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.

• Keywords
• MAPK pathway, eIF3, genetics, genomics, human, ribosomal proteins, ribosome, translation, translational control,
• MeSH
• Eukaryotic Initiation Factor-3 * metabolism   genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• MAP Kinase Signaling System * MeSH
• Protein Biosynthesis MeSH
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 * MeSH
• MAS1 protein, human MeSH Browser
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * 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.

• Keywords
• ES6(S), ES7(S), ES9(S), Trypanosoma cruzi, cryo-EM, eIF1, eIF2, eIF3, eIF5-CTD, k-DDX60, the 43S pre-initiation complex, translation initiation,
• 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.

• Keywords
• ATF4, GCN4, Ribo-seq, TCP-seq, UTR, co-translational assembly, eIF2, eIF3, gene expression, mRNA, ribosome, ribosome profiling, translational control,
• 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
• Names of Substances
• 5' Untranslated Regions MeSH
• ATF4 protein, human MeSH Browser
• Eukaryotic Initiation Factor-2 MeSH
• Eukaryotic Initiation Factor-3 MeSH
• GCN4 protein, S cerevisiae MeSH Browser
• Peptide Initiation Factors MeSH
• Codon, Initiator MeSH
• Multiprotein Complexes MeSH
• Saccharomyces cerevisiae Proteins MeSH
• Activating Transcription Factor 4 MeSH
• Basic-Leucine Zipper Transcription Factors 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
• Names of Substances
• EIF3C protein, human MeSH Browser
• EIF3D protein, human MeSH Browser
• EIF3K protein, human MeSH Browser
• EIF3L protein, human MeSH Browser
• Eukaryotic Initiation Factor-3 MeSH
• Formaldehyde MeSH
• RNA, Messenger MeSH
• Microtubule-Associated Proteins MeSH
• Cross-Linking Reagents 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
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• Ribosomal Proteins MeSH
• RNA, Transfer MeSH
• RPS3 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Codon, Terminator 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
• Names of Substances
• Eukaryotic Initiation Factor-1 MeSH
• Eukaryotic Initiation Factor-3 MeSH
• Eukaryotic Initiation Factor-5 MeSH
• Saccharomyces cerevisiae Proteins MeSH

The integrated stress response (ISR) is a homeostatic mechanism induced by endoplasmic reticulum (ER) stress. In acute/transient ER stress, decreased global protein synthesis and increased uORF mRNA translation are followed by normalization of protein synthesis. Here, we report a dramatically different response during chronic ER stress. This chronic ISR program is characterized by persistently elevated uORF mRNA translation and concurrent gene expression reprogramming, which permits simultaneous stress sensing and proteostasis. The program includes PERK-dependent switching to an eIF3-dependent translation initiation mechanism, resulting in partial, but not complete, translational recovery, which, together with transcriptional reprogramming, selectively bolsters expression of proteins with ER functions. Coordination of transcriptional and translational reprogramming prevents ER dysfunction and inhibits "foamy cell" development, thus establishing a molecular basis for understanding human diseases associated with ER dysfunction.

• Keywords
• ER stress, PERK, eIF2, eIF2B, eIF3, integrated stress response, mRNA translation, protein synthesis, stress signaling, unfolded protein response,
• MeSH
• Time Factors MeSH
• Eukaryotic Initiation Factor-3 genetics   metabolism   MeSH
• Phenotype MeSH
• Fibroblasts metabolism   pathology   MeSH
• Transcription, Genetic * MeSH
• HEK293 Cells MeSH
• Proteostasis MeSH
• eIF-2 Kinase genetics   metabolism   MeSH
• Humans MeSH
• RNA, Messenger biosynthesis   genetics   MeSH
• Mice MeSH
• Open Reading Frames MeSH
• Cellular Reprogramming MeSH
• Protein Biosynthesis * MeSH
• RNA Interference MeSH
• Signal Transduction MeSH
• Endoplasmic Reticulum Stress * MeSH
• Transfection MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• eIF-2 Kinase MeSH
• RNA, Messenger MeSH
• PERK kinase MeSH Browser

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.

• Keywords
• ATF4, GCN4, eIF3, mRNA, reinitiation, ribosome, translational control,
• MeSH
• Eukaryotic Initiation Factor-3 chemistry   metabolism   MeSH
• Peptide Chain Initiation, Translational * MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Open Reading Frames * MeSH
• Protein Biosynthesis MeSH
• Ribosomes metabolism   MeSH
• Mammals MeSH
• Activating Transcription Factor 4 chemistry   metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• RNA, Messenger MeSH
• Activating Transcription Factor 4 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
• Eukaryotic Initiation Factor-3 chemistry   genetics   metabolism   MeSH
• Protein Conformation * MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Models, Molecular MeSH
• Protein Subunits chemistry   genetics   metabolism   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes genetics   metabolism   MeSH
• Saccharomyces cerevisiae Proteins chemistry   genetics   metabolism   MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• RNA, Messenger MeSH
• Protein Subunits MeSH
• Saccharomyces cerevisiae Proteins MeSH