Transcription-Wide Mapping of Dihydrouridine (D) Reveals that mRNA Dihydrouridylation is Essential for Meiotic Chromosome Segregation

Finet, Olivier; Yague-Sanz, Carlo; Krüger, Lara Katharina; Migeot, Valérie; Ernst, Felix G.M.; Lafontaine, Denis L. J.; Tran, Phong T.; Wéry, Maxime; Morillon, Antonin; Hermand, Damien

doi:10.2139/ssrn.3569550

Cited by 2 publications

(5 citation statements)

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“…We did not detect any DHU in our purified mRNA samples. Recent sequencing based studies have reported the presence of DHU in mammalian and S. pombe mRNA 15,16 , but our findings indicate DHU either does not exist within S. cerevisiae mRNA or is incorporated at levels below our limit of detection. If dihydrouridine existed at levels just below our limit of detection (530 amol), (Supplemental Table S1) the maximum extent of DHU/U% retention in our purified mRNA would be 0.06% when calculated using the average digest uridine concentration in a sample of digested mRNA.…”

Section: Development Of Highly Sensitive Lc-ms/ms Methods For Simulta...contrasting

confidence: 84%

“…The significance of RNA modifications to cellular health is underscored by decades of observations implicating the mis-regulation of ncRNA modifying enzymes in cancer and other diseases [4][5][6][7][8][9] . Recent advances in next generation RNA sequencing (RNA-seq) [10][11][12][13][14][15][16][17][18][19] and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) technologies [20][21][22][23][24] enabled the detection of chemical modifications in protein encoding messenger RNAs (mRNA). Over 15 mRNA modifications have been reported, including N6-methyladensoine (m 6 A), inosine (I), N7-methylguanosine (m 7 G), and pseudouridine (Ψ) 1, 12,13,22,[25][26][27][28] .…”

Section: Introductionmentioning

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: Development Of Highly Sensitive Lc-ms/ms Methods For Simulta...contrasting

confidence: 84%

Section: Introductionmentioning

confidence: 99%

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

Jones

Franco

Smith

et al. 2022

Preprint

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“…Collectively the Dus enzymes are responsible for the presence of one or more DHUs in the d -loop of the overwhelming majority of tRNAs, located at positions 16, 17, 17a 20, or 20a [ 21 ], as well as the much rarer instances of DHU within the variable loop of some tRNA Tyr s [ 21 ]. One or more of the Dus enzymes appear to be responsible for most, if not all, of the DHUs recently found to be present at 143 sites within 125 fission yeast mRNAs [ 22 ]. A practical consequence is that DHU can be installed as a potential site for introduction of fluorophores not only into unmodified tRNA transcripts [ 23 , 24 ], but also for a wider variety of sites within RNAs in general.…”

Section: Exploitation Of Natural Posttranslational Modificationsmentioning

confidence: 99%

“…Their work made extensive use of proflavin derivatives of tRNA [ 25 ]. More recently rhodamine 110 [ 22 , 23 ] and hydrazide derivatives of both Cy dyes [ 26 , 27 ] and Alexa dyes [ 28 ] have been incorporated into tRNAs. Despite this rather extensive use of fluorescent tRNAs labeled at DHU positions, the precise chemistry of the reaction leading to fluorophore introduction into these positions was for a long time unclear.…”

Section: Exploitation Of Natural Posttranslational Modificationsmentioning

confidence: 99%

“…It has generally been assumed that fluorescent labeling via DHU, acp 3 U, and s 4 U chemistry would be restricted to tRNAs. The recent demonstration that DHU is also found in many mRNA sites [ 22 ] raises the possibility that fluorescent labeling via DHU chemistry could be extended to other RNA targets. The potential of this approach will become clearer as the preferred secondary structure [ 60 ] and/or sequence contexts for DHU formation are defined.…”

Section: Exploitation Of Natural Posttranslational Modificationsmentioning

confidence: 99%

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Site-Specific Fluorescent Labeling of RNA Interior Positions

Cooperman

2021

Molecules

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The introduction of fluorophores into RNA for both in vitro and in cellulo studies of RNA function and cellular distribution is a subject of great current interest. Here I briefly review methods, some well-established and others newly developed, which have been successfully exploited to site-specifically fluorescently label interior positions of RNAs, as a guide to investigators seeking to apply this approach to their studies. Most of these methods can be applied directly to intact RNAs, including (1) the exploitation of natural posttranslational modifications, (2) the repurposing of enzymatic transferase reactions, and (3) the nucleic acid-assisted labeling of intact RNAs. In addition, several methods are described in which specifically labeled RNAs are prepared de novo.

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Transcription-Wide Mapping of Dihydrouridine (D) Reveals that mRNA Dihydrouridylation is Essential for Meiotic Chromosome Segregation

Cited by 2 publications

References 0 publications

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

Methylated guanosine and uridine modifications inS. cerevisiaemRNAs modulate translation elongation

Site-Specific Fluorescent Labeling of RNA Interior Positions

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