title: Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs.
abstract: 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.
authors.affiliation: Laboratory of Regulation of Gene Expression, Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
authors.forename: Vanda
authors.initials: V
authors.lastname: Munzarová
authors.forename: Josef
authors.initials: J
authors.lastname: Pánek
authors.forename: Stanislava
authors.initials: S
authors.lastname: Gunišová
authors.forename: István
authors.initials: I
authors.lastname: Dányi
authors.forename: Béla
authors.initials: B
authors.lastname: Szamecz
authors.forename: Leoš Shivaya
authors.initials: LS
authors.lastname: Valášek
mesh_terms: D024506:5' Flanking Region; D020121:5' Untranslated Regions; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D039621:Eukaryotic Initiation Factor-3 / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D054682:Ribosome Subunits, Small, Eukaryotic / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D020121:5' Untranslated Regions; D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; D039621:Eukaryotic Initiation Factor-3; C501391:GCN4 protein, S cerevisiae; D012333:RNA, Messenger; C111889:RPG1 protein, S cerevisiae; C099412:RPS0A protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors