The context of the ribosome binding site in mRNAs defines specificity of action of kasugamycin, an inhibitor of translation initiation
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE185757 | GEO | 2022/02/02
REPOSITORIES: GEO
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Project description:The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable context. Previous findings suggest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and particular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak context. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at the NCCs initiating N-terminal extensions on GRS1 and ALA1 mRNAs, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons.
2019-10-18 | GSE138599 | GEO
Project description:eIF3 is a multi-subunit complex thought to execute numerous functions in canonical translation initiation, including mRNA recruitment to the 40S ribosome, scanning for the start codon, and inhibition of 60S subunit joining 1–3. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors 4, but a direct involvement in translation elongation has never been demonstrated. Using selective ribosome profiling, we made the unexpected observation that eIF3 remains bound to post-initiation 80S ribosomes, followed by release after translation of ~50 codons. Furthermore, eIF3 deficiency reduces early ribosomal elongation speed, particularly on mRNAs encoding proteins associated with membrane-associated functions, resulting in defective synthesis of their encoded proteins and abnormal mitochondrial and lysosomal physiology. Accordingly, heterozygous eIF3e+/- knockout mice accumulate giant mitochondria in skeletal muscle and show a progressive decline in muscle strength with age. Hence, in addition to its canonical role in translation initiation, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for normal muscle health.
2020-06-15 | GSE149697 | GEO
Project description:eIF3 is a multi-subunit complex thought to execute numerous functions in canonical translation initiation, including mRNA recruitment to the 40S ribosome, scanning for the start codon, and inhibition of 60S subunit joining 1–3. eIF3 was also found to interact with 40S and 60S ribosomal proteins and translation elongation factors 4, but a direct involvement in translation elongation has never been demonstrated. Using selective ribosome profiling, we made the unexpected observation that eIF3 remains bound to post-initiation 80S ribosomes, followed by release after translation of ~50 codons. Furthermore, eIF3 deficiency reduces early ribosomal elongation speed, particularly on mRNAs encoding proteins associated with membrane-associated functions, resulting in defective synthesis of their encoded proteins and abnormal mitochondrial and lysosomal physiology. Accordingly, heterozygous eIF3e+/- knockout mice accumulate giant mitochondria in skeletal muscle and show a progressive decline in muscle strength with age. Hence, in addition to its canonical role in translation initiation, eIF3 interacts with 80S ribosomes to enhance, at the level of early elongation, the synthesis of proteins with membrane-associated functions, an activity that is critical for normal muscle health.
2020-06-01 | GSE131074 | GEO
Project description:Firczuk2013 - Eukaryotic mRNA translation machinery
This is a model of Saccharomyces cerevisiae
mRNA translation which includes the initiation, elongation and termination phases. The model is for 20 condon mRNAs. The building of a multi-factor complex in initiation and also the different processes in elongation and termination are modelled in detail. The model takes into account that ribosomes cover more than one codon of mRNA so that the movement of ribosomes are effectively blocked by other ribosomes several codons downstream. It is assumed that 15 codons are occupied by each ribosome. This blocking effect is considered in reaction R18 in initiation and also reaction R26, the reaction where translocation of ribosomes takes place in elongation. The kinetic functions of these two reactions are based on MacDonald et al. 1968 and Heinrich & Rapaport 1980. All other kinetic functions follow mass-action kinetics. The concentrations of transfer RNA species (Met-tRNA, aa-tRNA and tRNA in the model) are kept constant, while the other species' concentrations can change in the course of the simulation. The model describes the translation of a short mRNA with 20 codons. Therefore, all reactions in the elongation cycle (R22, R23, R25, R26, R28 and R29) and the corresponding species are replicated accordingly to model the species with ribosomes bound at different positions. In summary, the model contains 165 different species and 141 reactions.
The value of the 56 rate constant parameters were estimated by fitting the model against a series of experimental data consisting of modulation of the various translation factors (Figures 2, 3 and S3). Overall the parameter estimation was carried out over 212 different data points (steady states).
This model is described in the article:
An in vivo control map for the eukaryotic mRNA translation machinery
Helena Firczuk, Shichina Kannambath, Jürgen Pahle, Amy Claydon, Robert Beynon, John Duncan, Hans Westerhoff, Pedro Mendes and John EG McCarthy
Molecular Systems Biology. 9:635
Abstract:
Rate control analysis defines the in vivo control map governing yeast protein synthesis and generates an extensively parameterized digital model of the translation pathway. Among other non-intuitive outcomes, translation demonstrates a high degree of functional modularity and comprises a non-stoichiometric combination of proteins manifesting functional convergence on a shared maximal translation rate. In exponentially growing cells, polypeptide elongation (eEF1A, eEF2, and eEF3) exerts the strongest control. The two other strong control points are recruitment of mRNA and tRNAi to the 40S ribosomal subunit (eIF4F and eIF2) and termination (eRF1; Dbp5). In contrast, factors that are found to promote mRNA scanning efficiency on a longer than-average 5′untranslated region (eIF1, eIF1A, Ded1, eIF2B, eIF3, and eIF5) exceed the levels required for maximal control. This is expected to allow the cell to minimize scanning transition times, particularly for longer 5′UTRs. The analysis reveals these and other collective adaptations of control shared across the factors, as well as features that reflect functional modularity and system robustness. Remarkably, gene duplication is implicated in the fine control of cellular protein synthesis.
