Yeast initiation factor eIF3 (eukaryotic initiation factor 3) has been implicated in multiple steps of translation initiation. Previously, we showed that the N-terminal domain (NTD) of eIF3a interacts with the small ribosomal protein RPS0A located near the mRNA exit channel, where eIF3 is proposed to reside. Here, we demonstrate that a partial deletion of the RPS0A-binding domain of eIF3a impairs translation initiation and reduces binding of eIF3 and associated eIFs to native preinitiation complexes in vivo. Strikingly, it also severely blocks the induction of GCN4 translation that occurs via reinitiation. Detailed examination unveiled a novel reinitiation defect resulting from an inability of 40S ribosomes to resume scanning after terminating at the first upstream ORF (uORF1). Genetic analysis reveals a functional interaction between the eIF3a-NTD and sequences 5 of uORF1 that is critically required to enhance reinitiation. We further demonstrate that these stimulatory sequences must be positioned precisely relative to the uORF1 stop codon and that reinitiation efficiency after uORF1 declines with its increasing length. Together, our results suggest that eIF3 is retained on ribosomes throughout uORF1 translation and, upon termination, interacts with its 5 enhancer at the mRNA exit channel to stabilize mRNA association with post-termination 40S subunits and enable resumption of scanning for reinitiation downstream.[Keywords: Translation initiation; reinitiation; eIF3; 40S ribosomal subunit; GCN4; short uORF] Supplemental material is available at http://www.genesdev.org.

Reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream uORFs to retain post-termination 40S subunits on mRNA. Its efficiency depends on surrounding cis-acting sequences, uORF elongation rates, various initiation factors, and the intercistronic distance. To unravel effects of cis-acting sequences, we investigated previously unconsidered structural properties of one such a cis-enhancer in the mRNA leader of GCN4 using yeast genetics and biochemistry. This leader contains four uORFs but only uORF1, flanked by two transferrable 5′ and 3′ cis-acting sequences, and allows efficient reinitiation. Recently we showed that the 5′ cis-acting sequences stimulate reinitiation by interacting with the N-terminal domain (NTD) of the eIF3a/TIF32 subunit of the initiation factor eIF3 to stabilize post-termination 40S subunits on uORF1 to resume scanning downstream. Here we identify four discernible reinitiation-promoting elements (RPEs) within the 5′ sequences making up the 5′ enhancer. Genetic epistasis experiments revealed that two of these RPEs operate in the eIF3a/TIF32-dependent manner. Likewise, two separate regions in the eIF3a/TIF32-NTD were identified that stimulate reinitiation in concert with the 5′ enhancer. Computational modeling supported by experimental data suggests that, in order to act, the 5′ enhancer must progressively fold into a specific secondary structure while the ribosome scans through it prior uORF1 translation. Finally, we demonstrate that the 5′ enhancer's stimulatory activity is strictly dependent on and thus follows the 3′ enhancer's activity. These findings allow us to propose for the first time a model of events required for efficient post-termination resumption of scanning. Strikingly, structurally similar RPE was predicted and identified also in the 5′ leader of reinitiation-permissive uORF of yeast YAP1. The fact that it likewise operates in the eIF3a/TIF32-dependent manner strongly suggests that at least in yeasts the underlying mechanism of reinitiation on short uORFs is conserved.

Background: AUG recognition is promoted by several initiation factors (eIFs).Results: eIF5 interacts with the extreme N terminus of eIF3c/Nip1 to promote pre-initiation complex assembly, and eIF1 binds the region that immediately follows.Conclusion: eIF1 binding to c/Nip1 is equally important for its 40 S ribosome recruitment and AUG selection.Significance: Understanding start codon selection that sets the reading frame for decoding is key in gene expression studies.

Human transcription co-regulator SNW1/SKIP is implicated in the regulation of both transcription elongation and alternative splicing. Prp45, the SNW/SKIP ortholog in yeast, is assumed to be essential for pre-mRNA processing. Here, we characterize prp45(1-169), a temperature sensitive allele of PRP45, which at permissive temperature elicits cell division defects and hypersensitivity to microtubule inhibitors. Using a synthetic lethality screen, we found that prp45(1-169) genetically interacts with alleles of NTC members SYF1, CLF1/SYF3, NTC20, and CEF1, and 2nd step splicing factors SLU7, PRP17, PRP18, and PRP22. Cwc2-associated spliceosomal complexes purified from prp45(1-169) cells showed decreased stoichiometry of Prp22, suggesting its deranged interaction with the spliceosome. In vivo splicing assays in prp45(1-169) cells revealed that branch point mutants accumulated more pre-mRNA whereas 5' and 3' splice site mutants showed elevated levels of lariat-exon intermediate as compared to wild-type cells. Splicing of canonical intron was unimpeded. Notably, the expression of Prp45(119-379) in prp45(1-169) cells restored Prp22 partition in the Cwc2-pulldowns and rescued temperature sensitivity and splicing phenotype of prp45(1-169) strain. Our data suggest that Prp45 contributes, in part through its interaction with the 2nd step-proofreading helicase Prp22, to splicing efficiency of substrates non-conforming to the consensus.