PRRC2 proteins are translation factors that promote leaky scanning
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE211440 | GEO | 2023/02/11
REPOSITORIES: GEO
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Project description:Translation regulation occurs largely during initiation. Currently, translation initiation can be studied in vitro, but these systems lack features present in vivo and on endogenous mRNAs. Here we develop selective 40S footprinting for visualizing initiating 40S ribosomes on endogenous mRNAs in vivo. It pinpoints where on an mRNA initiation factors join the ribosome to act, and where they leave. We discover that in human cells most scanning ribosomes remain attached to the 5’ cap. Consequently, only one ribosome scans a 5’UTR at a time, and 5’UTR length affects translation efficiency. We discover that eIF3B, eIF4G1 and eIF4E remain on translating 80S ribosomes with a decay half-length of ~12 codons. Hence ribosomes retain these initiation factors while translating short upstream Open Reading Frames (uORFs), providing an explanation for how ribosomes can re-initiate translation after uORFs in humans. This method will be of use for studying translation initiation mechanisms in vivo.
2020-06-25 | GSE139391 | 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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2011-04-30 | BIOMD0000000457 | BioModels
Project description:Microarray comparisons of transcript level in wild-type Arabidopsis and eif3h mutant plants. Goal:; To detect any change in transcript level between WT and eif3h mutant. BACKGROUND: The eukaryotic translation initiation factor eIF3 has multiple roles during the initiation of translation of cytoplasmic mRNAs. However, the contributions of individual subunits of eIF3 to the translation of specific mRNAs remain poorly understood. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana it was demonstrated that the h subunit of; eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5â leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant plants revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. Independently, eIF3h also boosted polysome loading of mRNAs with long coding sequences. Moreover, the lesion in eIF3h revealed a concerted upregulation of translation for specific functional subgroups of mRNAs, including ribosomal proteins and proteins involved in photosynthesis. CONCLUSIONS: The intact eIF3h protein contributes to efficient translation initiation on 5â leader sequences harboring multiple uORFs, although mRNA features independent of uORFs were also implicated. Moreover, our data suggest that regulons of translational control can be revealed by mutations in generic translation initiation factors. Experiment Overall Design: Total RNA samples were isolated from 10-day-old wild-type and eif3h mutant plants
2008-06-14 | E-GEOD-6025 | biostudies-arrayexpress
Project description:Translation is initiated by binding of the eIF4F complex to the 5' cap of the mRNA, which is followed by scanning of the initiation codon by scanning ribosomes. Here we demonstrate that the ASC-1 complex (ASCC), which was previously shown to promote the dissociation of colliding 80S ribosomes, associates with the scanning ribosomes to regulate translation initiation. Sel-TCP-seq analysis revealed that ASCC3, a subunit of ASCC with a helicase domain, localizes predominantly to the 5' untranslated region of mRNAs. Knockdown of ASCC3 resulted in reduced translation efficiency associated with reduced 43S preinitiation complex (PIC) loading and a reduced speed of scanning ribosomes. In addition, depletion of the ubiquitin ligase ZNF598, a sensor of collided 80S ribosomes, also reduces the PIC loading and speed of scanning ribosomes. Our results have thus revealed that ASCC is required not only for dissociation of colliding 80S ribosomes, but also for efficient translation initiation by scanning ribosomes.
2023-03-31 | GSE186502 | GEO
Project description:Microarray comparisons of polysome loading in wild-type Arabidopsis and eif3h mutant; Goal: ; To find the target mRNAs that are translationally regulated by eIF3h. BACKGROUND: The eukaryotic translation initiation factor eIF3 has multiple roles during the initiation of translation of cytoplasmic mRNAs. However, the contributions of individual subunits of eIF3 to the translation of specific mRNAs remain poorly understood. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana it was demonstrated that the h subunit of; eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5â leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant plants revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. Independently, eIF3h also boosted polysome loading of mRNAs with long coding sequences. Moreover, the lesion in eIF3h revealed a concerted upregulation of translation for specific functional subgroups of mRNAs, including ribosomal proteins and proteins involved in photosynthesis. CONCLUSIONS: The intact eIF3h protein contributes to efficient translation initiation on 5â leader sequences harboring multiple uORFs, although mRNA features independent of uORFs were also implicated. Moreover, our data suggest that regulons of translational control can be revealed by mutations in generic translation initiation factors. Experiment Overall Design: Polysomal and non-polysomal RNA samples were isolated from 10-day-old wild-type and eif3h mutant plants. The translation state (ratio between the polysome and non-polysome, PL/NP) for each gene in the WT and in the mutant was separately established, and then the translation states between the genotype were compared.