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2024-09-02 | BIOMD0000000457 | BioModels
Project description:Translation start site selection in eukaryotes is influenced by context nucleotides flanking the AUG codon and by levels of the eukaryotic translation initiation factors eIF1 and eIF5. In a search of human genes, we identified 5 Hox gene paralogs initiated by AUG codons in conserved suboptimal context as well as 13 Hox genes that contain evolutionarily conserved upstream open reading frames (uORFs) that initiate at AUG codons in poor sequence context. We analyzed published CAGE-seq data and generated CAGE-seq data from mRNAs from mouse somites. These data demonstrate that the 5’ leaders of Hox mRNAs of interest contain conserved uORFs, are much shorter than reported, and lack previously proposed IRES elements. We show that the conserved uORFs inhibit Hox reporter expression and that altering the stringency of start codon selection by overexpressing eIF1 or eIF5 modulates the expression of Hox reporters. We also show that modifying ribosome homeostasis by depleting a large ribosomal subunit protein or treating cells with sublethal concentrations of puromycin leads to lower stringency of start codon selection. Thus, altering global translation can confer gene-specific effects through altered start codon selection stringency.
2022-02-21 | GSE184515 | GEO
Project description:The conserved and essential DEAD-box RNA helicase Ded1p from yeast and its mammalian ortholog DDX3 are critical for translation initiation. Mutations in DDX3 are linked to tumorigenesis and intellectual disability, and the enzyme is targeted by diverse viruses. How Ded1p and its orthologs engage RNAs to impact translation initiation has been a longstanding, unresolved question. Here we show that Ded1p associates with the pre-initiation complex at the mRNA entry channel of the small ribosomal subunit and that the helicase unwinds mRNA structure ahead of the scanning pre-initiation complex. Defective Ded1p causes pervasive translation in 5’UTRs, starting from near-cognate initiation codons located 5' of mRNA structures and concomitant decrease of protein synthesis from of the main ORFs. The data indicate that Ded1p functions to suppress translation initiation on near-cognate codons proximal to mRNA structure and show how the helicase is targeted to specific RNA sites without common sequence signatures. Our results reveal a straightforward mechanism for the activation of upstream open reading frames and suggest that mRNA structure and proximal near-cognate initiation codons encode a widespread regulatory program for translation initiation that is sensitive to RNA helicase function.
2017-01-24 | GSE93959 | GEO
Project description:Unorthodox rules of extracting genetic information enable proteome expansion without increasing the genome size. The use of alternative translation initiation sites achieves this goal by allowing production of more than one protein from a single gene. Although several such examples have been serendipitously found in bacteria, genome-wide experimental mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin treatment followed by Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the annotated Escherichia coli genes but, strikingly, it also revealed putative alternative internal start sites in a number of genes. Experimental evidence demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose translation is initiated at an internal in-frame and out-of-frame start sites, can be functionally important and contribute to the ‘alternative’ bacterial proteome. In addition to proteome expansion, the internal start sites may play regulatory role in gene expression.
2019-03-04 | GSE122129 | GEO
Project description:The genetic code that specifies the identity of amino acids incorporated into proteins during protein synthesis is almost universally conserved. Mitochondrial translation deviates from the standard genetic code which includes the reassignment of two arginine codons into stop codons {Jukes, 1993 #438}. Translation termination at these non-canonical stop codons requires a protein factor to release the newly synthesized polypeptide chain, however, the identity of this factor is not known currently{Nadler, 2021 #406}. Here, we used gene editing and ribo-profiling in combination with cryo-electron microscopy to establish that the unusual mitochondrial release factor 1 (mtRF1) detects the non-canonical stop codons. We show that loss of mtRF1 leads to stalling of mitochondrial ribosomes on non-canonical stop codons and consequent reduced translation of cytochrome C oxidase subunit 1 that results in decreased mitochondrial respiration. We show that binding of mtRF1 to the decoding center of the ribosome stabilizes a highly unusual distortion in the mRNA conformation and that the ribosomal RNA importantly participates in the specific recognition of the non-canonical stop codons.
2023-03-21 | GSE216979 | GEO
Project description:Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation is regulated differently from canonical translation is poorly understood. We thus used start codon-selective reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of non-AUG reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA are resistant to cycloheximide (CHX), a translation inhibitor which slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes cause queuing of scanning pre-initiation complexes (PIC), preferentially enhancing otherwise poor recognition of non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. Moreover, PIC queuing can cause translation from an AUG codon in a poor context to become less sensitive to CHX. We further find that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence, but not those targeting newly initiated ribosomes. In total, these data indicate that ribosome queuing enables mRNAs with poor initiation context, namely those from non-AUG start codons, to be resistant to pharmacological inhibitors.
2019-06-05 | GSE125086 | GEO
Project description:Using quantitative profiling of initiating ribosomes, we found that ribosomal pausing at the start codon serves as a “brake” to restrain the translational output. In response to oncogenic RAS signaling, the initiation pausing relaxes and contributes to the increased translational flux. Intriguingly, mRNA m6A modification in the vicinity of start codons influences the behavior of initiating ribosomes. Under oncogenic RAS signaling, the reduced mRNA methylation leads to relaxed initiation pausing, thereby promoting malignant transformation and tumor growth. Restored initiation pausing by inhibiting m6A demethylases suppresses RAS-mediated oncogenic translation and subsequent tumorigenesis. Our findings unveil a new paradigm of translational control that is co-opted by RAS mutant cancer cells to drive malignant phenotypes.
2020-12-29 | GSE159210 | GEO