Gene expression regulation by upstream open reading frames in colorectal cancer

Silva, Joana; Romão, Luísa

http://hdl.handle.net/10400.18/6014

Use this identifier to reference this record.

Name:	Description:	Size:	Format:
Poster Joana Encontro Ciência 2018.pdf		1.76 MB	Adobe PDF	Download

Send Feedback

Authors

Silva, Joana

Romão, Luísa

Abstract(s)

Colorectal cancer (CRC) has a high incidence and mortality rates worldwide. Its carcinogenesis process is characterized by a continuous accumulation of genetic alterations that changes the overall gene expression profiles. Those alterations have been studied by microarray and RNA sequencing that measure the abundance of mRNA but do not provide information on protein synthesis, a step closer to the end-point of gene expression. Ribosome profiling (RiboSeq) emerges to monitor in vivo translation by deep sequencing of ribosome-protected mRNA fragments (RPFs). This technique detects ribosomes outside of known protein-coding regions, identifying translation of upstream open reading frames (uORFs) within 5’ untranslated regions (5’UTRs). The aim of this work is to determine the role of specific uORFs in CRC tumorigenesis. For that, we looked for potential uORFs-containing targets based in the 5’UTR RPFs occupancy from RiboSeq data from different cancer cell lines already available. We chose ABCE1, PAIP2, eIF4G2 and eIF2A as uORFs-containing mRNAs. Gene ontology analyses revealed an important role in translational control for the proteins encoded by these transcripts. By semi-quantitative RT-PCR, ABCE1 transcript is shown down-regulated in HCT116 cells in comparison to the non-neoplasic colorectal cell line (NCM460). To analyze the role of such uORFs in translational regulation and their biological function at the level of cell viability and proliferation, and acquisition of CRC features, a reporter plasmid was constructed carrying the ABCE1 5’UTR fused to the Firefly luciferase (Fluc) ORF (pGL2-ABCE1). Each one of the five upstream AUGs in ABCE15’UTR was mutated to obtained constructs with non-functinal uORFs and only one functional uORF. HCT116 cells were transiently transfected with pGL2-ABCE1 or each one of the above mentioned constructs. Fluc expression and activity was assessed by Western blot and luminometry assays, respectively. Results show a decrease in the translational efficiency of Fluc by pGL2-ABCE1. Moreover, the construct carrying only the uORF3 functional exhibits a stronger repression efficacy compared to pGL2-ABCE1 and the other constructs.