One of the extensively studied mechanisms of gene-specific translational regulation is reinitiation. It takes place on messenger RNAs (mRNAs) where main ORF is preceded by upstream ORF (uORF). Even though uORFs generally down-regulate main ORF expression, specific uORFs exist that allow high level of downstream ORF expression. The key is their ability to retain 40S subunits on mRNA upon termination of their translation to resume scanning for the next AUG. Here, we took advantage of the exemplary model system of reinitiation, the mRNA of yeast transcriptional activator GCN4 containing four short uORFs, and show that contrary to previous reports, not only the first but the first two of its uORFs allow efficient reinitiation. Strikingly, we demonstrate that they utilize a similar molecular mechanism relying on several cis-acting 5' reinitiation-promoting elements, one of which they share, and the interaction with the a/TIF32 subunit of translation initiation factor eIF3. Since a similar mechanism operates also on YAP1 uORF, our findings strongly suggest that basic principles of reinitiation are conserved. Furthermore, presence of two consecutive reinitiation-permissive uORFs followed by two reinitiation-non-permissive uORFs suggests that tightness of GCN4 translational control is ensured by a fail-safe mechanism that effectively prevents or triggers GCN4 expression under nutrient replete or deplete conditions, respectively.

Laboratory of Regulation of Gene Expression Institute of Microbiology ASCR Videnska 1083 142 20 Prague the Czech Republic

Zobrazit více v PubMed

Valášek L.S. ‘Ribozoomin’—translation initiation from the perspective of the ribosome-bound eukaryotic initiation factors (eIFs) Curr. Protein Pept. Sci.. 2012;13:305–330. PubMed PMC

Jackson R.J., Hellen C.U., Pestova T.V. Termination and post-termination events in eukaryotic translation. Adv. Protein Chem. Struct. Biol. 2012;86:45–93. PubMed

Hood H.M., Neafsey D.E., Galagan J., Sachs M.S. Evolutionary roles of upstream open reading frames in mediating gene regulation in fungi. Annu. Rev. Microbiol. 2009;63:385–409. PubMed

Calvo S.E., Pagliarini D.J., Mootha V.K. Upstream open reading frames cause widespread reduction of protein expression and are polymorphic among humans. PNAS. 2009;106:7507–7512. PubMed PMC

Iacono M., Mignone F., Pesole G. uAUG and uORFs in human and rodent 5′untranslated mRNAs. Gene. 2005;349:97–105. PubMed

Zhou F., Roy B., von Arnim A.G. Translation reinitiation and development are compromised in similar ways by mutations in translation initiation factor eIF3h and the ribosomal protein RPL24. BMC Plant Biol. 2010;10:193. PubMed PMC

Davuluri R.V., Suzuki Y., Sugano S., Zhang M.Q. CART classification of human 5′ UTR sequences. Genome Res. 2000;10:1807–1816. PubMed PMC

Kozak M. Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol. Cell. Biol. 1987;7:3438–3445. PubMed PMC

Pöyry T.A., Kaminski A., Jackson R.J. What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame? Genes Dev. 2004;18:62–75. PubMed PMC

Szamecz B., Rutkai E., Cuchalova L., Munzarova V., Herrmannova A., Nielsen K.H., Burela L., Hinnebusch A.G., Valášek L. eIF3a cooperates with sequences 5′ of uORF1 to promote resumption of scanning by post-termination ribosomes for reinitiation on GCN4 mRNA. Genes Dev. 2008;22:2414–2425. PubMed PMC

Roy B., Vaughn J.N., Kim B.-H., Zhou F., Gilchrist M.A., Von Arnim A.G. The h subunit of eIF3 promotes reinitiation competence during translation of mRNAs harboring upstream open reading frames. RNA. 2010;16:748–761. PubMed PMC

Munzarová V., Pánek J., Gunišová S., Dányi I., Szamecz B., Valášek L.S. Translation reinitiation relies on the interaction between eIF3a/TIF32 and progressively folded cis-acting mRNA elements preceding short uORFs. PLoS Genet. 2011;7:e1002137. PubMed PMC

Cuchalová L., Kouba T., Herrmannová A., Danyi I., Chiu W.-l., Valášek L. The RNA recognition motif of eukaryotic translation initiation factor 3g (eIF3g) is required for resumption of scanning of posttermination ribosomes for reinitiation on GCN4 and together with eIF3i stimulates linear scanning. Mol. Cell. Biol. 2010;30:4671–4686. PubMed PMC

Hinnebusch A.G. Translational regulation of GCN4 and the general amino acid control of yeast. Annu. Rev. Microbiol. 2005;59:407–450. PubMed

Mueller P.P., Hinnebusch A.G. Multiple upstream AUG codons mediate translational control of GCN4. Cell. 1986;45:201–207. PubMed

Hinnebusch A.G. Mechanisms of gene regulation in the general control of amino acid biosynthesis in Saccharomyces cerevisiae. Microbiol. Rev. 1988;52:248–273. PubMed PMC

Williams N.P., Mueller P.P., Hinnebusch A.G. The positive regulatory function of the 5′-proximal open reading frames in GCN4 mRNA can be mimicked by heterologous, short coding sequences. Mol. Cell. Biol. 1988;8:3827–3836. PubMed PMC

Miller P.F., Hinnebusch A.G. Sequences that surround the stop codons of upstream open reading frames in GCN4 mRNA determine their distinct functions in translational control. Genes Dev. 1989;3:1217–1225. PubMed

Grant C.M., Hinnebusch A.G. Effect of sequence context at stop codons on efficiency of reinitiation in GCN4 translational control. Mol. Cell. Biol. 1994;14:606–618. PubMed PMC

Grant C.M., Miller P.F., Hinnebusch A.G. Sequences 5′ of the first upstream open reading frame in GCN4 mRNA are required for efficient translational reinitiation. Nucleic Acids Res. 1995;23:3980–3988. PubMed PMC

Valášek L., Mathew A., Shin B.S., Nielsen K.H., Szamecz B., Hinnebusch A.G. The yeast eIF3 subunits TIF32/a and NIP1/c and eIF5 make critical connections with the 40S ribosome in vivo. Genes Dev. 2003;17:786–799. PubMed PMC

Kouba T., Danyi I., Gunišová S., Munzarová V., Vlčková V., Cuchalová L., Neueder A., Milkereit P., Valášek L.S. Small ribosomal protein RPS0 stimulates translation initiation by mediating 40S-binding of eIF3 via its direct contact with the eIF3a/TIF32 subunit. PLoS One. 2012;7:e40464. PubMed PMC

Rabl J., Leibundgut M., Ataide S.F., Haag A., Ban N. Crystal structure of the eukaryotic 40S ribosomal subunit in complex with initiation factor 1. Science. 2011;331:730–736. PubMed

Mueller P.P., Harashima S., Hinnebusch A.G. A segment of GCN4 mRNA containing the upstream AUG codons confers translational control upon a heterologous yeast transcript. Proc Natl. Acad. Sci. U.S.A. 1987;84:2863–2867. PubMed PMC

Ruiz-Echevarria M.J., Peltz S.W. The RNA binding protein Pub1 modulates the stability of transcripts containing upstream open reading frames. Cell. 2000;101:741–751. PubMed

Abastado J.P., Miller P.F., Jackson B.M., Hinnebusch A.G. Suppression of ribosomal reinitiation at upstream open reading frames in amino acid-starved cells forms the basis for GCN4 translational control. Mol. Cell. Biol. 1991;11:486–496. PubMed PMC

Grant C.M., Miller P.F., Hinnebusch A.G. Requirements for intercistronic distance and level of eIF-2 activity in reinitiation on GCN4 mRNA varies with the downstream cistron. Mol. Cell. Biol. 1994;14:2616–2628. PubMed PMC

Luttermann C., Meyers G. The importance of inter- and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mRNA. Genes Dev. 2009;23:331–344. PubMed PMC

Hashem Y., des Georges A., Dhote V., Langlois R., Liao H.Y., Grassucci R.A., Hellen C.U., Pestova T.V., Frank J. Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29. Cell. 2013;153:1108–1119. PubMed PMC

Khoshnevis S., Gunišová S., Vlčková V., Kouba T., Neumann P., Beznosková P., Ficner R., Valášek L.S. Structural integrity of the PCI domain of eIF3a/TIF32 is required for mRNA recruitment to the 43S pre-initiation complexes. Nucleic Acids Res. 2014 in press. PubMed PMC

Pöyry T.A., Kaminski A., Connell E.J., Fraser C.S., Jackson R.J. The mechanism of an exceptional case of reinitiation after translation of a long ORF reveals why such events do not generally occur in mammalian mRNA translation. Genes Dev. 2007;21:3149–3162. PubMed PMC

Mueller P.P., Jackson B.M., Miller P.F., Hinnebusch A.G. The first and fourth upstream open reading frames in}I}GCN4}i} mRNA have similar initiation efficiencies but respond differently in translational control to changes in length and sequence. Mol. Cell. Biol. 1988;8:5439–5447. PubMed PMC

Palam L.R., Baird T.D., Wek R.C. Phosphorylation of eIF2 facilitates ribosomal bypass of an inhibitory upstream ORF to enhance CHOP translation. J. Biol. Chem. 2011;286:10939–10949. PubMed PMC

Raveh-Amit H., Maissel A., Poller J., Marom L., Elroy-Stein O., Shapira M., Livneh E. Translational control of protein kinase Ceta by two upstream open reading frames. Mol. Cell. Biol. 2009;29:6140–6148. PubMed PMC

Lee Y.Y., Cevallos R.C., Jan E. An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation. J. Biol. Chem. 2009;284:6661–6673. PubMed PMC

Cvijović M., Dalevi D., Bilsland E., Kemp G.J., Sunnerhagen P. Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation. BMC Bioinformatics. 2007;8:1–15. PubMed PMC

Differential effects of 40S ribosome recycling factors on reinitiation at regulatory uORFs in GCN4 mRNA are not dictated by their roles in bulk 40S recycling

Stem-loop-induced ribosome queuing in the uORF2/ATF4 overlap fine-tunes stress-induced human ATF4 translational control

Impacts of yeast Tma20/MCTS1, Tma22/DENR and Tma64/eIF2D on translation reinitiation and ribosome recycling

eIF4G is retained on ribosomes elongating and terminating on short upstream ORFs to control reinitiation in yeast

Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor Complexes

Binding of eIF3 in complex with eIF5 and eIF1 to the 40S ribosomal subunit is accompanied by dramatic structural changes

Please do not recycle! Translation reinitiation in microbes and higher eukaryotes

Does eIF3 promote reinitiation after translation of short upstream ORFs also in mammalian cells?

Embraced by eIF3: structural and functional insights into the roles of eIF3 across the translation cycle

In vivo evidence that eIF3 stays bound to ribosomes elongating and terminating on short upstream ORFs to promote reinitiation

Human eIF3b and eIF3a serve as the nucleation core for the assembly of eIF3 into two interconnected modules: the yeast-like core and the octamer

Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex

In-depth analysis of cis-determinants that either promote or inhibit reinitiation on GCN4 mRNA after translation of its four short uORFs

Rules of UGA-N decoding by near-cognate tRNAs and analysis of readthrough on short uORFs in yeast