Fig. 2.

miR-122 binding to example NS5B and 3′UTR target sites. a Co-immunoprecipitation (co-IP) results with RNA 5B.2d (see Supplementary Figure S6) containing the first highly conserved miR-122 binding site in NS5B. HeLa cells were transfected with ³²P-labeled HCV RNA 5B.2d along with either miR-122 duplex RNA or with miR-124 as a control. Cells were lysed 6 h post-transfection, and Ago2/miRNA/target RNA complexes were precipitated with an anti-Ago2 antibody. The “Input” lane shows a small aliquot of the in vitro-transcript used for transfections as a size marker for the RNAs recovered from co-IPs. RNA was extracted and analyzed by denaturing gel electrophoresis and phosphorimaging (upper panel). RNA was isolated from an aliquot of the cell extract prior to co-IP to check RNA integrity after cell lysis (middle panel). Ago2 western blots show the pull-down of Ago2 protein by the immunoprecipitation (lower panel). b Co-IP results and controls as in a, but with RNA 5B.3c. c Co-IP results and controls as in a, but with RNA S3c containing the 3′UTR miR-122 binding site. d–f Mutants of the binding sites knock out miR-122 binding. The seed sequences of the miR-122 binding sites were mutated as indicated, and the RNAs were subjected to co-IP as in a–c