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 6 publications

(14 citation statements)

References 125 publications

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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%

“…Additionally, we investigated the possibility of cooperative affects between m 5 U containing mRNA codons and m 5 U54 in tRNA [19][20][21][22] . To accomplish this, we interrogated how amino acid addition on m 5 U-containing codons (1 st , 2 nd , and 3 rd position modified UUU or UUC codons) is impacted when decoded by tRNA Phe,-m5U .…”

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

confidence: 99%

“…mRNA was purified through a three-step purification pipeline 19 . First, small RNA (tRNA and small rRNA) was diminished using the MEGAclear Transcription Clean-Up Kit (Invitrogen) to purify RNA >200nt.…”

Section: S Cerevisiae Cell Growth and Mrna Purificationmentioning

confidence: 99%

“…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%

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

confidence: 99%

Section: S Cerevisiae Cell Growth and Mrna Purificationmentioning

confidence: 99%

Section: Qrt-pcrmentioning

confidence: 99%

Section: Rna -Seqmentioning

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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Why U matters: detection and functions of pseudouridine modifications in mRNAs

Rodell,

Robalin,

Martinez

2024

Trends in Biochemical Sciences

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N2-methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing

Wang

Herzel

Pythoud

et al. 2023

Nucleic Acids Research

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Modified nucleotides in non-coding RNAs, such as tRNAs and snRNAs, represent an important layer of gene expression regulation through their ability to fine-tune mRNA maturation and translation. Dysregulation of such modifications and the enzymes installing them have been linked to various human pathologies including neurodevelopmental disorders and cancers. Several methyltransferases (MTases) are regulated allosterically by human TRMT112 (Trm112 in Saccharomyces cerevisiae), but the interactome of this regulator and targets of its interacting MTases remain incompletely characterized. Here, we have investigated the interaction network of human TRMT112 in intact cells and identify three poorly characterized putative MTases (TRMT11, THUMPD3 and THUMPD2) as direct partners. We demonstrate that these three proteins are active N2-methylguanosine (m2G) MTases and that TRMT11 and THUMPD3 methylate positions 10 and 6 of tRNAs, respectively. For THUMPD2, we discovered that it directly associates with the U6 snRNA, a core component of the catalytic spliceosome, and is required for the formation of m2G, the last ‘orphan’ modification in U6 snRNA. Furthermore, our data reveal the combined importance of TRMT11 and THUMPD3 for optimal protein synthesis and cell proliferation as well as a role for THUMPD2 in fine-tuning pre-mRNA splicing.

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Methylated guanosine and uridine modifications in S. cerevisiae mRNAs modulate translation elongation

Abstract: Chemical modifications to protein encoding messenger RNAs (mRNAs) can modulate their localization, translation and stability within cells. Over 15 different types of mRNA modifications have been observed by sequencing and...

Cited by 6 publications

References 125 publications

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Conserved 5-methyluridine tRNA modification modulates ribosome translocation

Why U matters: detection and functions of pseudouridine modifications in mRNAs

N2-methylguanosine modifications on human tRNAs and snRNA U6 are important for cell proliferation, protein translation and pre-mRNA splicing

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