RESUMEN
Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S subunit from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation at short upstream open reading frames (uORFs) harboring penultimate codons that confer dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited reinitiation at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on reinitiation at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) reinitiation. We found that the Tma proteins generally impede reinitiation at native uORF4 and uORF4 variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on reinitiation at native uORF1, and equipping uORF1 with Tma-dependent penultimate codons generally did not confer Tma-dependent reinitiation; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the reinitiation potential of the uORF and the penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.
RESUMEN
Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.
Asunto(s)
Factor de Transcripción Activador 4 , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , Ribosomas , Factor de Transcripción Activador 4/metabolismo , Factor de Transcripción Activador 4/genética , Humanos , Ribosomas/metabolismo , Sistemas de Lectura Abierta/genética , Estrés Fisiológico , Células HEK293 , Secuencia de BasesRESUMEN
Bacteria have evolved structured RNAs that can associate with RNA polymerase (RNAP). Two of them have been known so far-6S RNA and Ms1 RNA but it is unclear if any other types of RNAs binding to RNAP exist in bacteria. To identify all RNAs interacting with RNAP and the primary σ factors, we have established and performed native RIP-seq in Bacillus subtilis, Corynebacterium glutamicum, Streptomyces coelicolor, Mycobacterium smegmatis and the pathogenic Mycobacterium tuberculosis. Besides known 6S RNAs in B. subtilis and Ms1 in M. smegmatis, we detected MTS2823, a homologue of Ms1, on RNAP in M. tuberculosis. In C. glutamicum, we discovered novel types of structured RNAs that associate with RNAP. Furthermore, we identified other species-specific RNAs including full-length mRNAs, revealing a previously unknown landscape of RNAs interacting with the bacterial transcription machinery.
Asunto(s)
Proteínas Bacterianas , ARN Polimerasas Dirigidas por ADN , ARN Bacteriano , Factor sigma , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Regulación Bacteriana de la Expresión Génica , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/enzimología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/metabolismo , ARN Bacteriano/genética , ARN no Traducido , Factor sigma/metabolismo , Factor sigma/genética , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo , Transcripción GenéticaRESUMEN
ATF4 is a master transcriptional regulator of the integrated stress response leading cells towards adaptation or death. ATF4's induction under stress was thought to be mostly due to delayed translation reinitiation, where the reinitiation-permissive uORF1 plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations, but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrate that the canonical ATF4 translation start site is substantially leaky-scanned. Thus, ATF4's translational control is more complex than originally described underpinning its key role in diverse biological processes.
RESUMEN
Under certain circumstances, any of the three termination codons can be read through by a near-cognate tRNA; i.e., a tRNA whose two out of three anticodon nucleotides base pair with those of the stop codon. Unless programed to synthetize C-terminally extended protein variants with expanded physiological roles, readthrough represents an undesirable translational error. On the other side of a coin, a significant number of human genetic diseases is associated with the introduction of nonsense mutations (premature termination codons [PTCs]) into coding sequences, where stopping is not desirable. Here, the tRNA's ability to induce readthrough opens up the intriguing possibility of mitigating the deleterious effects of PTCs on human health. In yeast, the UGA and UAR stop codons were described to be read through by four readthrough-inducing rti-tRNAs-tRNATrp and tRNACys, and tRNATyr and tRNAGln, respectively. The readthrough-inducing potential of tRNATrp and tRNATyr was also observed in human cell lines. Here, we investigated the readthrough-inducing potential of human tRNACys in the HEK293T cell line. The tRNACys family consists of two isoacceptors, one with ACA and the other with GCA anticodons. We selected nine representative tRNACys isodecoders (differing in primary sequence and expression level) and tested them using dual luciferase reporter assays. We found that at least two tRNACys can significantly elevate UGA readthrough when overexpressed. This indicates a mechanistically conserved nature of rti-tRNAs between yeast and human, supporting the idea that they could be used in the PTC-associated RNA therapies.
Asunto(s)
Cisteína , Saccharomyces cerevisiae , Humanos , Codón de Terminación/genética , Cisteína/genética , Cisteína/metabolismo , Células HEK293 , Saccharomyces cerevisiae/genética , ARN de Transferencia de Cisteína/metabolismo , ARN de Transferencia de Triptófano/metabolismo , ARN de Transferencia de Tirosina , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Anticodón , Codón sin Sentido/genética , Biosíntesis de ProteínasRESUMEN
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.
Asunto(s)
Anticodón , Codón de Terminación , Células Eucariotas , Código Genético , Mutación , Factores de Terminación de Péptidos , ARN de Transferencia , Anticodón/química , Anticodón/genética , Anticodón/metabolismo , Cilióforos/genética , Codón de Terminación/genética , Código Genético/genética , Factores de Terminación de Péptidos/genética , Factores de Terminación de Péptidos/metabolismo , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , ARN de Transferencia de Triptófano/genética , Saccharomyces cerevisiae/genética , ARN de Transferencia de Ácido Glutámico/genética , Trypanosoma brucei brucei/genéticaRESUMEN
Multiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.
Asunto(s)
Biosíntesis de Proteínas , Saccharomyces cerevisiae , Animales , Mamíferos/genética , ARN Mensajero/genética , Ribosomas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
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.
