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15 pages, 2821 KiB

Open AccessArticle

The Stress-Dependent Dynamics of Saccharomyces cerevisiae tRNA and rRNA Modification Profiles

by Yasemin Yoluç, Erik van de Logt and Stefanie Kellner-Kaiser

Genes 2021, 12(9), 1344; https://doi.org/10.3390/genes12091344 - 28 Aug 2021

Cited by 6 | Viewed by 2967

Abstract

RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling [...] Read more.

RNAs are key players in the cell, and to fulfil their functions, they are enzymatically modified. These modifications have been found to be dynamic and dependent on internal and external factors, such as stress. In this study we used nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) to address the question of which mechanisms allow the dynamic adaptation of RNA modifications during stress in the model organism S. cerevisiae. We found that both tRNA and rRNA transcription is stalled in yeast exposed to stressors such as H₂O₂, NaAsO₂ or methyl methanesulfonate (MMS). From the absence of new transcripts, we concluded that most RNA modification profile changes observed to date are linked to changes happening on the pre-existing RNAs. We confirmed these changes, and we followed the fate of the pre-existing tRNAs and rRNAs during stress recovery. For MMS, we found previously described damage products in tRNA, and in addition, we found evidence for direct base methylation damage of 2′O-ribose methylated nucleosides in rRNA. While we found no evidence for increased RNA degradation after MMS exposure, we observed rapid loss of all methylation damages in all studied RNAs. With NAIL-MS we further established the modification speed in new tRNA and 18S and 25S rRNA from unstressed S. cerevisiae. During stress exposure, the placement of modifications was delayed overall. Only the tRNA modifications 1-methyladenosine and pseudouridine were incorporated as fast in stressed cells as in control cells. Similarly, 2′-O-methyladenosine in both 18S and 25S rRNA was unaffected by the stressor, but all other rRNA modifications were incorporated after a delay. In summary, we present mechanistic insights into stress-dependent RNA modification profiling in S. cerevisiae tRNA and rRNA. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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12 pages, 1923 KiB

Open AccessArticle

SMITER—A Python Library for the Simulation of LC-MS/MS Experiments

by Manuel Kösters, Johannes Leufken and Sebastian A. Leidel

Genes 2021, 12(3), 396; https://doi.org/10.3390/genes12030396 - 11 Mar 2021

Cited by 6 | Viewed by 3858

Abstract

SMITER (Synthetic mzML writer) is a Python-based command-line tool designed to simulate liquid-chromatography-coupled tandem mass spectrometry LC-MS/MS runs. It enables the simulation of any biomolecule amenable to mass spectrometry (MS) since all calculations are based on chemical formulas. SMITER features a modular design, [...] Read more.

SMITER (Synthetic mzML writer) is a Python-based command-line tool designed to simulate liquid-chromatography-coupled tandem mass spectrometry LC-MS/MS runs. It enables the simulation of any biomolecule amenable to mass spectrometry (MS) since all calculations are based on chemical formulas. SMITER features a modular design, allowing for an easy implementation of different noise and fragmentation models. By default, SMITER uses an established noise model and offers several methods for peptide fragmentation, and two models for nucleoside fragmentation and one for lipid fragmentation. Due to the rich Python ecosystem, other modules, e.g., for retention time (RT) prediction, can easily be implemented for the tailored simulation of any molecule of choice. This facilitates the generation of defined gold-standard LC-MS/MS datasets for any type of experiment. Such gold standards, where the ground truth is known, are required in computational mass spectrometry to test new algorithms and to improve parameters of existing ones. Similarly, gold-standard datasets can be used to evaluate analytical challenges, e.g., by predicting co-elution and co-fragmentation of molecules. As these challenges hinder the detection or quantification of co-eluents, a comprehensive simulation can identify and thus, prevent such difficulties before performing actual MS experiments. SMITER allows the creation of such datasets easily, fast, and efficiently. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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12 pages, 1840 KiB

Open AccessArticle

Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides

by Marco Kristen, Johanna Plehn, Virginie Marchand, Kristina Friedland, Yuri Motorin, Mark Helm and Stephan Werner

Genes 2020, 11(8), 950; https://doi.org/10.3390/genes11080950 - 18 Aug 2020

Cited by 15 | Viewed by 4197

Abstract

Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for [...] Read more.

Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg²⁺ with Mn²⁺ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m¹A positions dropping from 82% down to 24%. Additionally, we observed a vast increase in nucleotide skipping events, with single positions rising from 4% to 49%, thus implying an enhanced read-through capability as an effect of Mn²⁺ on the reverse transcriptase, by promoting nucleotide skipping over synthesis abortion. While modifications such as m¹A, m²₂G, m¹G and m³C showed a clear influence of manganese ions on their RT signature, this effect was individual to the polymerase used. In summary, the results imply a supporting effect of Mn²⁺ on reverse transcription, thus overcoming blockades in the Watson-Crick face of modified ribonucleosides and improving both read-through rate and signal intensity in RT signature analysis. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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Review

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23 pages, 2279 KiB

Open AccessReview

RNA Modifications in Pathogenic Bacteria: Impact on Host Adaptation and Virulence

by Laura Antoine, Roberto Bahena-Ceron, Heemee Devi Bunwaree, Martin Gobry, Victor Loegler, Pascale Romby and Stefano Marzi

Genes 2021, 12(8), 1125; https://doi.org/10.3390/genes12081125 - 24 Jul 2021

Cited by 19 | Viewed by 5798

Abstract

RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as [...] Read more.

RNA modifications are involved in numerous biological processes and are present in all RNA classes. These modifications can be constitutive or modulated in response to adaptive processes. RNA modifications play multiple functions since they can impact RNA base-pairings, recognition by proteins, decoding, as well as RNA structure and stability. However, their roles in stress, environmental adaptation and during infections caused by pathogenic bacteria have just started to be appreciated. With the development of modern technologies in mass spectrometry and deep sequencing, recent examples of modifications regulating host-pathogen interactions have been demonstrated. They show how RNA modifications can regulate immune responses, antibiotic resistance, expression of virulence genes, and bacterial persistence. Here, we illustrate some of these findings, and highlight the strategies used to characterize RNA modifications, and their potential for new therapeutic applications. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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12 pages, 1241 KiB

Open AccessReview

An I for an A: Dynamic Regulation of Adenosine Deamination-Mediated RNA Editing

by Cornelia Vesely and Michael F. Jantsch

Genes 2021, 12(7), 1026; https://doi.org/10.3390/genes12071026 - 1 Jul 2021

Cited by 15 | Viewed by 7068

Abstract

RNA-editing by adenosine deaminases acting on RNA (ADARs) converts adenosines to inosines in structured RNAs. Inosines are read as guanosines by most cellular machineries. A to I editing has two major functions: first, marking endogenous RNAs as “self”, therefore helping the innate immune [...] Read more.

RNA-editing by adenosine deaminases acting on RNA (ADARs) converts adenosines to inosines in structured RNAs. Inosines are read as guanosines by most cellular machineries. A to I editing has two major functions: first, marking endogenous RNAs as “self”, therefore helping the innate immune system to distinguish repeat- and endogenous retrovirus-derived RNAs from invading pathogenic RNAs; and second, recoding the information of the coding RNAs, leading to the translation of proteins that differ from their genomically encoded versions. It is obvious that these two important biological functions of ADARs will differ during development, in different tissues, upon altered physiological conditions or after exposure to pathogens. Indeed, different levels of ADAR-mediated editing have been observed in different tissues, as a response to altered physiology or upon pathogen exposure. In this review, we describe the dynamics of A to I editing and summarize the known and likely mechanisms that will lead to global but also substrate-specific regulation of A to I editing. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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20 pages, 1678 KiB

Open AccessReview

Analysis of RNA Modifications by Second- and Third-Generation Deep Sequencing: 2020 Update

by Yuri Motorin and Virginie Marchand

Genes 2021, 12(2), 278; https://doi.org/10.3390/genes12020278 - 16 Feb 2021

Cited by 45 | Viewed by 6972

Abstract

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m⁶A methylation in mRNAs, [...] Read more.

