Chemically modified tRNA enhances the translation capacity of mRNA rich in cognate codons [ncRNA-Seq]
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PROVIDER: PRJNA1287742 | ENA |
REPOSITORIES: ENA
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Project description:Chemically modified tRNA enhances the translation capacity of mRNA rich in cognate codons [RNA-Seq]
| PRJNA1287745 | ENA
Project description:Chemically modified tRNA enhances the translation capacity of mRNA rich in cognate codons [Ribo-Seq]
| PRJNA1287737 | ENA
Project description:Although messenger RNA (mRNA) vaccines have been employed to prevent the spread of COVID-19, they are critically limited by instability and low translation capacity. Alterations in tRNA abundance and modification, associated with codon optimality, impact mRNA stability and protein output in a codon-dependent manner, implying tRNA as a potential translation enhancer. Here, we report a strategy named tRNA-plus to augment translation based on artificially modulating the tRNA availability. We demonstrated that overexpression of specific tRNAs enhanced the stability and translation efficiency of SARS-CoV-2 Spike mRNA, resulting in substantial improvements in protein levels by up to 4.7-fold. In addition, chemically synthesized tRNAs bearing multiple site-specific modifications, particularly at the anticodon loop and TΨC-loop, exhibited an average of ~4-fold higher decoding efficacy than unmodified tRNA, along with increased stability and reduced immune toxicity. We further leveraged an optimized lipid nanoparticles (LNP) system for codelivery of Spike mRNA vaccine and tRNA, which elicited enhanced spike-specific humoral and cellular immune responses in vivo. The methods presented here may become a general approach for elevating mRNA translation potency, which can be applied to various translation-based fields.
2025-08-27 | GSE301916 | GEO
Project description:Although messenger RNA (mRNA) vaccines have been employed to prevent the spread of COVID-19, they are critically limited by instability and low translation capacity. Alterations in tRNA abundance and modification, associated with codon optimality, impact mRNA stability and protein output in a codon-dependent manner, implying tRNA as a potential translation enhancer. Here, we report a strategy named tRNA-plus to augment translation based on artificially modulating the tRNA availability. We demonstrated that overexpression of specific tRNAs enhanced the stability and translation efficiency of SARS-CoV-2 Spike mRNA, resulting in substantial improvements in protein levels by up to 4.7-fold. In addition, chemically synthesized tRNAs bearing multiple site-specific modifications, particularly at the anticodon loop and TΨC-loop, exhibited an average of ~4-fold higher decoding efficacy than unmodified tRNA, along with increased stability and reduced immune toxicity. We further leveraged an optimized lipid nanoparticles (LNP) system for codelivery of Spike mRNA vaccine and tRNA, which elicited enhanced spike-specific humoral and cellular immune responses in vivo. The methods presented here may become a general approach for elevating mRNA translation potency, which can be applied to various translation-based fields.
2025-08-27 | GSE301911 | GEO
Project description:Although messenger RNA (mRNA) vaccines have been employed to prevent the spread of COVID-19, they are critically limited by instability and low translation capacity. Alterations in tRNA abundance and modification, associated with codon optimality, impact mRNA stability and protein output in a codon-dependent manner, implying tRNA as a potential translation enhancer. Here, we report a strategy named tRNA-plus to augment translation based on artificially modulating the tRNA availability. We demonstrated that overexpression of specific tRNAs enhanced the stability and translation efficiency of SARS-CoV-2 Spike mRNA, resulting in substantial improvements in protein levels by up to 4.7-fold. In addition, chemically synthesized tRNAs bearing multiple site-specific modifications, particularly at the anticodon loop and TΨC-loop, exhibited an average of ~4-fold higher decoding efficacy than unmodified tRNA, along with increased stability and reduced immune toxicity. We further leveraged an optimized lipid nanoparticles (LNP) system for codelivery of Spike mRNA vaccine and tRNA, which elicited enhanced spike-specific humoral and cellular immune responses in vivo. The methods presented here may become a general approach for elevating mRNA translation potency, which can be applied to various translation-based fields.
2025-08-27 | GSE301914 | GEO
Project description:Although ribosome-profiling and translation initiation sequencing (TI-seq) analyses have identified many noncanonical initiation codons, the precise detection of translation initiation sites (TISs) remains a challenge, mainly because of experimental artifacts of such analyses. Here we describe a new method, TISCA (TIS detection by translation Complex Analysis), for the accurate identification of TISs. TISCA proved to be more reliable for TIS detection compared with existing tools, and it identified a substantial number of near-cognate codons in Kozak-like sequence contexts. Analysis of proteomics data revealed the presence of methionine at the NH2-terminus of most proteins derived from near-cognate initiation codons. Although eukaryotic initiation factor 2 (eIF2), eIF2A, and eIF2D have previously been shown to contribute to translation initiation at near-cognate codons, we found that most noncanonical initiation events are dependent on eIF2, consistent with the initial amino acid being methionine. Comprehensive identification of TISs by TISCA should facilitate characterization of the mechanism of noncanonical initiation.
2021-07-01 | GSE174329 | 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:Some codons of the genetic code can be read not only by cognate, but also by near-cognate tRNAs. This flexibility is thought to be conferred mainly by a mismatch between the third base of the codon and the first of the anticodon (the so-called wobble position). However, this simplistic explanation underestimates the importance of nucleotide modifications in the decoding process. Using a system in which only near-cognate tRNAs can decode a specific codon, we investigated the role of six modifications of the anticodon, or adjacent nucleotides, of the tRNAs specific for Tyr, Gln, Lys, Trp, Cys and Arg in Saccharomyces cerevisiae. Modifications almost systematically rendered these tRNAs able to act as near-cognate tRNAs at stop codons, even though they involve non-canonical base-pairs, without markedly affecting their ability to decode cognate or near-cognate sense codons. These findings reveal an important effect of modifications to tRNA decoding with implications for understanding the flexibility of the genetic code.
2018-04-04 | GSE108772 | GEO
Project description:Combinatorial analyis of translation dynamics reveals eIF2 dependence of translation initiation at near-cognate codons
| PRJNA729461 | ENA
Project description:Nonsense mutations, responsible for ~11% of genetic diseases, create premature stop codons (PTCs) and lead to truncated and often non-functional proteins. Recently, programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress PTCs. However, current methods suffer from low efficiency and limited precision. Here, we develop RESTART v3, an updated version of RESTART, which utilizes near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold higher editing efficiency than RESTART v2 in cultured cells, currently the most active RNA pseudouridylation tool to mediate PTC readthrough. Moreover, RESTART v3 achieves functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense to nonsense codons. In line with the benign off-target effect of RESTART, RESTART v3 does not change the coding information nor the expression level of transcripts with off-target editing; except for the specifically overexpressed tRNA molecule, it does not alter the expression level of endogenous tRNA pool. Overall, RESTART v3 represents an enhanced RNA base editor with increased efficiency and accuracy.
2024-01-29 | GSE237632 | GEO