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.

2017-05-08Documento de conferência

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The potential function of alternative translation initiation of Argonaute 1 in cancer

Publication . Vieira da Silva, Verónica; Lacerda, Rafaela; Romão, Luísa

Translation is one of the most regulated and energy-consuming cellular processes crucial for proper cell function. Translation is initiated by the canonical cap-dependent mechanism. However, under stress conditions, the initiation of canonical translation is inhibited, which allows the translation of specific proteins via alternative mechanisms. This project aims to understand the biological relevance of alternative protein synthesis mechanisms in Argonaute 1 (AGO1) expression. The AGO1 protein is involved in microRNA regulation, gene expression modulation and inhibition. AGO1 is also involved in the regulation of gene expression by RNA interference (RNAi), and its deregulation can lead to the activation of oncogenes or the suppression of tumor suppressor genes, contributing to the development and progression of cancer. Our work has shown that AGO1 mRNA can be translated through a cap-independent initiation mechanism. An upstream open reading frame (uORF) has also been identified in its 5’ untranslated region (5’UTR), which may play a role in the initiation of AGO1 translation. The results showed that the 5’UTR of human AGO1 supports a cap-independent mechanism of translation initiation, which is maintained under stress conditions. However, our analyses did not provide conclusive evidence for a regulatory role of the uORF in this initiation process. To this end, the 5’UTR of human AGO1 was cloned into a bicistronic vector containing Renilla (RLuc) and Firefly luciferase (FLuc), with FLuc positioned downstream of the 5’UTR. Luminometry assays will be used to evaluate the relative FLuc/RLuc translation efficiency under the control of the AGO1 5’UTR. The same approach will be used with monocistronistic reporter vectors, contaning only FLuc. This project aims to understand how these alternative mechanisms of mRNA translation initiation can influence AGO1 expression and help explain their potential roles in certain pathologies and cancer progression, such as colorectal cancer.

2025-06-05Documento de conferência

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Número da atribuição

SFRH/BD/74778/2010