Surpassing limits of static RNA modification analysis with dynamic NAIL-MS

Reichle, Valentin F.; Kaiser, S.; Heiss, MM; Hagelskamp, Felix; Borland, Kayla; Kellner, Stefanie

doi:10.1016/j.ymeth.2018.10.025

Cited by 25 publications

(38 citation statements)

References 31 publications

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“…While NAIL-MS allows the observation of RNA modification adaptation processes 37,44 it was not possible to apply the technique in human cell culture due to the complexity of culture medium. 13 C6-glucose is a reasonable and economic option for stable isotope labeling (28 € per 50 mL medium) 45 but it suffers from the formation of multiple isotopomers which complicates its application especially when additional feeding with CD3-methionine is required. In such studies, the signals of partially 13 C-labeled nucleosides and CD3-methylated nucleosides can overlap and quantification becomes impossible.…”

Section: Discussionmentioning

confidence: 99%

“…NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) relies on the metabolic incorporation of stable isotope labeled nutrients into RNA and allows the distinction of original RNA and new RNA within a pulse chase experiment. With this tool, we studied the temporal placement of modified nucleosides in S. cerevisiae total tRNA 31 and the demethylation during tRNA repair in E. coli 37 . Both organisms are rather simple and they can be grown in minimal media with controlled availability of stable isotope labeled nutrients.…”

Section: Stable Isotope Labeling Of Rna In Human Cell Culturementioning

confidence: 99%

“…As recently described, it is possible to use glucose-free growth medium and supplement with 13 C6glucose 37 . The feeding with 13 C6-glucose leads to the formation of nucleosides with a variable number of ( 13 C) per nucleoside ( Figure S1C).…”

Section: Stable Isotope Labeling Of Rna In Human Cell Culturementioning

confidence: 99%

“…We have recently applied NAIL-MS to profile bacterial tRNA damage by methylating agents 29 and described the repair mechanisms in vivo 37 . With the goal to study the stress response in human cells, we determined the effect of methyl methanesulfonate (MMS) on growth of HEK 293 cells ( Figure S19).…”

Section: Impact Of Methylation Stress On Trna Modification Processesmentioning

confidence: 99%

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Cell culture NAIL-MS allows insight into human RNA modification dynamicsin vivo

Heiss

Hagelskamp

Kellner

2020

Preprint

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In the last years, studies about the dynamics of RNA modifications are among the most controversially discussed. As the main reason, we have identified the unavailability of a technique which allows to follow the temporal dynamics of RNA transcripts in human cell culture.Here, we present a NAIL-MS (nucleic acid isotope labeling coupled mass spectrometry) scheme for efficient stable isotope labeling in both RNA and DNA (>95% within 7 days) in common human cell lines and growth media. Validation experiments reveal that the labeling procedure itself does neither interfere with the isotope dilution MS quantification nor with RNA modification density. We design pulse chase NAIL-MS experiments and apply the new tool to study the temporal placement of modified nucleosides in e.g. tRNA Phe and 18S rRNA. In existing RNAs, we observe a constant loss of modified nucleosides over time which is masked by a post-transcriptional methylation mechanism and thus not detectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing transcripts.Overall, we present a fast and reliable stable isotope labeling strategy which allows a more detailed study of RNA modification dynamics in human cell culture. With cell culture NAIL-MS it is finally possible to study the speed of both modification and demethylation reactions inside human cells. Thus it will be possible to study the impact of external stimuli and stress on human RNA modification kinetics and processing of mature RNA.

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Section: Discussionmentioning

confidence: 99%

Section: Stable Isotope Labeling Of Rna In Human Cell Culturementioning

confidence: 99%

Section: Stable Isotope Labeling Of Rna In Human Cell Culturementioning

confidence: 99%

Section: Impact Of Methylation Stress On Trna Modification Processesmentioning

confidence: 99%

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Cell culture NAIL-MS allows insight into human RNA modification dynamicsin vivo

Heiss

Hagelskamp

Kellner

2020

Preprint

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“…is the prevailing approach used by numerous laboratories 4,[9][10][11][12][13][14][15][16][17][18][19][20] . In addition, bespoke methods have been developed using other stationary phases, such as HILIC 21,22 and porous graphitic carbon (PGC) 23,24 .…”

Section: Introductionmentioning

confidence: 99%

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

Gregorová

Sipari

Sarin

2020

Preprint

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Post-transcriptional RNA modifications play an important role in cellular metabolism with homeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukemia and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9 min sample-to-sample run. To validate the performance and versatility of our method, we analyzed tRNA modification patterns of representative microorganisms from each kingdom of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae) and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homeostasis studies of RNA modification in complex biological samples.

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Chemical Amination/Imination of Carbonothiolated Nucleosides During RNA Hydrolysis

et al. 2020

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Liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) has become the gold‐standard technique to study RNA and its various modifications. While most research on RNA nucleosides has been focused on their biological roles, discovery of new modifications remains of interest. With state‐of‐the‐art technology, the presence of artifacts can confound the identification of new modifications. Here, we report the characterization of a non‐natural mcm5isoC ribonucleoside in S. cerevisiae total tRNA hydrolysate by higher‐energy collisional dissociation (HCD)‐based fingerprints and isotope labeling of RNA. Its discovery revealed a class of amino/imino ribonucleoside artifacts that are generated during RNA hydrolysis under ammonium‐buffered mild basic conditions. We then identified digestion conditions that can reduce or eliminate their formation. These finding and method enhancements will improve the accurate detection of new RNA modifications.

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Surpassing limits of static RNA modification analysis with dynamic NAIL-MS

Cited by 25 publications

References 31 publications

Cell culture NAIL-MS allows insight into human RNA modification dynamicsin vivo

Cell culture NAIL-MS allows insight into human RNA modification dynamicsin vivo

Broad-range RNA modification analysis of complex biological samples using rapid C18-UPLC-MS

Chemical Amination/Imination of Carbonothiolated Nucleosides During RNA Hydrolysis

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