Asunto(s)
ARN Helicasas DEAD-box/genética , Factor 3 de Iniciación Eucariótica/genética , Factor 4G Eucariótico de Iniciación/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Codón Iniciador/genética , Factor 4E Eucariótico de Iniciación/genética , Humanos , Sistemas de Lectura Abierta/genética , Iniciación de la Cadena Peptídica Traduccional/genética , Biosíntesis de Proteínas/genética , Ribosomas/genética , Saccharomyces cerevisiae/genéticaRESUMEN
Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.
Asunto(s)
Codón de Terminación , Biosíntesis de Proteínas , ARN de Transferencia de Triptófano/metabolismo , ARN de Transferencia de Tirosina/metabolismo , Línea Celular , Genes Reporteros , Humanos , Mutación , Plásmidos/genética , Regiones Promotoras Genéticas , Proteínas/genética , ARN Nuclear Pequeño/genética , ARN de Transferencia de Triptófano/genética , ARN de Transferencia de Tirosina/genética , Triptófano-ARNt Ligasa/genética , Proteína p53 Supresora de Tumor/biosíntesis , Proteína p53 Supresora de Tumor/genética , Tirosina-ARNt Ligasa/genética , Proteínas Virales/genéticaRESUMEN
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.
Asunto(s)
Biosíntesis de Proteínas/inmunología , Trypanosomatina/patogenicidad , Animales , Mamíferos , Modelos MolecularesRESUMEN
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.
Asunto(s)
Complejos Multiproteicos/metabolismo , Factores de Iniciación de Péptidos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Regiones no Traducidas 5' , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Codón Iniciador , Factor 2 Eucariótico de Iniciación/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Factor 3 de Iniciación Eucariótica/genética , Factor 3 de Iniciación Eucariótica/metabolismo , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Factores de Iniciación de Péptidos/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/metabolismo , Ribosomas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
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.
Asunto(s)
Factor 3 de Iniciación Eucariótica/genética , Proteínas Asociadas a Microtúbulos/genética , Ribosomas/genética , Reactivos de Enlaces Cruzados/farmacología , Formaldehído/farmacología , Humanos , Unión Proteica , Biosíntesis de Proteínas/genética , ARN Mensajero/genética , Subunidades Ribosómicas Pequeñas de Eucariotas/genéticaRESUMEN
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.
Asunto(s)
Codón de Terminación/metabolismo , Factor 3 de Iniciación Eucariótica/metabolismo , Terminación de la Cadena Péptídica Traduccional , Proteínas Ribosómicas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Sitios de Unión/genética , Factor 3 de Iniciación Eucariótica/genética , Organismos Modificados Genéticamente , Terminación de la Cadena Péptídica Traduccional/genética , Unión Proteica , Biosíntesis de Proteínas/genética , ARN de Transferencia/metabolismo , Proteínas Ribosómicas/genética , Ribosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Reproduction success in angiosperm plants depends on robust pollen tube growth through the female pistil tissues to ensure successful fertilization. Accordingly, there is an apparent evolutionary trend to accumulate significant reserves during pollen maturation, including a population of stored mRNAs, that are utilized later for a massive translation of various proteins in growing pollen tubes. Here, we performed a thorough transcriptomic and proteomic analysis of stored and translated transcripts in three subcellular compartments of tobacco (Nicotiana tabacum), long-term storage EDTA/puromycin-resistant particles, translating polysomes, and free ribonuclear particles, throughout tobacco pollen development and in in vitro-growing pollen tubes. We demonstrated that the composition of the aforementioned complexes is not rigid and that numerous transcripts were redistributed among these complexes during pollen development, which may represent an important mechanism of translational regulation. Therefore, we defined the pollen sequestrome as a distinct and highly dynamic compartment for the storage of stable, translationally repressed transcripts and demonstrated its dynamics. We propose that EDTA/puromycin-resistant particle complexes represent aggregated nontranslating monosomes as the primary mediators of messenger RNA sequestration. Such organization is extremely useful in fast tip-growing pollen tubes, where rapid and orchestrated protein synthesis must take place in specific regions.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Polen/genética , Polen/metabolismo , Proteómica/métodos , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Polen/crecimiento & desarrollo , Tubo Polínico/genética , Tubo Polínico/crecimiento & desarrollo , Tubo Polínico/metabolismo , Polirribosomas/genética , Polirribosomas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Ribosomas/genética , Ribosomas/metabolismo , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismoRESUMEN
Protein production must be strictly controlled at its beginning and end to synthesize a polypeptide that faithfully copies genetic information carried in the encoding mRNA. In contrast to viruses and prokaryotes, the majority of mRNAs in eukaryotes contain only one coding sequence, resulting in production of a single protein. There are, however, many exceptional mRNAs that either carry short open reading frames upstream of the main coding sequence (uORFs) or even contain multiple long ORFs. A wide variety of mechanisms have evolved in microbes and higher eukaryotes to prevent recycling of some or all translational components upon termination of the first translated ORF in such mRNAs and thereby enable subsequent translation of the next uORF or downstream coding sequence. These specialized reinitiation mechanisms are often regulated to couple translation of the downstream ORF to various stimuli. Here we review all known instances of both short uORF-mediated and long ORF-mediated reinitiation and present our current understanding of the underlying molecular mechanisms of these intriguing modes of translational control.
Asunto(s)
Bacterias/genética , Eucariontes/genética , Biosíntesis de Proteínas/fisiología , Animales , Bacterias/metabolismo , Humanos , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas/genéticaRESUMEN
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.