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.
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 Helicases genetics MeSH
- Eukaryotic Initiation Factor-3 genetics MeSH
- Eukaryotic Initiation Factor-4E genetics MeSH
- Eukaryotic Initiation Factor-4G genetics MeSH
- Peptide Chain Initiation, Translational genetics MeSH
- Codon, Initiator genetics MeSH
- Humans MeSH
- Open Reading Frames genetics MeSH
- Protein Biosynthesis genetics MeSH
- Ribosomes genetics MeSH
- Saccharomyces cerevisiae Proteins genetics MeSH
- Saccharomyces cerevisiae genetics MeSH
- Basic-Leucine Zipper Transcription Factors genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- MeSH
- Cytoplasmic Granules physiology MeSH
- DNA-Binding Proteins genetics metabolism MeSH
- Endoplasmic Reticulum metabolism MeSH
- Eukaryotic Initiation Factor-3 chemistry genetics metabolism MeSH
- Humans MeSH
- RNA, Messenger genetics metabolism MeSH
- Mitochondria metabolism MeSH
- Heat-Shock Response * MeSH
- Saccharomyces cerevisiae Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae genetics growth & development metabolism MeSH
- Protein Stability MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Canonical mRNA translation in eukaryotes begins with the formation of the 43S pre-initiation complex (PIC). Its assembly requires binding of initiator Met-tRNAiMet and several eukaryotic initiation factors (eIFs) to the small ribosomal subunit (40S). Compared to their mammalian hosts, trypanosomatids present significant structural differences in their 40S, suggesting substantial variability in translation initiation. Here, we determine the structure of the 43S PIC from Trypanosoma cruzi, the parasite causing Chagas disease. Our structure shows numerous specific features, such as the variant eIF3 structure and its unique interactions with the large rRNA expansion segments (ESs) 9S, 7S, and 6S, and the association of a kinetoplastid-specific DDX60-like helicase. It also reveals the 40S-binding site of the eIF5 C-terminal domain and structures of key terminal tails of several conserved eIFs underlying their activities within the PIC. Our results are corroborated by glutathione S-transferase (GST) pull-down assays in both human and T. cruzi and mass spectrometry data.
- MeSH
- Models, Molecular MeSH
- Protein Biosynthesis immunology MeSH
- Mammals MeSH
- Trypanosomatina pathogenicity MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- MeSH
- 5' Untranslated Regions MeSH
- Eukaryotic Initiation Factor-2 genetics metabolism MeSH
- Eukaryotic Initiation Factor-3 genetics metabolism MeSH
- HEK293 Cells MeSH
- Peptide Initiation Factors genetics metabolism MeSH
- Codon, Initiator MeSH
- Humans MeSH
- Ribosome Subunits, Small, Eukaryotic genetics metabolism MeSH
- Multiprotein Complexes genetics metabolism MeSH
- Protein Biosynthesis * MeSH
- Ribosomes genetics metabolism MeSH
- Saccharomyces cerevisiae Proteins genetics metabolism MeSH
- Saccharomyces cerevisiae genetics MeSH
- Activating Transcription Factor 4 genetics metabolism MeSH
- Basic-Leucine Zipper Transcription Factors genetics metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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.
- MeSH
- Autoantigens immunology MeSH
- Autoantibodies blood MeSH
- Biomarkers blood MeSH
- Adult MeSH
- Eukaryotic Initiation Factor-3 blood immunology MeSH
- Mass Spectrometry methods MeSH
- Immunoprecipitation methods MeSH
- Immunosuppressive Agents administration & dosage MeSH
- Middle Aged MeSH
- Humans MeSH
- Polymyositis drug therapy immunology physiopathology MeSH
- Disease Progression * MeSH
- Reference Values MeSH
- Retrospective Studies MeSH
- Rheumatic Fever immunology physiopathology MeSH
- Sensitivity and Specificity MeSH
- Sjogren's Syndrome immunology physiopathology MeSH
- Case-Control Studies MeSH
- Severity of Illness Index MeSH
- Lupus Erythematosus, Systemic immunology physiopathology MeSH
- Blotting, Western methods MeSH
- Check Tag
- Adult MeSH
- Middle Aged MeSH
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Comparative Study 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
- Eukaryotic Initiation Factor-3 genetics MeSH
- Formaldehyde pharmacology MeSH
- Humans MeSH
- Ribosome Subunits, Small, Eukaryotic genetics MeSH
- RNA, Messenger genetics MeSH
- Microtubule-Associated Proteins genetics MeSH
- Protein Biosynthesis genetics MeSH
- Cross-Linking Reagents pharmacology MeSH
- Ribosomes genetics MeSH
- Protein Binding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Eukaryotic Initiation Factor-3 genetics metabolism MeSH
- Organisms, Genetically Modified MeSH
- Protein Biosynthesis genetics MeSH
- Ribosomal Proteins genetics physiology MeSH
- Ribosomes metabolism MeSH
- RNA, Transfer metabolism MeSH
- Saccharomyces cerevisiae Proteins genetics physiology MeSH
- Saccharomyces cerevisiae genetics metabolism MeSH
- Peptide Chain Termination, Translational * genetics MeSH
- Codon, Terminator metabolism MeSH
- Protein Binding MeSH
- Binding Sites genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't 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
- Cryoelectron Microscopy MeSH
- Eukaryotic Initiation Factor-1 chemistry genetics metabolism MeSH
- Eukaryotic Initiation Factor-3 chemistry genetics metabolism MeSH
- Eukaryotic Initiation Factor-5 chemistry genetics metabolism MeSH
- Peptide Chain Initiation, Translational * MeSH
- Ribosome Subunits, Small, Eukaryotic genetics metabolism MeSH
- Models, Molecular MeSH
- Protein Domains MeSH
- Saccharomyces cerevisiae Proteins chemistry genetics metabolism MeSH
- Saccharomyces cerevisiae genetics metabolism ultrastructure MeSH
- Protein Binding MeSH
- Binding Sites genetics MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
- MeSH
- Eukaryotic Initiation Factor-3 genetics MeSH
- Humans MeSH
- Actin Cytoskeleton genetics MeSH
- Mitochondria MeSH
- Mutation MeSH
- Myosin Type V genetics MeSH
- Protein Aggregates genetics MeSH
- HSP40 Heat-Shock Proteins genetics MeSH
- HSP70 Heat-Shock Proteins genetics MeSH
- Saccharomyces cerevisiae Proteins genetics MeSH
- Saccharomyces cerevisiae genetics growth & development MeSH
- Myosin Heavy Chains genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
