Pollen germination and pollen tube (PT) growth are extremely sensitive to high temperatures. During heat stress (HS), global translation shuts down and favors the maintenance of the essential cellular proteome for cell viability and protection against protein misfolding. Here, we demonstrate that under normal conditions, the Arabidopsis (Arabidopsis thaliana) eukaryotic translation initiation factor subunit eif3m1/eif3m2 double mutant exhibits poor pollen germination, loss of PT integrity and an increased rate of aborted seeds. Surprisingly, under HS at 37 °C, eif3m1 pollen germination outperformed wild-type Col-0, showing enhanced PT integrity. We established that the improved thermotolerance of the eif3m1 PT was due to increased expression of its putative paralog eIF3M2, which in turn upregulated Heat Shock protein 70 (HSP70) mRNA and protein levels. Indeed, eIF3M2 overexpression upregulated HSP70 expression, whereas eif3m2 knockdown showed reduced HSP70.1 promoter activity and increased in PT burst under HS conditions. Moreover, we show that eIF3M2 coimmunoprecipitates with HSP70 in PTs and directly interacts with cytoplasmic HSP70.1/2/4 and eIF4G in Nicotiana benthamiana pavement cells. Collectively, our data revealed that plants employ the eIF3M2-HSP70 module as a regulator of thermotolerance to maintain PT membrane integrity and improve fertilization and seed set adaptation under high temperatures.

• MeSH
• Arabidopsis * genetika   metabolismus   fyziologie   MeSH
• buněčná membrána metabolismus   MeSH
• eukaryotický iniciační faktor 3 metabolismus   genetika   MeSH
• klíčení genetika   MeSH
• proteiny huseníčku * metabolismus   genetika   MeSH
• proteiny tepelného šoku HSP70 * metabolismus   genetika   MeSH
• pylová láčka * růst a vývoj   genetika   metabolismus   MeSH
• reakce na tepelný šok * genetika   MeSH
• regulace genové exprese u rostlin MeSH
• upregulace genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 MeSH
• proteiny huseníčku * MeSH
• proteiny tepelného šoku HSP70 * MeSH

Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the proto-oncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5' UTRs that are implicated in homeostatic adaptation to stress and cancer.

• Klíčová slova
• MAPK pathway, eIF3, genetics, genomics, human, ribosomal proteins, ribosome, translation, translational control,
• MeSH
• eukaryotický iniciační faktor 3 * metabolismus   genetika   MeSH
• HeLa buňky MeSH
• lidé MeSH
• MAP kinasový signální systém * MeSH
• proteosyntéza MeSH
• protoonkogen Mas * MeSH
• ribozomální proteiny * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 * MeSH
• MAS1 protein, human MeSH Prohlížeč
• protoonkogen Mas * MeSH
• ribozomální proteiny * 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

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

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

OBJECTIVES: To describe the prevalence and clinical associations of autoantibodies to a novel autoantigen, eukaryotic initiation factor 3 (eIF3), detected in idiopathic inflammatory myositis. METHODS: Sera or plasma from 678 PM patients were analysed for autoantigen specificity by radio-labelled protein immunoprecipitation (IPP). Samples immunoprecipitating the same novel autoantigens were further analysed by indirect immunofluorescence and IPP using pre-depleted cell extracts. The autoantigen was identified through a combination of IPP and MALDI-TOF mass spectrometry, and confirmed using commercial antibodies and IPP-western blots. Additional samples from patients with DM (668), DM-overlap (80), PM-overlap (191), systemic sclerosis (150), systemic lupus erythematosus (200), Sjogren's syndrome (40), rheumatoid arthritis (50) and healthy controls (150) were serotyped by IPP as disease or healthy controls. RESULTS: IPP revealed a novel pattern in three PM patients (0.44%) that was not found in disease-specific or healthy control sera. Indirect immunofluorescence demonstrated a fine cytoplasmic speckled pattern for all positive patients. Mass spectrometry analysis of the protein complex identified the target autoantigen as eIF3, a cytoplasmic complex with a role in the initiation of translation. Findings were confirmed by IPP-Western blotting. The three anti-eIF3-positive patients had no history of malignancy or interstitial lung disease, and had a favourable response to treatment. CONCLUSION: We report a novel autoantibody in 0.44% of PM patients directed against a cytoplasmic complex of proteins identified as eIF3. Although our findings need further confirmation, anti-eIF3 appears to correlate with a good prognosis and a favourable response to treatment.

