GSE132007
Suppression of ribosomal pausing by eIF5A is necessary to maintain the fidelity of start codon selection (Ribosome profiling)
Sequences within 5' untranslated regions (UTRs) dictate the site and efficiency of translation initiation. In this study, an unbiased screen designed to interrogate the 5' UTR-mediated regulation of the growth-promoting gene MYC unexpectedly revealed the ribosomal pause-relief factor eIF5A as a regulator of translation initiation codon selection. Depletion of eIF5A enhanced upstream translation within 5' UTRs across yeast and human transcriptomes, including on the MYC transcript where this resulted in increased production of an N-terminally extended protein. Furthermore, ribosome profiling experiments established that the function of eIF5A as a suppressor of ribosomal pausing at sites of suboptimal peptide bond formation is conserved in human cells. We present evidence that proximal ribosomal pausing on a transcript triggers enhanced usage of upstream suboptimal or non-canonical initiation codons. Thus, we propose that eIF5A functions not only to maintain efficient translation elongation in eukaryotic cells, but also to maintain the fidelity of translation initiation. Overall design: Ribosome profiling (and complementary RNA-sequencing) was performed in HCT116 cells that received, via lentivirus, Cas9 and sgRNAs targeting either EIF5A, DOHH or non-targeting control sgRNAs. Two sgRNAs were used for each condition, and thus 6 biological samples were used for these experiments.