scholarly journals Cell culture NAIL-MS allows insight into human tRNA and rRNA modification dynamics in vivo

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Matthias Heiss ◽  
Felix Hagelskamp ◽  
Virginie Marchand ◽  
Yuri Motorin ◽  
Stefanie Kellner
Keyword(s):  
Cell Culture ◽  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Labeling ◽  
Modified Nucleosides ◽  
Rna Modification ◽  
Rna Transcripts ◽  
Temporal Placement ◽  
Depth Study

AbstractRecently, studies about RNA modification dynamics in human RNAs are among the most controversially discussed. As a main reason, we identified the unavailability of a technique which allows the investigation of the temporal processing of RNA transcripts. Here, we present nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS) for efficient, monoisotopic stable isotope labeling in both RNA and DNA in standard cell culture. We design pulse chase experiments and study the temporal placement of modified nucleosides in tRNAPhe and 18S rRNA. In existing RNAs, we observe a time-dependent constant loss of modified nucleosides which is masked by post-transcriptional methylation mechanisms and thus undetectable without NAIL-MS. During alkylation stress, NAIL-MS reveals an adaptation of tRNA modifications in new transcripts but not existing ones. Overall, we present a fast and reliable stable isotope labeling strategy which allows in-depth study of RNA modification dynamics in human cell culture.

scholarly journals Cell culture NAIL-MS allows insight into human RNA modification dynamics in vivo

2020 ◽  
Author(s):  
Matthias Heiss ◽  
Felix Hagelskamp ◽  
Stefanie Kellner
Keyword(s):  
Cell Culture ◽  
Stable Isotope ◽  
Human Cell ◽  
Isotope Labeling ◽  
Stable Isotope Labeling ◽  
Modified Nucleosides ◽  
Rna Modification ◽  
Growth Media ◽  
Human Cell Culture ◽  
The Impact

AbstractIn 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. tRNAPhe 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.

Benefits of stable isotope labeling in RNA analysis

2019 ◽  
Vol 400 (7) ◽  
pp. 847-865 ◽  
Author(s):  
Paria Asadi-Atoi ◽  
Pierre Barraud ◽  
Carine Tisne ◽  
Stefanie Kellner
Keyword(s):  
Mass Spectrometry ◽  
Stable Isotope ◽  
Rna Structure ◽  
Isotope Labeling ◽  
Stable Isotope Labeling ◽  
Modified Nucleosides ◽  
Rna Modification ◽  
Metabolic Labeling ◽  
Signal Loss ◽  
Labeled Rna

Abstract RNAs are key players in life as they connect the genetic code (DNA) with all cellular processes dominated by proteins. They contain a variety of chemical modifications and many RNAs fold into complex structures. Here, we review recent progress in the analysis of RNA modification and structure on the basis of stable isotope labeling techniques. Mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy are the key tools and many breakthrough developments were made possible by the analysis of stable isotope labeled RNA. Therefore, we discuss current stable isotope labeling techniques such as metabolic labeling, enzymatic labeling and chemical synthesis. RNA structure analysis by NMR is challenging due to two major problems that become even more salient when the size of the RNA increases, namely chemical shift overlaps and line broadening leading to complete signal loss. Several isotope labeling strategies have been developed to provide solutions to these major issues, such as deuteration, segmental isotope labeling or site-specific labeling. Quantification of modified nucleosides in RNA by MS is only possible through the application of stable isotope labeled internal standards. With nucleic acid isotope labeling coupled mass spectrometry (NAIL-MS), it is now possible to analyze the dynamic processes of post-transcriptional RNA modification and demodification. The trend, in both NMR and MS RNA analytics, is without doubt shifting from the analysis of snapshot moments towards the development and application of tools capable of analyzing the dynamics of RNA structure and modification profiles.

scholarly journals Intercellular Transfer of Proteins as Identified by Stable Isotope Labeling of Amino Acids in Cell Culture

2009 ◽  
Vol 285 (9) ◽  
pp. 6285-6297 ◽  
Author(s):  
Ming Li ◽  
Jason M. Aliotta ◽  
John M. Asara ◽  
Qian Wu ◽  
Mark S. Dooner ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Stable Isotope ◽  
Isotope Labeling ◽  
Stable Isotope Labeling ◽  
Intercellular Transfer

scholarly journals Characterization of the Primary Human Trophoblast Cell Secretome Using Stable Isotope Labeling With Amino Acids in Cell Culture

2021 ◽  
Vol 9 ◽  
Author(s):  
Fredrick J. Rosario ◽  
Sammy Pardo ◽  
Trond M. Michelsen ◽  
Kathryn Erickson ◽  
Lorna Moore ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Culture ◽  
Stable Isotope ◽  
Human Placenta ◽  
Isotope Labeling ◽  
Stable Isotope Labeling ◽  
Mass Spectrometry Analysis ◽  
Secreted Proteins ◽  
Placental Villus ◽  
Human Trophoblast

The placental villus syncytiotrophoblast, the nutrient-transporting and hormone-producing epithelium of the human placenta, is a critical regulator of fetal development and maternal physiology. However, the identities of the proteins synthesized and secreted by primary human trophoblast (PHT) cells remain unknown. Stable Isotope Labeling with Amino Acids in Cell Culture followed by mass spectrometry analysis of the conditioned media was used to identify secreted proteins and obtain information about their relative rates of synthesis in syncytialized multinucleated PHT cells isolated from normal term placental villus tissue (n = 4/independent placenta). A total of 1,344 proteins were identified, most of which have not previously been reported to be secreted by the human placenta or trophoblast. The majority of secreted proteins are involved in energy and carbon metabolism, glycolysis, biosynthesis of amino acids, purine metabolism, and fatty acid degradation. Histone family proteins and mitochondrial proteins were among proteins with the slowest synthesis rate whereas proteins associated with signaling and the plasma membrane were synthesized rapidly. There was a significant overlap between the PHT secretome and proteins known be secreted to the fetal circulation by the human placenta in vivo. The generated data will guide future experiments to determine the function of individual secreted proteins and will help us better understand how the placenta controls maternal and fetal physiology.

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