Fig. 5

Mass spectrometry validation of rlmH::ISS1 ribosomes and modification profile of the intron RNA. a. Isolation spectrum of the signal at 337 m/z from wild-type (WT) rRNA. The isolated precursor ion was submitted to collisional activation, and the observed fragmentation pattern was compared with those recorded for the standards (Additional File 1: Fig. S16). Gray highlighting indicates the presence of the unique 239 m/z signal corresponding to m3Ψ (panel C and Additional File 1: Fig. S16). Other prominent signals, such as 211 and 265 m/z correspond to characteristic fragments generated by methylated uridine/pseudouridine isobars (Additional File 1: Fig. S16). b. Isolation and fragmentation spectra of the signal at 337 m/z from RNA isolated from rlmH::ISS1 ribosomes. Gray highlighting indicates where the 239 m/z signal would fall, were the m3Ψ species present in the mutant rRNA. c. Fragmentation spectrum obtained from the m3Ψ standard, showing the prominent diagnostic signal at 239 m/z (gray), which is absent in the spectra of the other isobars (Additional File 1: Fig. S16). d. Relative abundance (AvP) of ribonucleotide variants detected in intron and/or 16S rRNA. Modifications detected in purified intron RNA (red), 16S rRNA (black), and in vitro-transcribed intron RNA (IVT, gray) are shown. Abbreviations: m-G = methylated guanosine, mm-A = dimethyl adenosine, ac4C = acetyl cytidine, mm-C = dimethyl cytidine, m-U/Ψ = methylated uridine or methylated pseudouridine, m-C = methylated cytidine