Dataset Information

Metabolic turnover and dynamics of modified ribonucleosides by ¹³C labeling.

ABSTRACT: Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a ¹³C labeling approach, called ¹³C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to resolve mRNA from ncRNA modifications in purified RNA or free ribonucleoside samples and showed the distinct kinetics of the N6-methyladenosine (m⁶A) versus 7-methylguanosine (m⁷G) modification in polyA+-purified RNA. We uncovered that N6,N6-dimethyladenosine (m⁶₂A) exhibits distinct turnover in small RNAs and free ribonucleosides when compared to known m⁶₂A-modified large rRNAs. Finally, combined measurements of turnover and abundance of these modifications informed on the transcriptional versus posttranscriptional sensitivity of modified ncRNAs and mRNAs, respectively, to stress conditions. Thus, ¹³C-dynamods enables studies of the origin of modified RNAs at steady-state and subsequent dynamics under nonstationary conditions. These results open new directions to probe the presence and biological regulation of modifications in particular RNAs.

SUBMITTER: Gameiro PA

PROVIDER: S-EPMC8567201 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling.

Gameiro Paulo A PA Encheva Vesela V Dos Santos Mariana Silva MS MacRae James I JI Ule Jernej J

The Journal of biological chemistry 20211009 5

Tandem mass spectrometry (MS/MS) is an accurate tool to assess modified ribonucleosides and their dynamics in mammalian cells. However, MS/MS quantification of lowly abundant modifications in non-ribosomal RNAs is unreliable, and the dynamic features of various modifications are poorly understood. Here, we developed a 13C labeling approach, called 13C-dynamods, to quantify the turnover of base modifications in newly transcribed RNA. This turnover-based approach helped to re ...[more]

PMID: 34634303

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Project description:BackgroundLiving cells maintain and adjust structural and functional integrity by continual synthesis and degradation of metabolites and macromolecules. The maintenance and adjustment of thylakoid membrane involve turnover of photosynthetic pigments along with subunits of protein complexes. Quantifying their turnover is essential to understand the mechanisms of homeostasis and long-term acclimation of photosynthetic apparatus. Here we report methods combining whole-plant long-term 13CO2 labeling and liquid chromatography - mass spectrometry (LC-MS) analysis to determine the size of non-labeled population (NLP) of carotenoids and chlorophylls (Chl) in leaf pigment extracts of partially 13C-labeled plants.ResultsThe labeling chamber enabled parallel 13CO2 labeling of up to 15 plants of Arabidopsis thaliana with real-time environmental monitoring ([CO2], light intensity, temperature, relative air humidity and pressure) and recording. No significant difference in growth or photosynthetic pigment composition was found in leaves after 7-d exposure to normal CO2 (~ 400 ppm) or 13CO2 in the labeling chamber, or in ambient air outside the labeling chamber (control). Following chromatographic separation of the pigments and mass peak assignment by high-resolution Fourier-transform ion cyclotron resonance MS, mass spectra of photosynthetic pigments were analyzed by triple quadrupole MS to calculate NLP. The size of NLP remaining after the 7-d 13CO2 labeling was ~ 10.3% and ~ 11.5% for all-trans- and 9-cis-β-carotene, ~ 21.9% for lutein, ~ 18.8% for Chl a and 33.6% for Chl b, highlighting non-uniform turnover of these pigments in thylakoids. Comparable results were obtained in all replicate plants of the 13CO2 labeling experiment except for three that were showing anthocyanin accumulation and growth impairment due to insufficient water supply (leading to stomatal closure and less 13C incorporation).ConclusionsOur methods allow 13CO2 labeling and estimation of NLP for photosynthetic pigments with high reproducibility despite potential variations in [13CO2] between the experiments. The results indicate distinct turnover rates of carotenoids and Chls in thylakoid membrane, which can be investigated in the future by time course experiments. Since 13C enrichment can be measured in a range of compounds, long-term 13CO2 labeling chamber, in combination with appropriate MS methods, facilitates turnover analysis of various metabolites and macromolecules in plants on a time scale of hours to days.

Dataset Information

Metabolic turnover and dynamics of modified ribonucleosides by ¹³C labeling.

Publications

Metabolic turnover and dynamics of modified ribonucleosides by <sup>13</sup>C labeling.

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Dataset Information

Metabolic turnover and dynamics of modified ribonucleosides by 13C labeling.

Publications

Metabolic turnover and dynamics of modified ribonucleosides by &lt;sup&gt;13&lt;/sup&gt;C labeling.

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OmicsDI is part of the ELIXIR infrastructure

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Metabolic turnover and dynamics of modified ribonucleosides by ¹³C labeling.

Metabolic turnover and dynamics of modified ribonucleosides by <sup>13</sup>C labeling.