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Essential Roles of Conserved Pseudouridines in Helix 69 for Ribosome Dynamics in Translation

ABSTRACT: The widespread distribution of pseudouridine (Ψ), an isomer of the canonical uridine base, in RNA indicates its functional importance to the cell. In eukaryotes, it is estimated that around 2% of ribosomal RNA nucleotides are pseudouridines, most of which are located in functional regions of the ribosome. Defects in RNA pseudouridylation induce a range of detrimental effects from compromised cellular protein biosynthesis to disease phenotypes in humans. However, genome-wide changes to mRNA translation profiles by ribosomes lacking specific conserved pseudouridines have not been extensively studied. Here, using a new genomic method called 5PSeq and in vitro biochemistry, we investigated changes in ribosome dynamics and cellular translation profiles upon loss of Ψ2258 and Ψ2260 in helix 69, the two most conserved pseudouridines in the ribosome in yeast cells. We found that inhibiting the formation of these two pseudouridines challenges ribosomes to maintain the correct open reading frame and causes generally faster ribosome dynamics in translation. Furthermore, mutant ribosomes are more prone to pause while translating a subset of GC-rich codons, especially rare codons such as Arg (CGA) and Arg (CGG). These results demonstrate the presence of Ψ2258 and Ψ2260 contributes to the dynamics of the H69 RNA stem-loop, and helps to maintain functional interactions with the tRNAs as they move within the ribosome. The optimality of this ribosome-tRNA interaction is likely to be more critical for those limited tRNAs that decode rare codons. Consistent with the changes in ribosome dynamics, we observe that IRES-mediated translation is compromised in the mutant ribosome. These results explain the importance of Ψ2258 and Ψ2260 in H69 to maintain cellular fitness. The strong conservation of Ψ2258 and Ψ2260 in the ribosomes from bacteria to humans indicates their functional significance in modulating ribosome functions. It's likely that the identified functions of these covalent modifications are conserved across species.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE295282 | GEO | 2025/04/22

REPOSITORIES: GEO

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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. This model is hosted on BioModels Database and identified by: BIOMD0000000457 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

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Essential Roles of Conserved Pseudouridines in Helix 69 for Ribosome Dynamics in Translation

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