Chemical modifications to mRNA nucleobases impact translation elongation and termination

Franco, Monika K; Koutmou, Kristin S.

doi:10.1016/j.bpc.2022.106780

Cited by 20 publications

(18 citation statements)

References 149 publications

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“…The three modifications we investigated alter translation differently depending on their location within a codon. Such a context dependence has been observed for every mRNA modification investigated to date 124 . Modifications have the capacity to change intra-molecular interactions with an mRNA, or interactions between rRNA and mRNA within the A site.…”

Section: Trm1 Trm2 Trm10 and Trm11 Incorporate Methylated Guanosine A...mentioning

confidence: 62%

“…This is the first evidence that m 5 U can influence amino acid addition when encountered by the ribosome. It is less clear how m 5 U and other modifications that do not change the Watson-Crick face of nucleobases (e.g., Ψ and 8-oxoG) impact translation 124 . It is possible that such modifications alter nucleobase ring electronics to perturb the strength of the hydrogen bond donors and acceptors involved in base pairing.…”

Section: Resultsmentioning

confidence: 99%

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Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

Jones

Franco

Smith

et al. 2022

Preprint

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Chemical modifications to protein encoding messenger RNA (mRNA) can modulate their localization, translation and stability within cells. Over 15 different types of mRNA modifications have been identified by sequencing and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technologies. While LC-MS/MS is arguably the most essential tool available for studying analogous protein post-translational modifications, the high-throughput discovery and quantitative characterization of mRNA modifications by LC-MS/MS has been hampered by the difficulty of obtaining sufficient quantities of pure mRNA and limited sensitivities for modified nucleosides. To overcome these challenges, we improved the mRNA purification and LC-MS/MS pipelines to identify new S. cerevisiae mRNA modifications and quantify 50 ribonucleosides in a single analysis. The methodologies we developed result in no detectable non-coding RNA modifications signals in our purified mRNA samples and provide the lowest limit of detection reported for ribonucleoside modification LC-MS/MS analyses. These advancements enabled the detection and quantification of 13 S. cerevisiae mRNA ribonucleoside modifications and revealed four new S. cerevisiae mRNA modifications at low to moderate levels (1-methyguanosine, N2-methylguanosine, N2, N2-dimethylguanosine, and 5-methyluridine). We identified four enzymes that incorporate these modifications into S. cerevisiae mRNAs (Trm10, Trm11, Trm1, and Trm2), though our results suggest that guanosine and uridine nucleobases are also non-enzymatically methylated at low levels. Regardless of whether they are incorporated in a programmed manner or as the result of RNA damage, we reasoned that the ribosome will encounter the modifications that we detect in cells and used a reconstituted translation system to discern the consequences of modifications on translation elongation. Our findings demonstrate that the introduction of 1-methyguanosine, N2-methylguanosine and 5-methyluridine into mRNA codons impedes amino acid addition in a position dependent manner. This work expands the repertoire of nucleoside modifications that the ribosome must decode in S. cerevisiae. Additionally, it highlights the challenge of predicting the effect of discrete modified mRNA sites on translation de novo because individual modifications influence translation differently depending on mRNA sequence context.

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Section: Trm1 Trm2 Trm10 and Trm11 Incorporate Methylated Guanosine A...mentioning

confidence: 62%

Section: Resultsmentioning

confidence: 99%

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

Jones

Franco

Smith

et al. 2022

Preprint

Self Cite

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“…Chemical modifications to nucleobases can change the propensity of the ribosome to incorporate alternative amino acids into a growing polypeptide chain (29)(30)(31)(32)(57)(58)(59)(60). In comparison to uridine, m 1 Ψ possesses a repositioned, methylated nitrogen in its pyrimidine ring (Figure 1A).…”

