GSE138599
eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide
The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at the NCCs initiating N-terminal extensions on GRS1 and ALA1 mRNAs, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself.  Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons. Overall design: We examined the effect of sui1-L96P on global translational efficiencies (TEs) by ribosome footprint profiling of isogenic WT and sui1-L96Pstrains. The study includes 8 samples, comprised of 4 mRNA-Seq samples and 4 ribosome footprint profiling samples, derived from 2 biological replicates of sui1? mutant strains harboring plasmid-borne sui1-L96P or the WT SUI1 allele. The 4 ribosome footprint profiling samples were deposited to GEO previously (GSM2895472-GSM2895475 in the GSE108334 records). Please note that wig files were generated from both replicates and are linked to the corresonding *_1 sample records.