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

Ribosome was long considered as a critical yet passive player in protein synthesis. Only recently the role of its basic components, ribosomal RNAs and proteins, in translational control has begun to emerge. Here we examined function of the small ribosomal protein uS3/Rps3, earlier shown to interact with eukaryotic translation initiation factor eIF3, in termination. We identified two residues in consecutive helices occurring in the mRNA entry pore, whose mutations to the opposite charge either reduced (K108E) or increased (R116D) stop codon readthrough. Whereas the latter increased overall levels of eIF3-containing terminating ribosomes in heavy polysomes in vivo indicating slower termination rates, the former specifically reduced eIF3 amounts in termination complexes. Combining these two mutations with the readthrough-reducing mutations at the extreme C-terminus of the a/Tif32 subunit of eIF3 either suppressed (R116D) or exacerbated (K108E) the readthrough phenotypes, and partially corrected or exacerbated the defects in the composition of termination complexes. In addition, we found that K108 affects efficiency of termination in the termination context-specific manner by promoting incorporation of readthrough-inducing tRNAs. Together with the multiple binding sites that we identified between these two proteins, we suggest that Rps3 and eIF3 closely co-operate to control translation termination and stop codon readthrough.

• MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• geneticky modifikované organismy MeSH
• proteosyntéza genetika   MeSH
• ribozomální proteiny genetika   fyziologie   MeSH
• ribozomy metabolismus   MeSH
• RNA transferová metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   fyziologie   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• terminace translace peptidového řetězce * genetika   MeSH
• terminační kodon metabolismus   MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• ribozomální proteiny MeSH
• RNA transferová MeSH
• RPS3 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• terminační kodon 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

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

Programmed stop codon readthrough is a post-transcription regulatory mechanism specifically increasing proteome diversity by creating a pool of C-terminally extended proteins. During this process, the stop codon is decoded as a sense codon by a near-cognate tRNA, which programs the ribosome to continue elongation. The efficiency of competition for the stop codon between release factors (eRFs) and near-cognate tRNAs is largely dependent on its nucleotide context; however, the molecular mechanism underlying this process is unknown. Here, we show that it is the translation initiation (not termination) factor, namely eIF3, which critically promotes programmed readthrough on all three stop codons. In order to do so, eIF3 must associate with pre-termination complexes where it interferes with the eRF1 decoding of the third/wobble position of the stop codon set in the unfavorable termination context, thus allowing incorporation of near-cognate tRNAs with a mismatch at the same position. We clearly demonstrate that efficient readthrough is enabled by near-cognate tRNAs with a mismatch only at the third/wobble position. Importantly, the eIF3 role in programmed readthrough is conserved between yeast and humans.

• MeSH
• aminoacyl-tRNA metabolismus   MeSH
• elongace translace peptidového řetězce * MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• HeLa buňky MeSH
• iniciace translace peptidového řetězce MeSH
• kvasinky genetika   MeSH
• lidé MeSH
• paromomycin farmakologie   MeSH
• regulace genové exprese MeSH
• ribozomy účinky léků   metabolismus   MeSH
• terminační kodon * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminoacyl-tRNA MeSH
• eukaryotický iniciační faktor 3 MeSH
• paromomycin MeSH
• terminační kodon * MeSH

The main role of the translation initiation factor 3 (eIF3) is to orchestrate formation of 43S-48S preinitiation complexes (PICs). Until now, most of our knowledge on eIF3 functional contribution to regulation of gene expression comes from yeast studies. Hence, here we developed several novel in vivo assays to monitor the integrity of the 13-subunit human eIF3 complex, defects in assembly of 43S PICs, efficiency of mRNA recruitment, and postassembly events such as AUG recognition. We knocked down expression of the PCI domain-containing eIF3c and eIF3a subunits and of eIF3j in human HeLa and HEK293 cells and analyzed the functional consequences. Whereas eIF3j downregulation had barely any effect and eIF3a knockdown disintegrated the entire eIF3 complex, eIF3c knockdown produced a separate assembly of the a, b, g, and i subunits (closely resembling the yeast evolutionary conserved eIF3 core), which preserved relatively high 40S binding affinity and an ability to promote mRNA recruitment to 40S subunits and displayed defects in AUG recognition. Both eIF3c and eIF3a knockdowns also severely reduced protein but not mRNA levels of many other eIF3 subunits and indeed shut off translation. We propose that eIF3a and eIF3c control abundance and assembly of the entire eIF3 and thus represent its crucial scaffolding elements critically required for formation of PICs.

