or absence of miR-124. (D, E) Initiation complexes formed with the radiolabelled short HCV RNA in the absence (D) or presence (E) of miR-122 and either in the absence of translation inhibitors, or in the presence of the GTP analogue GMP-PNP or the elongation inhibitor cycloheximide (CHX). (F) Ribosomal RNAs were re-extracted from a gradient run in parallel to localize ribosomal subunits. (G) Incorporation of radiolabelled miR-122 into ribosomal initiation complexes in the presence or absence of non-labelled short HCV RNA. (H) The full-length HCV RNA genome (NS5B polymerase GND mutant). The polyprotein reading frame is drawn interrupted to adjust image scale. (I) Initiation complexes formed after 10 min incubation in RRL with the full-length HCV RNA genome in the presence of miR-122 or miR-124.

miR-122 accelerates an early step of the association of the small ribosomal subunit with the HCV RNA. (A) The short HCV RNA used in (B). (B) Relative amounts of the short HCV RNA present in 48S and 80S complexes at the given times in the presence of miR-122 or miR-124. The data were obtained from the gradients shown in by summing up fractions 5–7 for 80S and 11–13 for 48S complexes. (C) The HCV Fluc 3′-UTR RNA used in (D–F). (D) Initiation complexes formed after 5 min incubation with the radiolabelled HCV Fluc 3′-UTR RNA in the presence of GMP-PNP or cycloheximide (CHX), respectively. The gradients in (D, E) were run only for 2.5 h to also resolve polysomes. (E) Initiation complexes formed with the HCV Fluc 3′-UTR RNA in the presence or absence of miR-122. Polysomes are marked by open arrows. Please note that the 48S peak is not resolved from the large peak of free RNA due to the shorter gradient run time. The profile shown represents the means of two experiments; two other experiments showed the same result. (F) Blow-up of fractions 1–16 of the gradient shown in (E).

Mutant miR-122 stimulates the formation of initiation complexes with the mutant HCV RNA in HeLa cells. (A) The short HCV RNA 5mut1,2 with the mutated miR-122 binding sites (open boxes) in the 5′-UTR. (B) Sucrose gradient analysis of translation initiation complexes harvested from HeLa cells 2 h after transfection of the mutant short HCV 5mut1,2 RNA in the absence of microRNA, or in the presence of miR-122 or the mutated miR-122 mu, respectively. As a control, cells transfected with the HCV RNA in the absence of microRNA were treated with 1 mM cycloheximide 30 min before harvesting (dotted line).