Section: Influences Aminoacyl-trna Selection By the Ribosome In A Con...mentioning

confidence: 99%

“…In cells, any changes to protein output observed when RNA modifying enzymes are removed are difficult to directly attribute to alterations in a particular mRNA’s modification status. Therefore, studies using reconstituted translation systems, where it is possible to uniformly change the modification status of an mRNA without impacting that of ncRNA, have been particularly useful for assessing the consequences of mRNA modifications on translation (29). Initial studies reveal that modifications commonly slow the ribosome, though some only do so only in particular mRNA sequence contexts (29).…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, studies using reconstituted translation systems, where it is possible to uniformly change the modification status of an mRNA without impacting that of ncRNA, have been particularly useful for assessing the consequences of mRNA modifications on translation (29). Initial studies reveal that modifications commonly slow the ribosome, though some only do so only in particular mRNA sequence contexts (29). Additionally, a subset of mRNA modifications, including pseudouridine (Ψ) and inosine (I), also impact the accuracy of mRNA decoding (30–32).…”

Section: Introductionmentioning

confidence: 99%

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N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

Monroe

Mitchell

Deb

et al. 2022

Preprint

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The ribosome relies on hydrogen bonding interactions between mRNA codons and incoming aminoacyl-tRNAs to ensure rapid and accurate protein production. The inclusion of chemically modified bases into mRNAs has the potential to alter the strength and pattern of hydrogen bonding between mRNAs and aminoacyl-tRNAs to alter protein synthesis. We investigated how the N1-methylpseudouridine (m1Ψ) modification, commonly incorporated into therapeutic and vaccine mRNA sequences, influences the ability of codons to react with cognate and near-cognate tRNAs and release factors. We find that the presence of a single m1Ψ does not substantially change the rate constants for amino acid addition by cognate tRNAs or termination by release factors. However, insertion of m1Ψ can affect the selection of near-cognate tRNAs both in vitro and in human cells. Our observations demonstrate that m1Ψ, and the related naturally occurring pseudouridine (Ψ) modification, exhibit the ability to both increase and decrease the extent of amino acid misincorporation in a codon-position and tRNA dependent manner. To ascertain the chemical logic for our biochemical and cellular observations, we computationally modeled tRNAIle(GAU) bound to unmodified and m1Ψ- or Ψ-modified phenylalanine codons (UUU). Our modeling suggests that changes in the energetics of mRNA:tRNA interactions largely correlate with the context specificity of Ile-miscoding events we observe on Ψ and m1Ψ containing Phe codons. These studies reveal that the sequence context of a given modification within an mRNA plays a large role in determining how (and if) the modification impacts the number and distribution of proteoforms synthesized by the ribosome.

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mRNA vaccines in the prevention and treatment of diseases

Duan

Yang

et al. 2022

MedComm

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Messenger ribonucleic acid (mRNA) vaccines made their successful public debut in the effort against the COVID‐19 outbreak starting in late 2019, although the history of mRNA vaccines can be traced back decades. This review provides an overview to discuss the historical course and present situation of mRNA vaccine development in addition to some basic concepts that underly mRNA vaccines. We discuss the general preparation and manufacturing of mRNA vaccines and also discuss the scientific advances in the in vivo delivery system and evaluate popular approaches (i.e., lipid nanoparticle and protamine) in detail. Next, we highlight the clinical value of mRNA vaccines as potent candidates for therapeutic treatment and discuss clinical progress in the treatment of cancer and coronavirus disease 2019. Data suggest that mRNA vaccines, with several prominent advantages, have achieved encouraging results and increasing attention due to tremendous potential in disease management. Finally, we suggest some potential directions worthy of further investigation and optimization. In addition to basic research, studies that help to facilitate storage and transportation will be indispensable for practical applications.

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Chemical modifications to mRNA nucleobases impact translation elongation and termination

Cited by 20 publications

References 149 publications

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

N1-Methylpseudouridine and pseudouridine modifications modulate mRNA decoding during translation

mRNA vaccines in the prevention and treatment of diseases

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