Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• MeSH
• iniciace translace peptidového řetězce MeSH
• malé podjednotky ribozomu eukaryotické * metabolismus   genetika   MeSH
• messenger RNA * metabolismus   genetika   MeSH
• otevřené čtecí rámce * MeSH
• proteosyntéza MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae * genetika   metabolismus   MeSH
• transkripční faktory bZIP * metabolismus   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• messenger RNA * MeSH
• Saccharomyces cerevisiae - proteiny * MeSH
• transkripční faktory bZIP * MeSH

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S subunit from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation at short upstream open reading frames (uORFs) harboring penultimate codons that confer dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited reinitiation at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on reinitiation at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) reinitiation. We found that the Tma proteins generally impede reinitiation at native uORF4 and uORF4 variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on reinitiation at native uORF1, and equipping uORF1 with Tma-dependent penultimate codons generally did not confer Tma-dependent reinitiation; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the reinitiation potential of the uORF and the penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• Klíčová slova
• DENR, MCTS1, Tma, eIF2D, reinitiation, ribosome recycling,
• Publikační typ
• časopisecké články MeSH
• preprinty 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
• Názvy látek
• DEAD-box RNA-helikasy MeSH
• eukaryotický iniciační faktor 3 MeSH
• eukaryotický iniciační faktor 4E MeSH
• eukaryotický iniciační faktor 4G MeSH
• kodon iniciační MeSH
• PRP2 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• TIF4631 protein, S cerevisiae MeSH Prohlížeč
• transkripční faktory bZIP MeSH

Translational control targeting the initiation phase is central to the regulation of gene expression. Understanding all of its aspects requires substantial technological advancements. Here we modified yeast translation complex profile sequencing (TCP-seq), related to ribosome profiling, and adapted it for mammalian cells. Human TCP-seq, capable of capturing footprints of 40S subunits (40Ss) in addition to 80S ribosomes (80Ss), revealed that mammalian and yeast 40Ss distribute similarly across 5'TRs, indicating considerable evolutionary conservation. We further developed yeast and human selective TCP-seq (Sel-TCP-seq), enabling selection of 40Ss and 80Ss associated with immuno-targeted factors. Sel-TCP-seq demonstrated that eIF2 and eIF3 travel along 5' UTRs with scanning 40Ss to successively dissociate upon AUG recognition; notably, a proportion of eIF3 lingers on during the initial elongation cycles. Highlighting Sel-TCP-seq versatility, we also identified four initiating 48S conformational intermediates, provided novel insights into ATF4 and GCN4 mRNA translational control, and demonstrated co-translational assembly of initiation factor complexes.

• Klíčová slova
• ATF4, GCN4, Ribo-seq, TCP-seq, UTR, co-translational assembly, eIF2, eIF3, gene expression, mRNA, ribosome, ribosome profiling, translational control,
• MeSH
• 5' nepřekládaná oblast MeSH
• eukaryotický iniciační faktor 2 genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• iniciační faktory genetika   metabolismus   MeSH
• kodon iniciační MeSH
• lidé MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• multiproteinové komplexy genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• transkripční faktor ATF4 genetika   metabolismus   MeSH
• transkripční faktory bZIP genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• ATF4 protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 2 MeSH
• eukaryotický iniciační faktor 3 MeSH
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• iniciační faktory MeSH
• kodon iniciační MeSH
• multiproteinové komplexy MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktor ATF4 MeSH
• transkripční faktory bZIP MeSH

eIF3 is a large multiprotein complex serving as an essential scaffold promoting binding of other eIFs to the 40S subunit, where it coordinates their actions during translation initiation. Perhaps due to a high degree of flexibility of multiple eIF3 subunits, a high-resolution structure of free eIF3 from any organism has never been solved. Employing genetics and biochemistry, we previously built a 2D interaction map of all five yeast eIF3 subunits. Here we further improved the previously reported in vitro reconstitution protocol of yeast eIF3, which we cross-linked and trypsin-digested to determine its overall shape in 3D by advanced mass-spectrometry. The obtained cross-links support our 2D subunit interaction map and reveal that eIF3 is tightly packed with its WD40 and RRM domains exposed. This contrasts with reported cryo-EM structures depicting eIF3 as a molecular embracer of the 40S subunit. Since the binding of eIF1 and eIF5 further fortified the compact architecture of eIF3, we suggest that its initial contact with the 40S solvent-exposed side makes eIF3 to open up and wrap around the 40S head with its extended arms. In addition, we mapped the position of eIF5 to the region below the P- and E-sites of the 40S subunit.

