Competitive Binding between miR-122 and p68 onto Hepatitis C Viral RNA

Background: Liver-specific microRNA (miR)-122 has been shown to be involved in regulating translation of hepatitis C viral (HCV) RNA. This study aimed to explore the molecular mechanism of miR-122 in regulating HCV RNA translation initiation.
Material and Methods: In human liver hepatocellular carcinoma cell line HepG2, UV cross-link assay was performed on a large scale to identify RNA-binding proteins with gradient concentrations of miR-122. Analytical ultracentrifugation was then used to separate the translation initiation complexes. All RNA-binding proteins were then identified by Western blotting.
Results: The binding of 68 kDa protein (p68) to HCV RNA was suppressed by the addition of miR-122 via the competitive binding assay. Such inhibition can be eliminated by the addition of 2’-O-methylated oligonucleotides. This binding suppression was determined to be specific for miR-122, which used the mature single-stranded RNA to suppress the binding of p68 onto HCV RNA. This binding inhibition was further validated by using authentic miR-122 with conserved regions and mutated sequences.
Conclusions: The binding of p68 onto HCV RNA can be specifically inhibited by miR-122 via a competitive binding process.

Keywords: Binding, Competitive, 5' Untranslated Regions, Hep G2 cells, Hepacivirus - metabolism, MicroRNAs - metabolism, Molecular Weight, Peptide Chain Initiation, Translational, Protein Binding, RNA, Viral - metabolism, RNA-Binding Proteins - metabolism