Translational regulation of RPA2 via IRES by UNR and e IF3a

OBJECTIVE To explore the mechanism of translation initiation regulation of RPA2 via IRES by UNR and e IF3a.METHODS Biotin pull down assay was taken to study the binding of RPA2 IRES and UNR.UNR was knocked down and overexpressed in H1299,A549 and SK-MES cell lines.Western blotting and real-time PCR were used to detect protein level and m RNA level respectively.CO-IP assay was conducted for the interaction of e IF3a and UNR.GST pul down assay was carried out to explore the exact domains.And the domains of e IF3a and UNR binding to RPA2 IRES were explored with EMSA assay.RESULTS NUR protein can bind to RPA2 IRES as well as e IF3a.UNR regulated the protein expression of RPA2 in H1299,A549 and SK-MES cells,and there was no change in RPA2 m RNA.UNR combined with e IF3a via the first domain of UNR and the first domain of e IF3a.UNR bound to RPA2 IRES with the first domain.And there was no sufficient evidence for the binding domain of e IF3a with RPA2 IRES yet.CONCLUSION UNR worked with eI F3a and co-regulate the RPA2 IRES activity and further regulate the expression of protein.This is the possible regulation mechanisms of cellular internal ribosomal entry site affect translation initiation.