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. 2011 Jun 7;39(17):7716–7729. doi: 10.1093/nar/gkr426

Figure 4.

Figure 4.

The sequence of nucleotides 2–4 of HCV RNA is important for translational regulation by miR-122. (A) Model for a miR-122 molecule binding to seed match A in HCV nucleotides 1–45 and making additional contacts with nucleotides 1–4. This was tested by mutating nucleotides 2–4 in 5′LUC3′ RNA with or without mutations at p3 + 4 of seed match B. (B) The mutant RNAs in (A) were introduced into Huh7 cells by transfection, with sequestration or overexpression of miR-122. Mutation of nucleotides 2–4 abolished regulation by miR122 via seed match A in the context of a p3 + 4 mutant seed match B (***P < 0.0001). (C) Model for miR-122 binding to seed match B in HCV nucleotides 1–45 and making additional contacts with the spacer and an unoccupied seed match A. The spacer was mutated as shown. (D) 5′m30-7LUC3′ RNA was introduced into Huh7 cells with sequestration or overexpression of miR-122 and did not show a significant difference in the response to miR-122 compared to wild-type 5′LUC3′ RNA. All values are firefly luciferase activity relative to Renilla luciferase activity from the transfection control, as a percentage of Rand-2′Ome values for each RNA. The average of three independent triplicate experiments is shown, with error bars representing standard deviation.