NON-CANONICAL INTERNAL RIBOSOME ENTRY SITE-MEDIATED TRANSLATION: IDENTIFICATION OF NEW PROTEINS INVOLVED IN COLORECTAL CANCER

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Human AGO1 5’UTR mediates an elF4G-enhanced but cap-independent mechanism of translation initiation

Publication . Lacerda, Rafaela; Menezes, Juliane; Romão, Luísa

Argonaute 1 (AGO1) is an essential effector in RNA-mediated gene silencing pathways. It regulates developmental control and stem cell maintenance, and is related to tumourigenesis. Such functions suggest its expression must be tightly regulated and, most likely, at protein synthesis level. Thus, we investigated whether AGO1 expression is controlled by alternative mechanisms of translation initiation. For that, we cloned the AGO1 5’UTR in a bicistronic luciferase vector upstream the downstream cistron (Firefly luciferase [FLuc]), and transfected HeLa cells with this construct. We observed a significant increase in FLuc expression levels compared to those from Renilla luciferase (upstream cistron) in cells transfected with the AGO1 5’UTR-containing constructs compared to those transfected with the empty transcript. Under cap-dependent translation initiation-impairing conditions, we saw that the identified cap-independent translation activity was enhanced upon knock-down of eukaryotic initiation factor (eIF) 4E, the cap-binding protein. However, inhibiting the eIF4G–eIF4E interaction significantly reduces such activity, suggesting AGO1 5’UTR-mediated translation may be dependent on eIF4G. Furthermore, in cells transfected with in vitro transcribed, capped and polyadenylated bicistronic AGO1 5’UTR-containing mRNA, the relative FLuc expression levels did not increase significantly, indicating that AGO1 5’UTR cannot mediate internal cap-independent translation initiation when it does not go through a nuclear experience. Nonetheless, in cells transfected with cap-lacking monocistronic transcripts, relative FLuc expression levels mediated by the AGO1 5’UTR were significantly increased. These results indicate that AGO1 5’UTR sequence mediates a non-canonical cap-independent eIF4G-dependent mechanism of translation initiation that seems to be enhanced by a free 5’ end.

2017-05-08Conference object

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