Stress granules (SGs) are membrane-less assemblies arising upon various stresses in eukaryotic cells. They sequester mRNAs and proteins from stressful conditions and modulate gene expression to enable cells to resume translation and growth after stress relief. SGs containing the translation initiation factor eIF3a/Rpg1 arise in yeast cells upon robust heat shock (HS) at 46 °C only. We demonstrate that the destabilization of Rpg1 within the PCI domain in the Rpg1-3 variant leads to SGs assembly already at moderate HS at 42 °C. These are bona fide SGs arising upon translation arrest containing mRNAs, which are components of the translation machinery, and associating with P-bodies. HS SGs associate with endoplasmatic reticulum and mitochondria and their contact sites ERMES. Although Rpg1-3-labeled SGs arise at a lower temperature, their disassembly is delayed after HS at 46 °C. Remarkably, the delayed disassembly of HS SGs after the robust HS is reversed by TDP-43, which is a human protein connected with amyotrophic lateral sclerosis. TDP-43 colocalizes with HS SGs in yeast cells and facilitates cell regrowth after the stress relief. Based on our results, we propose yeast HS SGs labeled by Rpg1 and its variants as a novel model system to study functions of TDP-43 in stress granules disassembly.

• Klíčová slova
• ER, ERMES, Hsp104, Rpg1, TDP-43, eIF3, heat shock, mitochondria, stress granules, yeast,
• MeSH
• cytoplazmatická granula fyziologie   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• endoplazmatické retikulum metabolismus   MeSH
• eukaryotický iniciační faktor 3 chemie   genetika   metabolismus   MeSH
• lidé MeSH
• messenger RNA genetika   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• reakce na tepelný šok * MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   růst a vývoj   metabolismus   MeSH
• stabilita proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• EIF3A protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 3 MeSH
• messenger RNA MeSH
• RPG1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• TARDBP protein, human MeSH Prohlížeč

Cells have elaborated a complex strategy to maintain protein homeostasis under physiological as well as stress conditions with the aim to ensure the smooth functioning of vital processes and producing healthy offspring. Impairment of one of the most important processes in living cells, translation, might have serious consequences including various brain disorders in humans. Here, we describe a variant of the translation initiation factor eIF3a, Rpg1-3, mutated in its PCI domain that displays an attenuated translation efficiency and formation of reversible assemblies at physiological growth conditions. Rpg1-3-GFP assemblies are not sequestered within mother cells only as usual for misfolded-protein aggregates and are freely transmitted from the mother cell into the bud although they are of non-amyloid nature. Their bud-directed transmission and the active movement within the cell area depend on the intact actin cytoskeleton and the related molecular motor Myo2. Mutations in the Rpg1-3 protein render not only eIF3a but, more importantly, also the eIF3 core complex prone to aggregation that is potentiated by the limited availability of Hsp70 and Hsp40 chaperones. Our results open the way to understand mechanisms yeast cells employ to cope with malfunction and aggregation of essential proteins and their complexes.

• Klíčová slova
• Actin, Aggregation, Asymmetric segregation, Hsp40, Hsp70, Myo2, Rpg1/eIF3a, Yeast,
• MeSH
• eukaryotický iniciační faktor 3 genetika   MeSH
• lidé MeSH
• mikrofilamenta genetika   MeSH
• mitochondrie MeSH
• mutace MeSH
• myosin typu V genetika   MeSH
• proteinové agregáty genetika   MeSH
• proteiny tepelného šoku HSP40 genetika   MeSH
• proteiny tepelného šoku HSP70 genetika   MeSH
• Saccharomyces cerevisiae - proteiny genetika   MeSH
• Saccharomyces cerevisiae genetika   růst a vývoj   MeSH
• těžké řetězce myosinu genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• MYO2 protein, S cerevisiae MeSH Prohlížeč
• myosin typu V MeSH
• proteinové agregáty MeSH
• proteiny tepelného šoku HSP40 MeSH
• proteiny tepelného šoku HSP70 MeSH
• RPG1 protein, S cerevisiae MeSH Prohlížeč
• Saccharomyces cerevisiae - proteiny MeSH
• těžké řetězce myosinu 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

The 12-subunit mammalian eIF3 is the largest and most complex translation initiation factor and has been implicated in numerous steps of translation initiation, termination and ribosomal recycling. Imbalanced eIF3 expression levels are observed in various types of cancer and developmental disorders, but the consequences of altered eIF3 subunit expression on its overall structure and composition, and on translation in general, remain unclear. We present the first complete in vivo study monitoring the effects of RNAi knockdown of each subunit of human eIF3 on its function, subunit balance and integrity. We show that the eIF3b and octameric eIF3a subunits serve as the nucleation core around which other subunits assemble in an ordered way into two interconnected modules: the yeast-like core and the octamer, respectively. In the absence of eIF3b neither module forms in vivo, whereas eIF3d knock-down results in severe proliferation defects with no impact on eIF3 integrity. Disrupting the octamer produces an array of subcomplexes with potential roles in translational regulation. This study, outlining the mechanism of eIF3 assembly and illustrating how imbalanced expression of eIF3 subunits impacts the factor's overall expression profile, thus provides a comprehensive guide to the human eIF3 complex and to the relationship between eIF3 misregulation and cancer.

• MeSH
• down regulace MeSH
• eukaryotický iniciační faktor 3 fyziologie   MeSH
• HeLa buňky MeSH
• lidé MeSH
• multimerizace proteinu MeSH
• multiproteinové komplexy metabolismus   MeSH
• proliferace buněk MeSH
• Saccharomyces cerevisiae MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• EIF3A protein, human MeSH Prohlížeč
• EIF3B protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 3 MeSH
• multiproteinové komplexy 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

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

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
• Názvy látek
• eukaryotické iniciační faktory MeSH

The Schizosaccharomyces pombe eIF3a ortholog (SpeIF3a) was shown to be unable to substitute for S. cerevisiae eIF3a (SceIF3a) in its essential function in the initiation of translation. Overproduction of SpeIF3a altered the distribution of SceIF3a but formation of the endogenous eIF3 complex was not affected. SpeIF3a was found to be more tightly bound to S. cerevisiae ribosomes than SceIF3a and other eIF3 subunits (eIF3g, eIF3i, eIF3j). The host cells displayed aberrant morphology and altered chitin deposition. SpeIF3a probably competes with SceIF3a for binding to either ribosomes or yet to be identified substrates.

• MeSH
• cytoplazma chemie   MeSH
• delece genu MeSH
• eukaryotický iniciační faktor 3 genetika   fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• klonování DNA MeSH
• konfokální mikroskopie MeSH
• proteosyntéza genetika   MeSH
• ribozomy metabolismus   MeSH
• Saccharomyces cerevisiae genetika   růst a vývoj   fyziologie   MeSH
• Schizosaccharomyces pombe - proteiny genetika   fyziologie   MeSH
• Schizosaccharomyces genetika   MeSH
• testy genetické komplementace MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• Schizosaccharomyces pombe - proteiny MeSH