Protein production must be strictly controlled at its beginning and end to synthesize a polypeptide that faithfully copies genetic information carried in the encoding mRNA. In contrast to viruses and prokaryotes, the majority of mRNAs in eukaryotes contain only one coding sequence, resulting in production of a single protein. There are, however, many exceptional mRNAs that either carry short open reading frames upstream of the main coding sequence (uORFs) or even contain multiple long ORFs. A wide variety of mechanisms have evolved in microbes and higher eukaryotes to prevent recycling of some or all translational components upon termination of the first translated ORF in such mRNAs and thereby enable subsequent translation of the next uORF or downstream coding sequence. These specialized reinitiation mechanisms are often regulated to couple translation of the downstream ORF to various stimuli. Here we review all known instances of both short uORF-mediated and long ORF-mediated reinitiation and present our current understanding of the underlying molecular mechanisms of these intriguing modes of translational control.

• MeSH
• Bacteria genetika   metabolismus   MeSH
• Eukaryota genetika   MeSH
• lidé MeSH
• otevřené čtecí rámce genetika   MeSH
• proteosyntéza genetika   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Intramural MeSH

Reinitiation after translation of short upstream ORFs (uORFs) represents one of the means of regulation of gene expression on the mRNA-specific level in response to changing environmental conditions. Over the years it has been shown-mainly in budding yeast-that its efficiency depends on cis-acting features occurring in sequences flanking reinitiation-permissive uORFs, the nature of their coding sequences, as well as protein factors acting in trans. We earlier demonstrated that the first two uORFs from the reinitiation-regulated yeast GCN4 mRNA leader carry specific structural elements in their 5' sequences that interact with the translation initiation factor eIF3 to prevent full ribosomal recycling post their translation. Actually, this interaction turned out to be instrumental in stabilizing the mRNA·40S post-termination complex, which is thus capable to eventually resume scanning and reinitiate on the next AUG start site downstream. Recently, we also provided important in vivo evidence strongly supporting the long-standing idea that to stimulate reinitiation, eIF3 has to remain bound to ribosomes elongating these uORFs until their stop codon has been reached. Here we examined the importance of eIF3 and sequences flanking uORF1 of the human functional homolog of yeast GCN4, ATF4, in stimulation of efficient reinitiation. We revealed that the molecular basis of the reinitiation mechanism is conserved between yeasts and humans.

• MeSH
• eukaryotický iniciační faktor 3 chemie   metabolismus   MeSH
• iniciace translace peptidového řetězce * MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• otevřené čtecí rámce * MeSH
• proteosyntéza MeSH
• ribozomy metabolismus   MeSH
• savci MeSH
• transkripční faktor ATF4 chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Translational control in eukaryotes is exerted by many means, one of which involves a ribosome translating multiple cistrons per mRNA as in bacteria. It is called reinitiation (REI) and occurs on mRNAs where the main ORF is preceded by a short upstream uORF(s). Some uORFs support efficient REI on downstream cistrons, whereas some others do not. The mRNA of yeast transcriptional activator GCN4 contains four uORFs of both types that together compose an intriguing regulatory mechanism of its expression responding to nutrients' availability and various stresses. Here we subjected all GCN4 uORFs to a comprehensive analysis to identify all REI-promoting and inhibiting cis-determinants that contribute either autonomously or in synergy to the overall efficiency of REI on GCN4. We found that the 3' sequences of uORFs 1-3 contain a conserved AU1-2A/UUAU2 motif that promotes REI in position-specific, autonomous fashion such as the REI-promoting elements occurring in 5' sequences of uORF1 and uORF2. We also identified autonomous and transferable REI-inhibiting elements in the 3' sequences of uORF2 and uORF3, immediately following their AU-rich motif. Furthermore, we analyzed contributions of coding triplets and terminating stop codon tetranucleotides of GCN4 uORFs showing a negative correlation between the efficiency of reinitiation and efficiency of translation termination. Together we provide a complex overview of all cis-determinants of REI with their effects set in the context of the overall GCN4 translational control.

• MeSH
• iniciace translace peptidového řetězce MeSH
• messenger RNA genetika   metabolismus   MeSH
• otevřené čtecí rámce MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza RNA MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• 5' nepřekládaná oblast MeSH
• 5' přiléhající oblast DNA MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• otevřené čtecí rámce genetika   MeSH
• regulační oblasti nukleových kyselin MeSH
• ribozomální proteiny genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• zesilovače transkripce MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Translation reinitiation is a gene-specific translational control mechanism characterized by the ability of some short upstream ORFs to prevent recycling of the post-termination 40S subunit in order to resume scanning for reinitiation downstream. Its efficiency decreases with the increasing uORF length, or by the presence of secondary structures, suggesting that the time taken to translate a uORF is more critical than its length. This led to a hypothesis that some initiation factors needed for reinitiation are preserved on the 80S ribosome during early elongation. Here, using the GCN4 mRNA containing four short uORFs, we developed a novel in vivo RNA-protein Ni2+-pull down assay to demonstrate for the first time that one of these initiation factors is eIF3. eIF3 but not eIF2 preferentially associates with RNA segments encompassing two GCN4 reinitiation-permissive uORFs, uORF1 and uORF2, containing cis-acting 5΄ reinitiation-promoting elements (RPEs). We show that the preferred association of eIF3 with these uORFs is dependent on intact RPEs and the eIF3a/TIF32 subunit and sharply declines with the extended length of uORFs. Our data thus imply that eIF3 travels with early elongating ribosomes and that the RPEs interact with eIF3 in order to stabilize the mRNA-eIF3-40S post-termination complex to stimulate efficient reinitiation downstream.

• MeSH
• 5' nepřekládaná oblast MeSH
• elongace translace peptidového řetězce MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• genetické techniky MeSH
• iniciace translace peptidového řetězce * MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• otevřené čtecí rámce * MeSH
• regulace genové exprese * MeSH
• ribozomy metabolismus   MeSH
• terminace translace peptidového řetězce MeSH
• terminační kodon MeSH
• Publikační typ
• časopisecké články MeSH

Recent reports have begun unraveling the details of various roles of individual eukaryotic translation initiation factor 3 (eIF3) subunits in translation initiation. Here we describe functional characterization of two essential Saccharomyces cerevisiae eIF3 subunits, g/Tif35 and i/Tif34, previously suggested to be dispensable for formation of the 48S preinitiation complexes (PICs) in vitro. A triple-Ala substitution of conserved residues in the RRM of g/Tif35 (g/tif35-KLF) or a single-point mutation in the WD40 repeat 6 of i/Tif34 (i/tif34-Q258R) produces severe growth defects and decreases the rate of translation initiation in vivo without affecting the integrity of eIF3 and formation of the 43S PICs in vivo. Both mutations also diminish induction of GCN4 expression, which occurs upon starvation via reinitiation. Whereas g/tif35-KLF impedes resumption of scanning for downstream reinitiation by 40S ribosomes terminating at upstream open reading frame 1 (uORF1) in the GCN4 mRNA leader, i/tif34-Q258R prevents full GCN4 derepression by impairing the rate of scanning of posttermination 40S ribosomes moving downstream from uORF1. In addition, g/tif35-KLF reduces processivity of scanning through stable secondary structures, and g/Tif35 specifically interacts with Rps3 and Rps20 located near the ribosomal mRNA entry channel. Together these results implicate g/Tif35 and i/Tif34 in stimulation of linear scanning and, specifically in the case of g/Tif35, also in proper regulation of the GCN4 reinitiation mechanism.

• MeSH
• aminokyselinové motivy genetika   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• fungální RNA metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteosyntéza MeSH
• ribozomální proteiny chemie   genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekundární struktura proteinů MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• vazba proteinů MeSH
• western blotting MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF's cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1-RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.

