Methylated guanosine and uridine modifications in <i>S. cerevisiae</i> mRNAs modulate translation elongation

Jones, Joshua D.; Franco, Monika K; Smith, T.J.; Snyder, L.R.; Anders, Anna G; Ruotolo, Brandon T.; Kennedy, Robert T.; Koutmou, Kristin S.

doi:10.1039/d2cb00229a

Cited by 11 publications

(14 citation statements)

References 125 publications

(330 reference statements)

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“…These findings were compared with assays we previously published investigating amino acid addition on m 5 U-modified codons using a fully modified tRNA Phe19 . As on the unmodified codons, the rate constants for amino acid addition were essentially unchanged when tRNA Phe and tRNA Phe,-m5U were used in translation assays (Figure 4C) 19 . Our data collectively demonstrate that loss of m 5 U54 in tRNA Phe,-m5U does not alter the overall rate constant for Phe addition on unmodified or m 5 U modified codons.…”

Section: Mrna M 5 U Modification Does Not Drive Trm2-dependent Change...mentioning

confidence: 88%

“…The RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA) was used to reverse transcribe DNase I-treated total RNA and three-stage purified mRNA (200 ng) using the random hexamer primer. The resulting cDNA was diluted 5,000-fold and 1 μL of the resulting mixture was analyzed using the Luminaris Color HiGreen qPCR Master Mix (Thermo Scientific, USA) with gene-specific primers 19 .…”

Section: Qrt-pcrmentioning

confidence: 99%

“…The WT S. cerevisiae total RNA and mRNA were analyzed by RNA-seq as previously described by paired-end sequencing using 2.5% of an Illumina NovaSeq (S4) 300 cycle sequencing platform flow cell (0.625% of flow cell for each sample) 19 . All sequence data are paired-end 150 bp reads.…”

Section: Rna -Seqmentioning

confidence: 99%

“…In vitro translation studies using m 5 U-depleted tRNAs demonstrate that loss of m 5 U54 from tRNAs does not slow peptide elongation by the ribosome 16–18 . m 5 U has also recently been discovered in eukaryotic mRNAs 19–22 , providing the modification a possible non-tRNA mediated avenue to influence protein synthesis. Nevertheless, the low level of m 5 U in mRNAs (at least 10-fold less than N6-methyladenosine (m 6 A) and pseudouridine (Ψ) modifications), and its minor (0 to 2-fold) effect on the rate constant for amino acid addition makes m 5 U in mRNAs unlikely to broadly enhance cellular fitness 19 .…”

Section: Introductionmentioning

confidence: 99%

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Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Jones,

Franco,

Tardu

et al. 2023

Preprint

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While the centrality of post-transcriptional modifications to RNA biology has long been acknowledged, the function of the vast majority of modified sites remains to be discovered. Illustrative of this, there is not yet a discrete biological role assigned for one the most highly conserved modifications, 5-methyluridine at position 54 in tRNAs (m5U54). Here, we uncover contributions of m5U54 to both tRNA maturation and protein synthesis. Our mass spectrometry analyses demonstrate that cells lacking the enzyme that installs m5U in the T-loop (TrmA inE. coli, Trm2 inS. cerevisiae) exhibit altered tRNA modifications patterns. Furthermore, m5U54 deficient tRNAs are desensitized to small molecules that prevent translocationin vitro.This finding is consistent with our observations that, relative to wild-type cells,trm2Δ cell growth and transcriptome-wide gene expression are less perturbed by translocation inhibitors. Together our data suggest a model in which m5U54 acts as an important modulator of tRNA maturation and translocation of the ribosome during protein synthesis.

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Section: Mrna M 5 U Modification Does Not Drive Trm2-dependent Change...mentioning

confidence: 88%

Section: Qrt-pcrmentioning

confidence: 99%

Section: Rna -Seqmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Jones,

Franco,

Tardu

et al. 2023

Preprint

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“…Furthermore, it is plausible that direct incorporation of 5FU metabolite FUTP in the open-reading frame (ORF) of the mRNAs or 5FU-induced changes in nucleotide modifications within the ORF could also impede ribosomes and result in stalling. While the ability of fluorinated uridines to directly impede ribosomes is not known, other types of modified uridine within the ORF including pseudouridine 16 and 5-methyluridine 51 were shown to stall ribosomes and triggers RQC 17 . Incorporation of 5FU-derived metabolites into non-coding RNAs, such as ribosomal RNAs (rRNAs) or transfer RNAs (tRNAs) may also contribute to the 5FU-induced ribosome stalling.…”

Section: Discussionmentioning

confidence: 99%

Ribosome Quality Control Mechanism Mitigates the Cytotoxic Impacts of Ribosome Collisions Induced by 5-Fluorouracil

Chatterjee,

Naeli,

Simms

et al. 2023

Preprint

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Translation of aberrant or damaged mRNAs results in ribosome stalling and collisions. The Ribosome Quality Control (RQC) mechanism detects collided ribosomes and removes aberrant mRNAs and nascent peptides, thus preventing their cytotoxic effects. Conversely, excessive or unresolved ribosome collisions can induce apoptosis. 5-Fluorouracil (5FU) forms the backbone of standard-of-care chemotherapeutic regimens for several types of cancer. Although best known for its incorporation into DNA and inhibition of thymidylate synthase, a major determinant of 5FU’s anticancer activity is its incorporation into RNAs. Nevertheless, the mechanism(s) underlying RNA-dependent 5FU cytotoxicity and the cellular response to its impact on RNA metabolism remain unclear. Here, we report a key role for RQC in mitigating the cytotoxic effects of 5FU-induced dysregulation of mRNA translation. We show that acute 5FU treatment results in the rapid induction of the mTOR signalling pathway, an enhanced rate of mRNA translation initiation, and increased ribosome collisions that trigger RQC. We also found that RQC deficiency, caused by the depletion of ZNF598, results in increased 5FU-induced cell death, a phenotype that is reversed by inhibition of mTOR or repression of mRNA translation initiation. Importantly, 5FU treatment enhances the expression of key RQC factors, including ZNF598 and GIGYF2, via an mTOR-dependent post-translational regulation mechanism. This acute adaptation likely mitigates the cytotoxic consequences of increased ribosome collisions upon 5FU treatment. Overall, our data indicate a heretofore unknown mTOR-dependent mechanism that augments the RQC process, mitigating the cytotoxicity of 5FU and undermining its anticancer efficacy.

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