2008-06-14 | E-GEOD-6024 | biostudies-arrayexpress
Project description:Alternative translation initiation mechanisms such as leaky scanning and reinitiation potentiate the polycistronic nature of transcripts. By allowing for reprogrammed translation, these mechanisms can mediate biological responses to stress stimuli. We combined proteomics with ribosome profiling and mRNA sequencing to identify the biological targets of translation control triggered by eukaryotic translation initiation factor 1 (eIF1), a protein implicated in the stringency of start codon selection. We quantified expression changes of over 4,000 proteins and 10,000 actively translated transcripts, leading to the identification of 245 transcripts undergoing translational control mediated by upstream open reading frames (uORFs) upon eIF1 deprivation. The stringency of start codon selection and preference for optimal nucleotide context were largely diminished leading to translational regulation of uORFs with sub-optimal start sites. Affected genes were implicated in energy production and sensing of metabolic stress. Interestingly, knockdown of eIF1 elicited a synergic response from eIF5 and eIF1B.
2017-01-25 | GSE87328 | GEO
Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation.
2018-08-30 | GSE108942 | GEO
Project description:Alternative translation initiation mechanisms such as leaky scanning and re-initiation potentiate the polycistronic nature of transcripts. By allowing for reprogrammed translation, these mechanisms can mediate biological responses to stress stimuli. We combined proteomics with ribosome profiling and mRNA sequencing to identify the biological targets of translation control triggered by the eukaryotic translation initiation factor 1 (eIF1), a protein implicated in the stringency of start codon selection. We quantified expression changes of over 4,000 proteins and 10,000 actively translated transcripts, leading to the identification of 245 transcripts undergoing translational control mediated by upstream open reading frames (uORFs) upon eIF1 deprivation. The stringency of start codon selection and preference for an optimal nucleotide context were largely diminished leading to translational upregulation of uORFs with sub-optimal start sites. Affected genes were implicated in energy production and sensing of metabolic stress. Interestingly, knockdown of eIF1 elicited a synergic response from eIF5 and eIF1B.
2017-09-13 | PXD004980 | Pride
Project description:Microarray comparisons of polysome loading in wild-type Arabidopsis and eif3h mutant Goal: To find the target mRNAs that are translationally regulated by eIF3h. BACKGROUND: The eukaryotic translation initiation factor eIF3 has multiple roles during the initiation of translation of cytoplasmic mRNAs. However, the contributions of individual subunits of eIF3 to the translation of specific mRNAs remain poorly understood. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana it was demonstrated that the h subunit of eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5’ leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant plants revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. Independently, eIF3h also boosted polysome loading of mRNAs with long coding sequences. Moreover, the lesion in eIF3h revealed a concerted upregulation of translation for specific functional subgroups of mRNAs, including ribosomal proteins and proteins involved in photosynthesis. CONCLUSIONS: The intact eIF3h protein contributes to efficient translation initiation on 5’ leader sequences harboring multiple uORFs, although mRNA features independent of uORFs were also implicated. Moreover, our data suggest that regulons of translational control can be revealed by mutations in generic translation initiation factors. Keywords: mutant, polysome, non-polysome
2007-05-01 | GSE6024 | GEO
Project description:Microarray comparisons of transcript level in wild-type Arabidopsis and eif3h mutant plants. Goal: To detect any change in transcript level between WT and eif3h mutant. BACKGROUND: The eukaryotic translation initiation factor eIF3 has multiple roles during the initiation of translation of cytoplasmic mRNAs. However, the contributions of individual subunits of eIF3 to the translation of specific mRNAs remain poorly understood. RESULTS: Working with stable reporter transgenes in Arabidopsis thaliana it was demonstrated that the h subunit of eIF3 contributes to the efficient translation initiation of mRNAs harboring upstream open reading frames (uORFs) in their 5’ leader sequence. uORFs, which can function as devices for translational regulation, are present in over 30% of Arabidopsis mRNAs, and are enriched among mRNAs for transcriptional regulators and protein modifying enzymes. Microarray comparisons of polysome loading in wild-type and eif3h mutant plants revealed that eIF3h generally helps to maintain efficient polysome loading of mRNAs harboring multiple uORFs. Independently, eIF3h also boosted polysome loading of mRNAs with long coding sequences. Moreover, the lesion in eIF3h revealed a concerted upregulation of translation for specific functional subgroups of mRNAs, including ribosomal proteins and proteins involved in photosynthesis. CONCLUSIONS: The intact eIF3h protein contributes to efficient translation initiation on 5’ leader sequences harboring multiple uORFs, although mRNA features independent of uORFs were also implicated. Moreover, our data suggest that regulons of translational control can be revealed by mutations in generic translation initiation factors. Keywords: mutant, total RNA
2007-05-01 | GSE6025 | GEO