Global discovery of RNA modifications and functional analysis of m5C methylome in cyanobacteria

RNA modifications have been found in all domains of life and play regulatory roles in diverse biological processes. However, their distribution, function, and regulation in cyanobacteria remain unexplored. Here, we have employed a quantitative RNA profiling strategy based on mass spectrometry (MS) analysis to identify 21 different RNA modifications in the model cyanobacterium Synechocystis sp. PCC 6803 ( Synechocystis ). MS analyses reveal a dynamic pattern of these RNA modifications under different culture conditions. We subsequently perform transcriptome-wide 5-methylcytosine (m 5 C) profiling in Synechocystis by using bisulfite sequencing. In total, we identify 824 high-confidence m 5 C sites in 382 mRNAs, with majority of m 5 C-modified genes participating in ribosome, RNA degradation, carbon metabolism and photosynthesis. Combined with m 5 C RNA immunoprecipitation (m 5 C-RIP) detection method, 40.17% (331) m 5 C sites were validated and located within 129 m 5 C-RIP peaks on 145 mRNAs. Notably, integrated transcriptomic, proteomic and m 5 C methylome analysis shows that m 5 C modification is negatively associated with protein abundance and contributes to the RNA–protein discordance, implying the importance of m 5 C on post-transcriptional regulation in Synechocystis . Collectively, our study provides a holistic view of RNA modifications and the first mRNA m 5 C map in cyanobacteria, which present a critical database for functional analyses of RNA modifications in cyanobacteria. The method used in this study is applicable to any sequenced prokaryotes and could be applied as a standard part of transcriptomic analysis.