• Klíčová slova
• autoantibodies, autoantigens, myositis,
• MeSH
• autoantigeny imunologie   MeSH
• autoprotilátky krev   MeSH
• biologické markery krev   MeSH
• dospělí MeSH
• eukaryotický iniciační faktor 3 krev   imunologie   MeSH
• hmotnostní spektrometrie metody   MeSH
• imunoprecipitace metody   MeSH
• imunosupresiva aplikace a dávkování   MeSH
• lidé středního věku MeSH
• lidé MeSH
• polymyozitida farmakoterapie   imunologie   patofyziologie   MeSH
• progrese nemoci * MeSH
• referenční hodnoty MeSH
• retrospektivní studie MeSH
• revmatická horečka imunologie   patofyziologie   MeSH
• senzitivita a specificita MeSH
• Sjögrenův syndrom imunologie   patofyziologie   MeSH
• studie případů a kontrol MeSH
• stupeň závažnosti nemoci MeSH
• systémový lupus erythematodes imunologie   patofyziologie   MeSH
• western blotting metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• autoantigeny MeSH
• autoprotilátky MeSH
• biologické markery MeSH
• eukaryotický iniciační faktor 3 MeSH
• imunosupresiva MeSH

One of the key roles of the 12-subunit eukaryotic translation initiation factor 3 (eIF3) is to promote the formation of the 43S and 48S pre-initiation complexes (PICs). However, particular contributions of its individual subunits to these two critical initiation reactions remained obscure. Here, we adapted formaldehyde gradient cross-linking protocol to translation studies and investigated the efficiency of the 43S and 48S PIC assembly in knockdowns of individual subunits of human eIF3 known to produce various partial subcomplexes. We revealed that eIF3d constitutes an important intermolecular bridge between eIF3 and the 40S subunit as its elimination from the eIF3 holocomplex severely compromised the 43S PIC assembly. Similarly, subunits eIF3a, c and e were found to represent an important binding force driving eIF3 binding to the 40S subunit. In addition, we demonstrated that eIF3c, and eIF3k and l subunits alter the efficiency of mRNA recruitment to 43S PICs in an opposite manner. Whereas the eIF3c knockdown reduces it, downregulation of eIF3k or eIF3l increases mRNA recruitment, suggesting that the latter subunits possess a regulatory potential. Altogether this study provides new insights into the role of human eIF3 in the initial assembly steps of the translational machinery.

• MeSH
• eukaryotický iniciační faktor 3 genetika   MeSH
• formaldehyd farmakologie   MeSH
• lidé MeSH
• malé podjednotky ribozomu eukaryotické genetika   MeSH
• messenger RNA genetika   MeSH
• proteiny asociované s mikrotubuly genetika   MeSH
• proteosyntéza genetika   MeSH
• reagencia zkříženě vázaná farmakologie   MeSH
• ribozomy genetika   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• EIF3C protein, human MeSH Prohlížeč
• EIF3D protein, human MeSH Prohlížeč
• EIF3K protein, human MeSH Prohlížeč
• EIF3L protein, human MeSH Prohlížeč
• eukaryotický iniciační faktor 3 MeSH
• formaldehyd MeSH
• messenger RNA MeSH
• proteiny asociované s mikrotubuly MeSH
• reagencia zkříženě vázaná 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

Regulation of translation represents a critical step in the regulation of gene expression. In plants, the translation regulation plays an important role at all stages of development and, during stress responses, functions as a fast and flexible tool which not only modulates the global translation rate but also controls the production of specific proteins. Regulation of translation is mostly focused on the initiation phase. There, one of essential initiation factors is the large multisubunit protein complex of eukaryotic translation initiation factor 3 (eIF3). In all eukaryotes, the general eIF3 function is to scaffold the formation of the translation initiation complex and to enhance the accuracy of scanning mechanism for start codon selection. Over the past decades, additional eIF3 functions were described as necessary for development in various eukaryotic organisms, including plants. The importance of the eIF3 complex lies not only at the global level of initiation event, but also in the precise translation regulation of specific transcripts. This review gathers the available information on functions of the plant eIF3 complex.

• Klíčová slova
• Plant development, Translation, Translation regulation, Upstream open reading frame (uORF),
• MeSH
• eukaryotický iniciační faktor 3 * metabolismus   MeSH
• kodon iniciační MeSH
• proteosyntéza genetika   MeSH
• regulace genové exprese u rostlin * genetika   MeSH
• rostliny genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• eukaryotický iniciační faktor 3 * MeSH
• kodon iniciační 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