Absolute quantification of exogenous stimuli-induced nucleic acid modification dynamics with LC-MS

Modifications of nucleic acids comply different functions and are involved in genome or-ganization, cell differentiation, silencing, structure stability and enzyme recognition. Modi-fication abundances can be regulated intrinsically, like the incorporation of cap modifica-tions on viral RNA to evade the host immune response, but also extrinsically as a cause of damage, which can result in mutations or translational defects. Either way, modifications are highly dynamic. It is of great importance to trace and quantify these changes in order to understand the underlying mechanisms, which may offer a more divers applica-bility of RNA therapeutics and even facilitate the establishment of personalized medicine. Mass Spectrometry is a common technique to examine nucleic acids. However, mass spectrometry per se offers solely a static insight into the versatile dynamics of nucleic acid modifications. In order to circumvent this obstacle, Nucleic Acid Isotope Labeling coupled Mass Spectrometry (NAIL-MS) was developed. This powerful technique allows for absolute quantification on the one hand and on the other hand for examination of modification dynamics originating from endogenous or exogenous actuators. In 2010, the stress-induced reprogramming of tRNA modification in S. cerevisiae was reported. However, the underlying mechanisms remained to be elucidated. Few years later, the dynamics of RNA modifications and mechanisms like dilution, degradation and (de-)modification could be identified by the application of NAIL-MS. The first part of my dissertation deals with the examination of the extent of damage-induced alterations on nucleic acids. Therefore, a novel biosynthetically produced stable isotope labeled internal standard (SILIS) was established, to avoid the interference of signals with isotopologues generated in the stable isotope labeled pulse-chase experiments. Furthermore, the L-methionine-[2H3]-methyl labeling in S. cerevisiae was optimized to achieve full efficient labeling and thus again avoiding signal interferences with isotopologues due to inefficient labeling. Additionally, the tandem size exclusion chromatography was developed, allowing the time efficient purification of 28S/25S, 18S rRNA and tRNA in a single step. The appli-cation of improved stable isotope labeling and the facilitated purification of RNA popula-tions allowed for the examination of the stress-induced alterations in the RNA modifica-tion profile of S. cerevisiae. Thereby, the knowledge on stress-induced reprogramming of tRNA modifications in yeast could be expanded. Original and new transcripts could be discerned and in addition endogenous methylation could be differentiated from damage induced methylation. It was shown, that stress-induced alterations occur on original tRNA transcripts, whereas new transcripts were not affected. Moreover, the fast decrease of damage-induced methylations on 25S, 18S rRNA and tRNA in S. cerevisiae was demon-strated. Additionally, the formation of base damage on 2’-O-methylated nucleosides in rRNA upon methyl methanesulfonate (MMS) exposure were detected and thereby novel damage products of MMS could be identified. Furthermore, the application of NAIL-MS was expanded to study the endogenous and damage-induced methylome on the genomic levels in S. cerevisiae and E. coli. In parallel to the aforementioned findings, the fast dis-appearance of damage-induced methylations in the genome and transcriptome of S. cerevisiae and E. coli was shown. Apart from that, m7dG and m7G could be identified as the main damage products in the genome and transcriptome of both organisms. In parallel to prokaryotes and eukaryotes, the modifications in viral RNA are highly dy-namic. RNA viruses have high mutation rates and their modification abundances can vary during infection. So, several mutants and variants of the RNA virus SARS-CoV-2 emerged since 2019. It is necessary to understand the characteristics of the viral genome and the differences in mutants and variants in order to identify novel drug targets and optimize the application of available therapeutics and vaccines. Our previous work on absolute quantification of nucleic acid modifications in various organisms showed the strength of our LC-MS based approach. In the course of this study it was aimed at inves-tigating the viral RNA modification profile in the different mutants and variants of SARS-CoV-2. The absolute quantification of RNA modifications and the comparison to pub-lished reports lead to the assumption that observed modification densities are highly de-pendent on the cultivation and infection conditions as well as the purification method and verification of sample integrity is crucial for valid analysis. As outlined above, less is known about the genome of SARS-CoV-2 in terms of internal modifications. While the cap modification of the 5’ end of the SARS-CoV-2 genome is confirmed from many sides and is ascribed to regulate the host innate immune response and the viral replication. Hence, a better understanding of the viral capping mechanism is required in order to limit its contagiousness. Besides the interest in biological capping processes, the investigations on cap modifications become more relevant nowadays be-cause of mRNA therapeutics. The cap modification on engineered mRNA is necessary to prevent immunogenicity, improve intercellular stability and translation efficiency. Thus, therapeutic mRNA is engineered to resemble mature and processed eukaryotic mRNA, including the 5’ cap and the 3’ poly A tail. Currently, there are only a few published LC-MS methods for detection of cap modifications. Nevertheless, these methods include labor intensive sample preparation, long analyses times and have moderate sensitivity. In the course of my dissertation, the development and optimization of a time efficient and highly sensitive LC-MS method for absolute quantification of cap modifications is pre-sented. It includes an extensive method development, optimizing chromatographic and mass spectrometric parameters under consideration of short analysis time, low detection and quantification limits. For absolute quantification of cap modifications, an in vitro tran-scribed cap-SILIS was generated. Furthermore, limits of detection and quantification as well as the dynamic range for size and amount of macromolecules to be analyzed were determined. The high sensitivity allows for the analysis of RNA from synthetic but also from biological sources. The time efficiency is aspirational for ecologic and economic rea-sons, thus making this method suitable for high throughput analyses and industry. The identification and quantification of RNA modifications is getting more important with the significance of RNA therapeutics. In this work, efficient LC-MS based tools to study the extent of nucleic acid modifications are described. Insight into the stress-dependent regulation of the genome and transcriptome of common model organisms is given and a powerful method to quantify cap modifications is presented. These techniques can be used to study nucleic acid dynamics in clinical studies but also for quality control of RNA therapeutics