The precise mapping and quantification of the numerous RNA modifications that are present in tRNAs, rRNAs, ncRNAs/miRNAs, and mRNAs remain a major challenge and a top priority of the epitranscriptomics field. After the keystone discoveries of massive m⁶A methylation in mRNAs, dozens of deep sequencing-based methods and protocols were proposed for the analysis of various RNA modifications, allowing us to considerably extend the list of detectable modified residues. Many of the currently used methods rely on the particular reverse transcription signatures left by RNA modifications in cDNA; these signatures may be naturally present or induced by an appropriate enzymatic or chemical treatment. The newest approaches also include labeling at RNA abasic sites that result from the selective removal of RNA modification or the enhanced cleavage of the RNA ribose-phosphate chain (perhaps also protection from cleavage), followed by specific adapter ligation. Classical affinity/immunoprecipitation-based protocols use either antibodies against modified RNA bases or proteins/enzymes, recognizing RNA modifications. In this survey, we review the most recent achievements in this highly dynamic field, including promising attempts to map RNA modifications by the direct single-molecule sequencing of RNA by nanopores. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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19 pages, 2230 KiB

Open AccessReview

Post-Transcriptional Modifications of Conserved Nucleotides in the T-Loop of tRNA: A Tale of Functional Convergent Evolution

by Martine Roovers, Louis Droogmans and Henri Grosjean

Genes 2021, 12(2), 140; https://doi.org/10.3390/genes12020140 - 22 Jan 2021

Cited by 16 | Viewed by 3152

Abstract

The high conservation of nucleotides of the T-loop, including their chemical identity, are hallmarks of tRNAs from organisms belonging to the three Domains of Life. These structural characteristics allow the T-loop to adopt a peculiar intraloop conformation able to interact specifically with other [...] Read more.

The high conservation of nucleotides of the T-loop, including their chemical identity, are hallmarks of tRNAs from organisms belonging to the three Domains of Life. These structural characteristics allow the T-loop to adopt a peculiar intraloop conformation able to interact specifically with other conserved residues of the D-loop, which ultimately folds the mature tRNA in a unique functional canonical L-shaped architecture. Paradoxically, despite the high conservation of modified nucleotides in the T-loop, enzymes catalyzing their formation depend mostly on the considered organism, attesting for an independent but convergent evolution of the post-transcriptional modification processes. The driving force behind this is the preservation of a native conformation of the tRNA elbow that underlies the various interactions of tRNA molecules with different cellular components. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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Other

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20 pages, 421 KiB

Open AccessPerspective

The Regulation of RNA Modification Systems: The Next Frontier in Epitranscriptomics?

by Matthias R. Schaefer

Genes 2021, 12(3), 345; https://doi.org/10.3390/genes12030345 - 26 Feb 2021

Cited by 26 | Viewed by 4447

Abstract

RNA modifications, long considered to be molecular curiosities embellishing just abundant and non-coding RNAs, have now moved into the focus of both academic and applied research. Dedicated research efforts (epitranscriptomics) aim at deciphering the underlying principles by determining RNA modification landscapes and investigating [...] Read more.

RNA modifications, long considered to be molecular curiosities embellishing just abundant and non-coding RNAs, have now moved into the focus of both academic and applied research. Dedicated research efforts (epitranscriptomics) aim at deciphering the underlying principles by determining RNA modification landscapes and investigating the molecular mechanisms that establish, interpret and modulate the information potential of RNA beyond the combination of four canonical nucleotides. This has resulted in mapping various epitranscriptomes at high resolution and in cataloguing the effects caused by aberrant RNA modification circuitry. While the scope of the obtained insights has been complex and exciting, most of current epitranscriptomics appears to be stuck in the process of producing data, with very few efforts to disentangle cause from consequence when studying a specific RNA modification system. This article discusses various knowledge gaps in this field with the aim to raise one specific question: how are the enzymes regulated that dynamically install and modify RNA modifications? Furthermore, various technologies will be highlighted whose development and use might allow identifying specific and context-dependent regulators of epitranscriptomic mechanisms. Given the complexity of individual epitranscriptomes, determining their regulatory principles will become crucially important, especially when aiming at modifying specific aspects of an epitranscriptome both for experimental and, potentially, therapeutic purposes. Full article

(This article belongs to the Special Issue Functions and Dynamics of RNA Modifications)

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