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

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 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.

Single methylation of 23S rRNA triggers late steps of 50S ribosomal subunit assembly

“…Given that most 50S subunits in cells are associated with 30S subunits at sufficient concentrations of Mg 2+ , it is reasonable to speculate that the physiological substrate for RlmE is not the 50S subunit, but rather the 45S precursor, because Um2552 in H92 of 23S rRNA is located on the interface side of the 50S subunit (24)(25)(26). Quantitative analyses of rRNA modifications by HPLC/MS have shown that the level of Um2552 surges during late steps of 50S assembly (32,33), and these results are consistent with our observations. To obtain insights into the function of an RNA modification responsible for a specific biological process, it is necessary to determine whether the RNA-modifying enzyme or the RNA modification itself plays a pivotal role in the process.…”

Pseudo-Seq

“…The bulky Ψ-CMC adduct acts as a strong stop to reverse transcriptase (RT) one nucleotide 3 0 of the Ψ, allowing Ψ locations to be mapped using gene-specific primer extension assays (Bakin & Ofengand, 1993). More recently, techniques have been developed to allow site-specific identification and quantification of Ψ using thin-layer chromatography (Zhao & Yu, 2004), as well as mass spectrometry-based techniques that allow detection of normally mass silent Ψ (Durairaj & Limbach, 2008;Popova & Williamson, 2014). While powerful in some contexts, these assays are low throughput and are best suited to identification of Ψs in highly abundant RNAs.…”

Pseudouridine: Still mysterious, but never a fake (uridine)!

“…Incomplete reactions, side products, and unstable response factors prevent quantitative analysis by this approach. In consequence there is an increasing interest in derivatization-free MS/ MS-based approaches that allow quantitative analysis, 26,27 and which have recently included isotope labeling 28,29 So called hypermodified C derivatives are formed via further modification of C. At present, they comprise Cm, m 1 C, (found in all domains of life, predominantly in tRNA 1 ), m 3 C (in rRNA of Eubacteria 1 ), as well as 3-(3-amino-3-carboxypropyl)-C .axap3C/ 30,31 and m 1 acap 3 C 1 in rRNA of eukaryotes (Fig. 1).…”