• MeSH
• buněčné linie MeSH
• eukaryotický iniciační faktor 3 genetika   metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• iniciace translace peptidového řetězce genetika   MeSH
• lidé MeSH
• malá interferující RNA MeSH
• proliferace buněk MeSH
• proteiny vázající RNA genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• ribozomální proteiny genetika   metabolismus   MeSH
• RNA interference MeSH
• RNA ribozomální genetika   MeSH
• vazba proteinů genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EIF3A protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 3 MeSH
• malá interferující RNA MeSH
• proteiny vázající RNA MeSH
• ribozomální proteiny MeSH
• RNA ribozomální MeSH

Transfer of genetic information from genes into proteins is mediated by messenger RNA (mRNA) that must be first recruited to ribosomal pre-initiation complexes (PICs) by a mechanism that is still poorly understood. Recent studies showed that besides eIF4F and poly(A)-binding protein, eIF3 also plays a critical role in this process, yet the molecular mechanism of its action is unknown. We showed previously that the PCI domain of the eIF3c/NIP1 subunit of yeast eIF3 is involved in RNA binding. To assess the role of the second PCI domain of eIF3 present in eIF3a/TIF32, we performed its mutational analysis and identified a 10-Ala-substitution (Box37) that severely reduces amounts of model mRNA in the 43-48S PICs in vivo as the major, if not the only, detectable defect. Crystal structure analysis of the a/TIF32-PCI domain at 2.65-Å resolution showed that it is required for integrity of the eIF3 core and, similarly to the c/NIP1-PCI, is capable of RNA binding. The putative RNA-binding surface defined by positively charged areas contains two Box37 residues, R363 and K364. Their substitutions with alanines severely impair the mRNA recruitment step in vivo suggesting that a/TIF32-PCI represents one of the key domains ensuring stable and efficient mRNA delivery to the PICs.

• MeSH
• alanin genetika   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• fenotyp MeSH
• iniciace translace peptidového řetězce * MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• messenger RNA metabolismus   MeSH
• molekulární modely MeSH
• mutace MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• substituce aminokyselin MeSH
• terciární struktura proteinů MeSH
• transkripční faktory bZIP genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alanin MeSH
• eukaryotický iniciační faktor 3 MeSH
• GCN4 protein, S cerevisiae MeSH Prohlížeč
• messenger RNA MeSH
• NIP1 protein, S cerevisiae MeSH Prohlížeč
• RPG1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• transkripční faktory bZIP MeSH

In mammals, double-stranded RNA (dsRNA) can mediate sequence-specific RNA interference, activate sequence-independent interferon response, or undergo RNA editing by adenosine deaminases. We showed that long hairpin dsRNA expression had negligible effects on mammalian somatic cells--expressed dsRNA was slightly edited, poorly processed into siRNAs, and it did not activate the interferon response. At the same time, we noticed reduced reporter expression in transient co-transfections, which was presumably induced by expressed dsRNA. Since transient co-transfections are frequently used for studying gene function, we systematically explored the role of expressed dsRNA in this silencing phenomenon. We demonstrate that dsRNA expressed from transiently transfected plasmids strongly inhibits the expression of co-transfected reporter plasmids but not the expression of endogenous genes or reporters stably integrated in the genome. The inhibition is concentration-dependent, it is found in different cell types, and it is independent of transfection method and dsRNA sequence. The inhibition occurs at the level of translation and involves protein kinase R, which binds the expressed dsRNA. Thus, dsRNA expression represents a hidden danger in transient transfection experiments and must be taken into account during interpretation of experimental results.

• MeSH
• buňky 3T3 MeSH
• dvouvláknová RNA metabolismus   MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• imunoprecipitace MeSH
• lidé MeSH
• malá interferující RNA genetika   MeSH
• myši MeSH
• plazmidy genetika   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• průtoková cytometrie MeSH
• regulace genové exprese genetika   MeSH
• reportérové geny genetika   MeSH
• transfekce metody   MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• dvouvláknová RNA MeSH
• malá interferující RNA MeSH
• protein-serin-threoninkinasy MeSH

Translation is divided into initiation, elongation, termination and ribosome recycling. Earlier work implicated several eukaryotic initiation factors (eIFs) in ribosomal recycling in vitro. Here, we uncover roles for HCR1 and eIF3 in translation termination in vivo. A substantial proportion of eIF3, HCR1 and eukaryotic release factor 3 (eRF3) but not eIF5 (a well-defined "initiation-specific" binding partner of eIF3) specifically co-sediments with 80S couples isolated from RNase-treated heavy polysomes in an eRF1-dependent manner, indicating the presence of eIF3 and HCR1 on terminating ribosomes. eIF3 and HCR1 also occur in ribosome- and RNA-free complexes with both eRFs and the recycling factor ABCE1/RLI1. Several eIF3 mutations reduce rates of stop codon read-through and genetically interact with mutant eRFs. In contrast, a slow growing deletion of hcr1 increases read-through and accumulates eRF3 in heavy polysomes in a manner suppressible by overexpressed ABCE1/RLI1. Based on these and other findings we propose that upon stop codon recognition, HCR1 promotes eRF3·GDP ejection from the post-termination complexes to allow binding of its interacting partner ABCE1/RLI1. Furthermore, the fact that high dosage of ABCE1/RLI1 fully suppresses the slow growth phenotype of hcr1Δ as well as its termination but not initiation defects implies that the termination function of HCR1 is more critical for optimal proliferation than its function in translation initiation. Based on these and other observations we suggest that the assignment of HCR1 as a bona fide eIF3 subunit should be reconsidered. Together our work characterizes novel roles of eIF3 and HCR1 in stop codon recognition, defining a communication bridge between the initiation and termination/recycling phases of translation.

• MeSH
• ABC transportéry genetika   MeSH
• eukaryotický iniciační faktor 3 genetika   MeSH
• iniciační faktory genetika   MeSH
• mutace MeSH
• proteosyntéza * MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   MeSH
• sekvence aminokyselin MeSH
• terminace translace peptidového řetězce * MeSH
• terminační kodon genetika   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• ABC transportéry MeSH
• eukaryotický iniciační faktor 3 MeSH
• HCR1 protein, S cerevisiae MeSH Prohlížeč
• iniciační faktory MeSH
• RLI1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• terminační kodon MeSH

The ribosome translates information encoded by mRNAs into proteins in all living cells. In eukaryotes, its small subunit together with a number of eukaryotic initiation factors (eIFs) is responsible for locating the mRNA's translational start to properly decode the genetic message that it carries. This multistep process requires timely and spatially coordinated placement of eIFs on the ribosomal surface. In our long-standing pursuit to map the 40S-binding site of one of the functionally most complex eIFs, yeast multisubunit eIF3, we identified several interactions that placed its major body to the head, beak and shoulder regions of the solvent-exposed side of the 40S subunit. Among them is the interaction between the N-terminal domain (NTD) of the a/TIF32 subunit of eIF3 and the small ribosomal protein RPS0A, residing near the mRNA exit channel. Previously, we demonstrated that the N-terminal truncation of 200 residues in tif32-Δ8 significantly reduced association of eIF3 and other eIFs with 40S ribosomes in vivo and severely impaired translation reinitiation that eIF3 ensures. Here we show that not the first but the next 200 residues of a/TIF32 specifically interact with RPS0A via its extreme C-terminal tail (CTT). Detailed analysis of the RPS0A conditional depletion mutant revealed a marked drop in the polysome to monosome ratio suggesting that the initiation rates of cells grown under non-permissive conditions were significantly impaired. Indeed, amounts of eIF3 and other eIFs associated with 40S subunits in the pre-initiation complexes in the RPS0A-depleted cells were found reduced; consistently, to the similar extent as in the tif32-Δ8 cells. Similar but less pronounced effects were also observed with the viable CTT-less mutant of RPS0A. Together we conclude that the interaction between the flexible RPS0A-CTT and the residues 200-400 of the a/TIF32-NTD significantly stimulates attachment of eIF3 and its associated eIFs to small ribosomal subunits in vivo.

• MeSH
• eukaryotický iniciační faktor 3 metabolismus   MeSH
• genový knockout MeSH
• iniciace translace peptidového řetězce * MeSH
• interakční proteinové domény a motivy MeSH
• malé podjednotky ribozomu eukaryotické metabolismus   MeSH
• podjednotky proteinů metabolismus   MeSH
• ribozomální proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• podjednotky proteinů MeSH
• ribozomální proteiny MeSH
• RPG1 protein, S cerevisiae MeSH Prohlížeč
• RPS0A protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH