Study of the function of translating ribosomes within mRNA 5’ untranslated regions in colorectal cancer tumorigenesis

Colorectal cancer (CRC) has a high incidence and mortality rates worldwide. Its carcinogenenic process is based in a continuous accumulation of genetic alterations with concomitant variations in the profiles of gene expression. In order to study the variations in the gene expression profiles involved in cancer progression, genome-wide analyses have so far focused on the abundance of mRNA as measured either by microarray or RNA sequencing. However, neither approach provides information on the rate of protein synthesis, a step closer to the end-point of gene expression. Furthermore, the correlation between transcript and protein abundance is underestimated, as it does not account for the translational regulation mechanisms, thus limiting and masking the analysis of gene expression. To this end, ribosome profiling (Ribo-seq) emerges to monitor in vivo translation, providing global and quantitative measurements of translation by deep sequencing of ribosome-protected mRNA fragments (RPFs). The advent of this technique led to the identification of translation beyond the known annotated coding sequences. For instance, Ribo-seq analysis is informative about other start sites relative to the annotated canonical start codon leading to alternative open reading frames (AltORFs). In addition, this approach detects translating ribosomes within the 5’ untranslated regions (5’UTRs) consistent with the translation of upstream ORFs (uORFs), as well as ribosomes at the 3’UTR. Our aim is to determine the biological role of specific uORFs in the process of CRC tumorigenesis. For that, we will use already available Ribo-seq data from different cancer cell lines to get the 5’UTR translation profiles and choose potential uORFs-containing targets for further study. Then, we will analyze the role of such uORFs in translational regulation and study the biological function of those translatable uORFs at the level of cell viability and proliferation, and acquisition of malignant features to understand their involvement in CRC development. We analyzed the 5’UTR ribosome occupancy profiles obtained by available Ribo-seq data using a selection criteria based on a higher number of RPFs at the 5’UTR of cancer cells compared to the non-neoplasic cell lines as a proxy of uORFs translation. Then, those targets were characterized in terms of molecular function and biological process by gene ontology analysis in order to choose the ones with a cancer-related function. We are currently mapping the exact 5’-end of each transcript 5’UTR by circular rapid amplification of cDNA ends (cRACE) to finally clone them in a reporter plasmid and study their function in translational control.