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Ac4C does not impair in vitro transcription with T7 polymerase

ABSTRACT: Synthetic mRNA therapeutics offer a versatile platform with promising applications in a range of diseases, including cancer and infectious diseases. These mRNAs are delivered to cells encapsulated in lipid nanoparticles (LNPs) and commonly incorporate modified ribonucleotides to improve their stability and translation while mitigating the immune response to foreign RNA1. N1-methylpseudouridine (m1Ψ) has become the industry standard for synthetic mRNAs due to its effectiveness in enhancing translation and reducing immunogenicity2. However, recent studies have shown that m1Ψ can compromise translational fidelity, leading to errors such as ribosomal frameshifting3,4. Here, we reveal N4-acetylcytidine (ac4C) as a promising alternative to m1Ψ for use in therapeutic mRNAs. In comparing synthetic mRNA translation in cell lines, primary human monocyte-derived dendritic cells and murine liver, we found that while both ac4C and m1Ψ suppress the inflammatory response to synthetic mRNA, ac4C consistently results in higher protein yields. Using single-molecule imaging of translation, we observed that ribosome densities per RNA were similar between ac4C and m1Ψ, but translation elongation rate was nearly doubled in the presence of ac4C —providing a rationale for increased net translation. Importantly, a synthetic mRNA that caused ribosomal frameshifting in the presence of m1Ψ did not induce frameshifting with ac4C. These findings underscore the importance of context in designing therapeutic mRNAs and position translation elongation rate as a key determinant of the efficacy of modified ribonucleotides.

ORGANISM(S): synthetic construct

PROVIDER: GSE301223 | GEO | 2026/05/14

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.

Project description:Generation of the "epitranscriptome through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here we describe N4-acetylcytidine (ac4C) as an mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were enriched within coding sequences. Ablation of NAT10 reduced ac4C detection at the mapped mRNA sites and was globally associated with target mRNA down-regulation. Analysis of mRNA half-lives revealed a NAT10-dependent increase in stability in the cohort of acetylated mRNAs. mRNA acetylation was further demonstrated to enhance substrate translation in vitro and in vivo. Codon content analysis within ac4C peaks uncovered a biased representation of cytidine within wobble sites that was empirically determined to influence mRNA decoding efficiency. These findings expand the repertoire of mRNA modifications to include an acetylated residue and establish a role for ac4C in the regulation of mRNA translation. Generation of the “epitranscriptome” through post-transcriptional ribonucleoside modification embeds a layer of regulatory complexity into RNA structure and function. Here we describe N4-acetylcytidine (ac4C) as a novel mRNA modification that is catalyzed by the acetyltransferase NAT10. Transcriptome-wide mapping of ac4C revealed discretely acetylated regions that were distributed across target mRNAs with the majority of peaks occurring within coding regions. Depletion of ac4C through NAT10 ablation revealed a relationship to gene expression wherein loss of ac4C was globally associated with transcript downregulation. The presence of ac4C within coding sequences was associated with elevated ribosome density and enhanced translation, as assessed in vivo and in vitro. In addition to expanding the repertoire of mRNA modifications to include an acetylated residue, these findings highlight a role for ac4C in the control of mRNA metabolism at the level of translation.

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Ac4C does not impair in vitro transcription with T7 polymerase

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