The stress-dependent dynamics of Saccharomyces cerevisiae tRNA and rRNA modification profiles

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

Different modification pathways for m1A58 incorporation in yeast elongator and initiator tRNAs

As essential components of the cellular protein synthesis machineries, tRNAs undergo a tightly controlled biogenesis process, which include the incorporation of a large number of posttranscriptional chemical modifications. Maturation defaults resulting in lack of modifications in the tRNA core may lead to the degradation of hypomodified tRNAs by the rapid tRNA decay (RTD) and nuclear surveillance pathways. Although modifications are typically introduced in tRNAs independently of each other, several modification circuits have been identified in which one or more modifications stimulate or repress the incorporation of others. We previously identified m1A58 as a late modification introduced after more initial modifications, such as Ѱ55 and T54 in yeast elongator tRNAPhe. However, previous reports suggested that m1A58 is introduced early along the tRNA modification process, with m1A58 being introduced on initial transcripts of initiator tRNAiMet, and hence preventing its degradation by the nuclear surveillance and RTD pathways. Here, aiming to reconcile this apparent inconsistency on the temporality of m1A58 incorporation, we examined the m1A58 modification pathways in yeast elongator and initiator tRNAs. For that, we first implemented a generic approach enabling the preparation of tRNAs containing specific modifications. We then used these specifically modified tRNAs to demonstrate that the incorporation of T54 in tRNAPhe is directly stimulated by Ѱ55, and that the incorporation of m1A58 is directly and individually stimulated by Ѱ55 and T54, thereby reporting on the molecular aspects controlling the Ѱ55 → T54 → m1A58 modification circuit in yeast elongator tRNAs. We also show that m1A58 is efficiently introduced on unmodified tRNAiMet, and does not depend on prior modifications. Finally, we show that the m1A58 single modification has tremendous effects on the structural properties of yeast tRNAiMet, with the tRNA elbow structure being properly assembled only when this modification is present. This rationalizes on structural grounds the degradation of hypomodified tRNAiMet lacking m1A58 by the nuclear surveillance and RTD pathways

Instrumental analysis of RNA modifications

International audienceOrganisms from all domains of life invest a substantial amount of energy for the introduction of RNA modifications into nearly all transcripts studied to date. Instrumental analysis of RNA can focus on the modified residues and reveal the function of these epitranscriptomic marks. Here, we will review recent advances and breakthroughs achieved by NMR spectroscopy, sequencing, and mass spectrometry of the epitranscriptome

Strategies to avoid artifacts in mass spectrometry-based epitranscriptome analyses

In this report, we perform structure validation of recently reported RNA phosphorothioate (PT) modifications, a new set of epitranscriptome marks found in bacteria and eukaryotes including humans. By comparing synthetic PT-containing diribonucleotides with native species in RNA hydrolysates by high-resolution mass spectrometry (MS), metabolic stable isotope labeling, and PT-specific iodine-desulfurization, we disprove the existence of PTs in RNA from E. coli, S. cerevisiae, human cell lines, and mouse brain. Furthermore, we discuss how an MS artifact led to the initial misidentification of 2′-O-methylated diribonucleotides as RNA phosphorothioates. To aid structure validation of new nucleic acid modifications, we present a detailed guideline for MS analysis of RNA hydrolysates, emphasizing how the chosen RNA hydrolysis protocol can be a decisive factor in discovering and quantifying RNA modifications in biological samples