Fig. 3.

miR-122 dependent interaction of Ago2 protein with target RNAs containing miR-122 target site 5B.2. Left panels The structures of the RNAs are shown schematically with the miR-122 target sites boxed (the complete sequences and predicted structures of RNAs are shown in Supplementary Figures S6 to S15). In the enlargement, miR-122 target sites are shown in detail (boxes). The average single strand probability (ssp) for the miR-122 seed target site is indicated. Middle panels miR-122 dependent Ago2 protein binding to target RNAs. HeLa cells were transfected with miR-122 duplex RNA and with ³²P-labeled target RNAs including the miR-122 target site sequence (named RNA 5B.2a, 5B.2b or 5B.2c, respectively) but with different lengths of upstream and downstream flanking regions. 6 h post transfection, Ago2/miRNA/target RNA complexes were subjected to co-IP with either an anti-Ago2 or a Flag antibody as negative control. 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. Right panels RNAs recovered after cells lysis but prior to co-IP to check RNA integrity after cell lysis