Combinatorial analysis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons

Abstract

Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here, we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are most probably dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.

PEPseq Quantifies Transcriptome-Wide Changes in Protein Occupancy and Reveals Selective Translational Repression After Translational Stress

“…In yeast and human cells it was demonstrated that in ribosome profiling (Ribo-seq) the length of a ribosome footprint informs about the conformation of the ribosome during its ratcheting motion on mRNA (21). To find further evidence that arsenite stalled ribosomes in the CDS, we therefore first turned to a published Ribo-seq dataset investigating arsenite-induced translational arrest in human HEK293 cells (22). Indeed, we observed a strong 2.5 fold shift from 21 nucleotide reads (pre-translocation) to 28 nucleotide reads (post-translocation) upon arsenite treatment in the published Ribo-seq data (two-sided Kolmogorov-Smirnov test p<2E-16, Figure S3A).…”

“…In order to validate the observations we had made for protein coding transcripts, where certain groups of mRNA increased their protein occupancy during arsenite stress, we cross-referenced them with published data from different cell lines. Therefore, we first asked if stalling ribosomes might be the cause for this increased occupancy and re-analyzed Ribo-seq data from arsenite-treated HEK293 cells (22). In this dataset, translation efficiencies of iPO mRNAs decreased strongly upon arsenite (p=2.7E-7, two-sided Kolmogorov-Smirnoff test, Figure S5A).…”

“…In order to validate that certain groups of transcripts increased their protein occupancy during arsenite stress we cross-referenced them with the published Ribo-seq data from arsenite-treated HEK293 cells [21]. In this dataset, translation efficiencies of iPO mRNAs or mRNAs coding for cytosolic ribosomal proteins decreased strongly upon arsenite (p<2.7E-7, two-sided Kolmogorov-Smirnoff test, Figure 5A).…”

“…For our re-analysis of the Ribo-seq data from arsenite-treated HEK293T cells (Ichihara et al, 2021), raw data with the accession number PRJNA729461 corresponding to SRR14510024, SRR14510025 (Ribo-seq), and SRR14510056, SRR14510057 (Ribo-seq, arsenite treatment) were downloaded from the NCBI BioProject database. Reads aligning to a custom bowtie2 v2.3.0 (Langmead and Salzberg, 2012) ribosomal index were discarded.…”

IRES-independent, eIF2A/eIF2D-dependent translation of the enterovirus genome

“…Initiation of translation in mammals is no longer thought to be as specific as it once was. Use of non-AUG codons under conditions in which eIF2α is active is now well documented [28, 39] ( Fig. 9A ).…”