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 ov...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.展开更多
基金The project supported by National Natural Science Foundation of China(81573463)
文摘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.
