A miRNA-responsive cell-free translation system facilitates isolation of hepatitis C virus miRNP complexes.
Micro(mi)RNAs are 21- to 23-nt RNAs that regulate multiple biological processes. In association with Argonaute (Ago) proteins and other factors that form the RNA-induced silencing complex (RISC), miRNAs typically bind mRNA 3' untranslated regions (UTRs) and repress protein production through antagonizing translation and transcript stability. For a given mRNA-miRNA interaction, cis-acting RNA elements and trans-acting RNA-binding proteins (RBPs) may influence mRNA fate. This is particularly true of the hepatitis C virus (HCV) genome which interacts with miR-122, an abundant liver miRNA. miR-122 binding to HCV RNA considerably stimulates virus replication in cultured cells and primates, but the mechanism(s) and associated host factors required for enhancement of HCV replication have not been fully elucidated. We recapitulated miR-122-HCV RNA interactions in a cell-free translation system derived from cells that express miR-122. Specifically, lysates produced from HEK-293 cells that inducibly transcribe and process pri-miR-122 were characterized alongside those from isogenic cells lacking miR-122 expression. We observed a stimulatory effect of miR-122 on HCV reporter mRNAs in a manner that depended on expression of miR-122 and intact target sites within the HCV 5' UTR. We took advantage of this system to affinity-purify miR-122-HCV RNP complexes. Similar to functional assays, we found that association of immobilized HCV internal ribosome entry site (IRES) RNA with endogenous Ago2 requires both miR-122 expression and intact miR-122 target sites in cis. This combined approach may be generalizable to affinity purification of miRNP complexes for selected target mRNAs, allowing identification of miRNP components and RBPs that may contribute to regulation.
Bradrick, SS; Nagyal, S; Novatt, H
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