Quantitative Analysis of rRNA Modifications Using Stable Isotope Labeling and Mass Spectrometry

Post-transcriptional RNA modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. The current lack of a comprehensive method for a fast quantitative analysis of rRNA modifications significantly limits our understanding of how individual...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2014-02, Vol.136 (5), p.2058-2069
Main Authors: Popova, Anna M, Williamson, James R
Format: Article
Language:English
Subjects:
Chromatography, Liquid - methods
Escherichia coli
Escherichia coli - enzymology
Isotope Labeling
Isotopes
Marking
Mass Spectrometry - methods
Monitoring
Nitrogen Isotopes
Protein synthesis
Proteins
Quantitative analysis
Residues
Ribonucleic acids
RNA Processing, Post-Transcriptional
RNA, Ribosomal, 16S - chemistry
RNA, Ribosomal, 16S - metabolism
RNA, Ribosomal, 23S - chemistry
RNA, Ribosomal, 23S - metabolism
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Summary:Post-transcriptional RNA modifications that are introduced during the multistep ribosome biogenesis process are essential for protein synthesis. The current lack of a comprehensive method for a fast quantitative analysis of rRNA modifications significantly limits our understanding of how individual modification steps are coordinated during biogenesis inside the cell. Here, an LC-MS approach has been developed and successfully applied for quantitative monitoring of 29 out of 36 modified residues in the 16S and 23S rRNA from Escherichia coli. An isotope labeling strategy is described for efficient identification of ribose and base methylations, and a novel metabolic labeling approach is presented to allow identification of MS-silent pseudouridine modifications. The method was used to measure relative abundances of modified residues in incomplete ribosomal subunits compared to a mature 15N-labeled rRNA standard, and a number of modifications in both 16S and 23S rRNA were present in substoichiometric amounts in the preribosomal particles. The RNA modification levels correlate well with previously obtained profiles for the ribosomal proteins, suggesting that RNA is modified in a schedule comparable to the association of the ribosomal proteins. Importantly, this study establishes an efficient workflow for a global monitoring of ribosomal modifications that will contribute to a better understanding of mechanisms of RNA modifications and their impact on intracellular processes in the future.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja412084b