• MeSH
• elektronová kryomikroskopie MeSH
• eukaryotický iniciační faktor 1 chemie   genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• eukaryotický iniciační faktor 5 chemie   genetika   metabolismus   MeSH
• iniciace translace peptidového řetězce * MeSH
• malé podjednotky ribozomu eukaryotické genetika   metabolismus   MeSH
• molekulární modely MeSH
• proteinové domény MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   ultrastruktura   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 1 MeSH
• eukaryotický iniciační faktor 3 MeSH
• eukaryotický iniciační faktor 5 MeSH
• Saccharomyces cerevisiae - proteiny MeSH

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.

• Klíčová slova
• ATF4, GCN4, eIF3, mRNA, reinitiation, ribosome, translational control,
• 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
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• messenger RNA MeSH
• transkripční faktor ATF4 MeSH

Protein synthesis is mediated via numerous molecules including the ribosome, mRNA, tRNAs, as well as translation initiation, elongation and release factors. Some of these factors play several roles throughout the entire process to ensure proper assembly of the preinitiation complex on the right mRNA, accurate selection of the initiation codon, errorless production of the encoded polypeptide and its proper termination. Perhaps, the most intriguing of these multitasking factors is the eukaryotic initiation factor eIF3. Recent evidence strongly suggests that this factor, which coordinates the progress of most of the initiation steps, does not come off the initiation complex upon subunit joining, but instead it remains bound to 80S ribosomes and gradually falls off during the first few elongation cycles to: (1) promote resumption of scanning on the same mRNA molecule for reinitiation downstream-in case of translation of upstream ORFs short enough to preserve eIF3 bound; or (2) come back during termination on long ORFs to fine tune its fidelity or, if signaled, promote programmed stop codon readthrough. Here, we unite recent structural views of the eIF3-40S complex and discus all known eIF3 roles to provide a broad picture of the eIF3's impact on translational control in eukaryotic cells.

• MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• konformace proteinů * MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• molekulární modely MeSH
• podjednotky proteinů chemie   genetika   metabolismus   MeSH
• proteosyntéza * MeSH
• ribozomy genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• messenger RNA MeSH
• podjednotky proteinů MeSH
• Saccharomyces cerevisiae - proteiny MeSH

For many years initiation and termination of mRNA translation have been studied separately. However, a direct link between these 2 isolated stages has been suggested by the fact that some initiation factors also control termination and can even promote ribosome recycling; i.e. the last stage where post-terminating 80S ribosomes are split to start a new round of initiation. Notably, it is now firmly established that, among other factors, ribosomal recycling critically requires the NTPase ABCE1. However, several earlier reports have proposed that ABCE1 also somehow participates in the initiation complex assembly. Based on an extended analysis of our recently published late-stage 48S initiation complex from rabbit, here we provide new mechanistic insights into this putative role of ABCE1 in initiation. This point of view represents the first structural evidence in which the regulatory role of the recycling factor ABCE1 in initiation is discussed and establishes a corner stone for elucidating the interplay between ABCE1 and several initiation factors during the transit from ribosomal recycling to formation of the elongation competent 80S initiation complex.

• Klíčová slova
• ABCE1, cryo-EM, recycling, ribosome, translation,
• MeSH
• ABC transportéry chemie   metabolismus   MeSH
• elongační faktory MeSH
• hydrolýza MeSH
• iniciace translace peptidového řetězce * MeSH
• iniciační faktory metabolismus   MeSH
• králíci MeSH
• molekulární modely MeSH
• nukleosidy chemie   MeSH
• ribozomy metabolismus   MeSH
• terminace translace peptidového řetězce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• králíci MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• ABC transportéry MeSH
• elongační faktory MeSH
• iniciační faktory MeSH
• nukleosidy 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
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• eukaryotický iniciační faktor 3 MeSH
• terminační kodon MeSH