Analysis of the Role of EIF5A in Mammalian Translation

MYC is a critical growth-promoting gene that is subject to tight post-transcriptional control. However, the genes and mechanisms that mediate this regulation at the mRNA level are poorly understood. In order to identify regulators of MYC that function through the 5' UTR of the transcript, we performed a fluorescent reporter-coupled genome-scale CRISPR/Cas9-mediated loss of function screen. Analysis of screening data identified eukaryotic initiation factor 5A (EIF5A) as novel regulator of MYC translation. eIF5A is a highly conserved translation factor that has been demonstrated to relieve ribosome pauses during translation elongation at 'difficult to translate' peptide sequences in yeast and bacteria. We observed that eIF5A regulates protein isoform distribution of MYC, and that loss of function of this gene results in enhanced upstream non-canonical translation initiation on this transcript. Upon performing ribosome profiling in cells where eIF5A or its upstream activating enzyme were ablated, we discovered that the protein's function as a ribosome pause relief factor is conserved in mammalian cells. Importantly, analysis of ribosome profiling data under conditions of eIF5A depletion revealed not only evidence of enhanced ribosome pausing within coding sequences at elongation stall sites, but also an increase in non-canonical/sub-optimal translation initiation events in 5' UTRs in both yeast and human cells. These data lead us to formulate and test the hypothesis that ribosome pausing resulting from loss of eIF5A increases non-canonical translation initiation at pause-proximal upstream sub-optimal initiation codons. We present data from ribosome profiling experiments in yeast and human cells, as well as luciferase reporter assays that are consistent with this model. Thus, we propose a novel role for the translation elongation factor eIF5A in maintaining appropriate start codon selection during initiation in eukaryotic cells.