• MeSH
• DEAD-box RNA-helikasy genetika   MeSH
• eukaryotický iniciační faktor 3 genetika   MeSH
• eukaryotický iniciační faktor 4E genetika   MeSH
• eukaryotický iniciační faktor 4G genetika   MeSH
• iniciace translace peptidového řetězce genetika   MeSH
• kodon iniciační genetika   MeSH
• lidé MeSH
• otevřené čtecí rámce genetika   MeSH
• proteosyntéza genetika   MeSH
• ribozomy genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• transkripční faktory bZIP genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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.

• MeSH
• 5' nepřekládaná oblast MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• fungální RNA genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce * MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• otevřené čtecí rámce MeSH
• regulace genové exprese u hub MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Protein synthesis is a fundamental biological mechanism bringing the DNA-encoded genetic information into life by its translation into molecular effectors - proteins. The initiation phase of translation is one of the key points of gene regulation in eukaryotes, playing a role in processes from neuronal function to development. Indeed, the importance of the study of protein synthesis is increasing with the growing list of genetic diseases caused by mutations that affect mRNA translation. To grasp how this regulation is achieved or altered in the latter case, we must first understand the molecular details of all underlying processes of the translational cycle with the main focus put on its initiation. In this review I discuss recent advances in our comprehension of the molecular basis of particular initiation reactions set into the context of how and where individual eIFs bind to the small ribosomal subunit in the pre-initiation complex. I also summarize our current knowledge on how eukaryotic initiation factor eIF3 controls gene expression in the gene-specific manner via reinitiation.

• MeSH
• Eukaryota metabolismus   MeSH
• eukaryotické iniciační faktory metabolismus   MeSH
• iniciace translace peptidového řetězce genetika   MeSH
• lidé MeSH
• molekulární modely MeSH
• ribozomy metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Little is known about the impact of trans-acting genetic variation on the rates with which proteins are synthesized by ribosomes. Here, we investigate the influence of such distant genetic loci on the efficiency of mRNA translation and define their contribution to the development of complex disease phenotypes within a panel of rat recombinant inbred lines. RESULTS: We identify several tissue-specific master regulatory hotspots that each control the translation rates of multiple proteins. One of these loci is restricted to hypertrophic hearts, where it drives a translatome-wide and protein length-dependent change in translational efficiency, altering the stoichiometric translation rates of sarcomere proteins. Mechanistic dissection of this locus across multiple congenic lines points to a translation machinery defect, characterized by marked differences in polysome profiles and misregulation of the small nucleolar RNA SNORA48. Strikingly, from yeast to humans, we observe reproducible protein length-dependent shifts in translational efficiency as a conserved hallmark of translation machinery mutants, including those that cause ribosomopathies. Depending on the factor mutated, a pre-existing negative correlation between protein length and translation rates could either be enhanced or reduced, which we propose to result from mRNA-specific imbalances in canonical translation initiation and reinitiation rates. CONCLUSIONS: We show that distant genetic control of mRNA translation is abundant in mammalian tissues, exemplified by a single genomic locus that triggers a translation-driven molecular mechanism. Our work illustrates the complexity through which genetic variation can drive phenotypic variability between individuals and thereby contribute to complex disease.

• MeSH
• biogeneze organel MeSH
• genetická variace MeSH
• iniciace translace peptidového řetězce * MeSH
• kardiomegalie genetika   metabolismus   patologie   MeSH
• krysa rodu rattus MeSH
• lokus kvantitativního znaku * MeSH
• malá jadérková RNA genetika   metabolismus   MeSH
• messenger RNA genetika   metabolismus   MeSH
• myokard metabolismus   patologie   MeSH
• myši inbrední C57BL MeSH
• myši knockoutované MeSH
• myši MeSH
• potkani inbrední SHR MeSH
• potkani transgenní MeSH
• regulace genové exprese MeSH
• ribozomální proteiny genetika   metabolismus   MeSH
• ribozomy genetika   metabolismus   patologie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• sarkomery metabolismus   patologie   MeSH
• stanovení celkové genové exprese MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH