UNLABELLED: Trypanosomatids are among the most extensively studied protists due to their parasitic interactions with insects, vertebrates, and plants. Recently, Blastocrithidia nonstop was found to depart from the canonical genetic code, with all three stop codons reassigned to encode amino acids (UAR for glutamate and UGA for tryptophan), and UAA having dual meaning also as a termination signal (glutamate and stop). To explore features linked to this phenomenon, we analyzed the genomes of four Blastocrithidia and four Obscuromonas species, the latter representing a sister group employing the canonical genetic code. We found that all Blastocrithidia species encode cognate tRNAs for UAR codons, possess a distinct 4 bp anticodon stem tRNATrpCCA decoding UGA, and utilize UAA as the only stop codon. The distribution of in-frame reassigned codons is consistently non-random, suggesting a translational burden avoided in highly expressed genes. Frame-specific enrichment of UAA codons immediately following the genuine UAA stop codon, not observed in Obscuromonas, points to a specific mode of termination. All Blastocrithidia species possess specific mutations in eukaryotic release factor 1 and a unique acidic region following the prion-like N-terminus of eukaryotic release factor 3 that may be associated with stop codon readthrough. We infer that the common ancestor of the genus Blastocrithidia already exhibited a GC-poor genome with the non-canonical genetic code. Our comparative analysis highlights features associated with this extensive stop codon reassignment. This cascade of mutually dependent adaptations, driven by increasing AU-richness in transcripts and frequent emergence of in-frame stops, underscores the dynamic interplay between genome composition and genetic code plasticity to maintain vital functionality. IMPORTANCE: The genetic code, assigning amino acids to codons, is almost universal, yet an increasing number of its alterations keep emerging, mostly in organelles and unicellular eukaryotes. One such case is the trypanosomatid genus Blastocrithidia, where all three stop codons were reassigned to amino acids, with UAA also serving as a sole termination signal. We conducted a comparative analysis of four Blastocrithidia species, all with the same non-canonical genetic code, and their close relatives of the genus Obscuromonas, which retain the canonical code. This across-genome comparison allowed the identification of key traits associated with genetic code reassignment in Blastocrithidia. This work provides insight into the evolutionary steps, facilitating an extensive departure from the canonical genetic code that occurred independently in several eukaryotic lineages.

• Keywords
• AT-rich genomes, eukaryotic release factors, nuclear genetic code, reassigned codon, tRNA structure, termination of translation,
• MeSH
• Cell Nucleus * genetics   MeSH
• Phylogeny MeSH
• Genetic Code * MeSH
• Genome, Protozoan * MeSH
• Genomics MeSH
• Evolution, Molecular MeSH
• RNA, Transfer genetics   MeSH
• Codon, Terminator genetics   MeSH
• Trypanosomatina * genetics   classification   MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH
• Names of Substances
• RNA, Transfer MeSH
• Codon, Terminator MeSH

Transfer RNAs (tRNAs) serve as a dictionary for the ribosome translating the genetic message from mRNA into a polypeptide chain. In addition to this canonical role, tRNAs are involved in other processes such as programmed stop codon readthrough (SC-RT). There, tRNAs with near-cognate anticodons to stop codons must outcompete release factors and incorporate into the ribosomal decoding center to prevent termination and allow translation to continue. However, not all near-cognate tRNAs promote efficient SC-RT. Here, with the help of Saccharomyces cerevisiae and Trypanosoma brucei, we demonstrate that those tRNAs that promote efficient SC-RT establish critical contacts between their anticodon stem (AS) and ribosomal proteins Rps30/eS30 and Rps25/eS25 forming the decoding site. Unexpectedly, the length and well-defined nature of the AS determine the strength of these contacts, which is reflected in organisms with reassigned stop codons. These findings open an unexplored direction in tRNA biology that should facilitate the design of artificial tRNAs with specifically altered decoding abilities.

• MeSH
• Anticodon metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Protein Biosynthesis * MeSH
• Ribosomal Proteins metabolism   MeSH
• Ribosomes * metabolism   MeSH
• RNA, Transfer * metabolism   genetics   chemistry   MeSH
• Saccharomyces cerevisiae genetics   metabolism   MeSH
• Codon, Terminator * genetics   metabolism   MeSH
• Trypanosoma brucei brucei genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Anticodon MeSH
• Ribosomal Proteins MeSH
• RNA, Transfer * MeSH
• Codon, Terminator * 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.

• 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

Cognate tRNAs deliver specific amino acids to translating ribosomes according to the standard genetic code, and three codons with no cognate tRNAs serve as stop codons. Some protists have reassigned all stop codons as sense codons, neglecting this fundamental principle1-4. Here we analyse the in-frame stop codons in 7,259 predicted protein-coding genes of a previously undescribed trypanosomatid, Blastocrithidia nonstop. We reveal that in this species in-frame stop codons are underrepresented in genes expressed at high levels and that UAA serves as the only termination codon. Whereas new tRNAsGlu fully cognate to UAG and UAA evolved to reassign these stop codons, the UGA reassignment followed a different path through shortening the anticodon stem of tRNATrpCCA from five to four base pairs (bp). The canonical 5-bp tRNATrp recognizes UGG as dictated by the genetic code, whereas its shortened 4-bp variant incorporates tryptophan also into in-frame UGA. Mimicking this evolutionary twist by engineering both variants from B. nonstop, Trypanosoma brucei and Saccharomyces cerevisiae and expressing them in the last two species, we recorded a significantly higher readthrough for all 4-bp variants. Furthermore, a gene encoding B. nonstop release factor 1 acquired a mutation that specifically restricts UGA recognition, robustly potentiating the UGA reassignment. Virtually the same strategy has been adopted by the ciliate Condylostoma magnum. Hence, we describe a previously unknown, universal mechanism that has been exploited in unrelated eukaryotes with reassigned stop codons.

• MeSH
• Anticodon * chemistry   genetics   metabolism   MeSH
• Ciliophora genetics   MeSH
• Eukaryotic Cells * MeSH
• Genetic Code * genetics   MeSH
• Mutation * MeSH
• Peptide Termination Factors * genetics   metabolism   MeSH
• RNA, Transfer, Glu genetics   MeSH
• RNA, Transfer, Trp genetics   MeSH
• RNA, Transfer * genetics   metabolism   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Codon, Terminator * genetics   MeSH
• Trypanosoma brucei brucei genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anticodon * MeSH
• Peptide Termination Factors * MeSH
• RNA, Transfer, Glu MeSH
• RNA, Transfer, Trp MeSH
• RNA, Transfer * MeSH
• Codon, Terminator * 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.

• Keywords
• ER, ERMES, Hsp104, Rpg1, TDP-43, eIF3, heat shock, mitochondria, stress granules, yeast,
• 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
• Names of Substances
• DNA-Binding Proteins MeSH
• EIF3A protein, human MeSH Browser
• Eukaryotic Initiation Factor-3 MeSH
• RNA, Messenger MeSH
• RPG1 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• TARDBP protein, human MeSH Browser

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

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.

• Keywords
• Actin, Aggregation, Asymmetric segregation, Hsp40, Hsp70, Myo2, Rpg1/eIF3a, Yeast,
• 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
• Names of Substances
• Eukaryotic Initiation Factor-3 MeSH
• MYO2 protein, S cerevisiae MeSH Browser
• Myosin Type V MeSH
• Protein Aggregates MeSH
• HSP40 Heat-Shock Proteins MeSH
• HSP70 Heat-Shock Proteins MeSH
• RPG1 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• Myosin Heavy Chains MeSH