Suppression of Ribosomal Pausing by eIF5A Is Necessary to Maintain the Fidelity of Start Codon Selection

Abstract

SUMMARY Sequences within 5′ 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 enhances upstream translation within 5′ UTRs across yeast and human transcriptomes, including on the MYC transcript, where this results 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 use 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.

Ribosome Recycling by ABCE1 Links Lysosomal Function and Iron Homeostasis to 3ʹ UTR-Directed Regulation and Nonsense-Mediated Decay

“…The EGFP PTC-231 and EGFP PTC-35 constructs were then placed under the control of a strong constitutive promoter and integrated into the AAVS1 locus, a region of open chromatin that allows high-level transgene expression ( Hockemeyer et al, 2009 ; Lamartina et al, 2000 ), in HCT116 cells, a stably diploid human cell line amenable to pooled CRISPR screening ( Golden et al, 2017 ; Manjunath et al, 2019 ). As expected, EGFP PTC-231 cells exhibited approximately 10-fold lower GFP fluorescence compared with EGFP PTC-35 cells ( Figure 1C ).…”

“…A DNA fragment containing HBB exon 2 (231bp), intron 2, and exon 3 was amplified from human genomic DNA by PCR with Phusion High-Fidelity DNA Polymerase (New England Biolabs) and cloned into pAAVS- EGFP -DONOR ( Manjunath et al, 2019 ) XbaI and MfeI sites using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) to generate pAAVS- EGFP PTC-231 . A sub-fragment of HBB , beginning 35 bp upstream of the of the 3′ end of exon 2, was amplified from pAAVS- EGFP PTC-231 and cloned into pAAVS- EGFP -DONOR XbaI and MfeI sites to generate pAAVS- EGFP PTC-35 .…”

“…sgRNAs targeting genes of interest, as well as non-target (NT) sgRNAs (sequences provided in Table S3 ), were cloned into the lenti-CRISPR v2 vector (Addgene #52961) as previously described ( Sanjana et al, 2014 ). Knockout pools were generated as described previously ( Manjunath et al, 2019 ) with minor modifications. To generate ABCE1 -knockout pools, transduced cells were selected with 1 μg/ml puromycin for 4 days (HCT116, Huh7, and HeLa) or 5 days (HEK293T) before analysis.…”

“…Only transcripts with CPM > 0.5 were used for CDF analyses. Ribosome profiling data were analyzed as previously described ( Manjunath et al, 2019 ).…”

“…We recently developed a highly effective genome-wide CRISPR screening approach that utilizes fluorescent reporters coupled with a single round of high-stringency sorting that allows recovery of essential genes as hits ( Golden et al, 2017 ; Manjunath et al, 2019 ). Here we describe application of this strategy to identify components of the NMD pathway in mammalian cells.…”