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J Am Chem Soc. 2014 Feb 5;136(5):2058-69. doi: 10.1021/ja412084b. Epub 2014 Jan 27.

Quantitative analysis of rRNA modifications using stable isotope labeling and mass spectrometry.

Author information

1
Department of Integrative Structural and Computational Biology and ‡Department of Chemistry, The Scripps Research Institute , La Jolla, California 92037, United States.

Abstract

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 (15)N-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.

PMID:
24422502
PMCID:
PMC3985470
DOI:
10.1021/ja412084b
[Indexed for MEDLINE]
Free PMC Article

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