Mass spectrometry profiling analysis enables the identification of new modifications in ribosomal RNA

Ribosomal RNAs (rRNAs) provide the structural framework of ribosomes and play critical roles in protein translation. In ribosome biogenesis, rRNAs acquire various modifications that can influence the struc-ture and catalytic activity of ribosomes. However, rRNA modifications in plants have yet to be fully de-fined. Herein, we proposed a method to purify rRNAs by a successive isolation with different strategies, including polyA-based mRNA depletion and agarose gel electrophoresis-based purification, with which highly pure rRNAs could be obtained. In addition, we developed a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method to systematically profile and characterize modifications from the isolated highly pure plant 18S rRNA and 25S rRNA. LC-ESI-MS/MS analysis showed that 10 and 12 kinds of modifications were present in plant 18S rRNA and 25S rRNA, respectively. No-tably, among these identified modifications, 2 kinds of modifications of N2,N2-dimethylguanosine (m 2,2 G) and N6,N6-dimethyladenosine (m6,6A) in 18S rRNA, and 4 kinds of modifications of m2,2G, m6,6A, N7-methylguanosine (m7G) and 3-methyluridin (m3U) in 25S rRNA, were first reported to be present in plants. Moreover, exposure of Arabidopsis thaliana to cadmium (Cd) led to significant changes of mod-ifications in both 18S rRNA and 25S rRNA of plants, indicating that rRNA modifications play important roles in response to environmental stress. The discovery of new modifications in plant rRNAs improves the spectra of plant rRNA modifications and may promote the investigation of the functional roles of plant ribosomes in regulating gene expression.(c) 2023 Published by Elsevier B.V. on behalf of Chinese Chemical Society and Institute of Materia Medica, Chinese Academy of Medical Sciences.

Automated summary

In this study, a method for purifying plant ribosomal RNA (rRNA) was proposed and modifications in plant 18S rRNA and 25S rRNA were characterized using LC-ESI-MS/MS. The study revealed the presence of new modifications in plant rRNAs, including 2 modifications in 18S rRNA and 4 modifications in 25S rRNA. Exposure to cadmium induced significant changes in rRNA modifications, highlighting the importance of rRNA modifications in response to environmental stress. The discovery of new modifications expands our understanding of plant rRNA modifications and enhances the investigation of the functional roles of plant ribosomes in gene expression regulation.