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

TL;DR

This study identifies RNA modifications in cyanobacteria and shows how 5-methylcytosine (m5C) affects gene regulation and protein production.

Contribution

The first comprehensive map of mRNA m5C modifications in cyanobacteria and a new method for RNA modification analysis in prokaryotes.

Findings

Identified 21 RNA modifications in Synechocystis under varying conditions.

Mapped 824 m5C sites in mRNAs linked to ribosome, RNA degradation, carbon metabolism, and photosynthesis.

m5C modification is negatively correlated with protein abundance, indicating post-transcriptional regulation.

Abstract

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 analysis to identify 21 different RNA modifications in the model cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). Mass spectrometry analyses reveal a dynamic pattern of these RNA modifications under different culture conditions. We subsequently perform transcriptome-wide 5-methylcytosine (m5C) profiling in Synechocystis by using bisulfite sequencing. In total, we identify 824 high-confidence m5C sites in 382 mRNAs, with the majority of m5C-modified genes participating in ribosome, RNA degradation, carbon metabolism, and photosynthesis. Combined with the m5C-RNA immunoprecipitation detection method, 40.17% (331) m5C sites were validated and located within 129 m5C-RNA immunoprecipitation peaks on 145 mRNAs. Notably, integrated transcriptomic, proteomic, and m5C methylome analysis shows that m5C modification is negatively associated with protein abundance and contributes to the RNA–protein discordance, implying the importance of m5C on post-transcriptional regulation in Synechocystis. Collectively, our study provides a holistic view of RNA modifications and the first mRNA m5C 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.

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