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The RNA helicase Ded1p suppresses translation initiation from near-cognate start codons

ABSTRACT: The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian ortholog DDX3 are critical for translation initiation. Mutations in DDX3 are linked to tumorigenesis and intellectual disability, and the enzyme is targeted by diverse viruses. How Ded1p and its orthologs engage RNAs to impact translation initiation has been a longstanding, unresolved question. Here we show that Ded1p associates with the pre-initiation complex at the mRNA entry channel of the small ribosomal subunit and that the helicase unwinds mRNA structure ahead of the scanning pre-initiation complex. Defective Ded1p causes pervasive translation in 5’UTRs, starting from near-cognate initiation codons located 5' of mRNA structures and concomitant decrease of protein synthesis from of the main ORFs. The data indicate that Ded1p functions to suppress translation initiation on near-cognate codons proximal to mRNA structure and show how the helicase is targeted to specific RNA sites without common sequence signatures. Our results reveal a straightforward mechanism for the activation of upstream open reading frames and suggest that mRNA structure and proximal near-cognate initiation codons encode a widespread regulatory program for translation initiation that is sensitive to RNA helicase function.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE93959 | GEO | 2017/01/24

SECONDARY ACCESSION(S): PRJNA362935

REPOSITORIES: GEO

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Saccharomyces cerevisiae

Project description:The RNA helicase Ded1p suppresses translation initiation from near-cognate start codons

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Project description: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.

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