Prevalence of N6-methyladenosine (m6A) in mycoplasma mRNA:Epitranscriptomic regulation in minimal genomes

Messenger RNA N6 methyladenosine (m6A) modification has been considered as the main post-transcriptional modification of eukaryotic mRNA; however, its role in the regulation of prokaryotic mRNA transcription remains unclear. The N6 methyladenosine (m6A) modifications in prokaryotic mRNA has been found in Pseudomonas aeruginosa and Escherichia coli so far. In this study, ultra-high-pressure liquid chromatography coupled with triple quadrupole tandem mass spectroscopy (UHPLC-QQQ-MS/MS) was used to calculate the m6A/A ratio in multiple mRNA from a wide range of mycoplasma species, representing as a category of genomically minimal prokaryote. The results showed that mycoplasma mRNA has a higher m6A/A ratio than other prokaryotes and eukaryotes reported previously, varying in the range of 0.07-4.56 %. Furthermore, Nano UMI meRIP-seq analysis (a high-resolution long-read sequencing approach integrating unique molecular identifiers (UMIs) to map RNA methylation at the transcriptome level across eight different mycoplasma species. It showed that most m6A peaks are located in the protein coding region with unique "GGAGG" motif, which is different from those described in eukaryotes and other prokaryotes previously. Gene Ontology (GO) analysis showed that the genes regulated by this methylation modification system was involved in the ribosome, pyrimidine metabolism, purine metabolism, pyruvate metabolism and other metabolic pathways required for mycoplasma growth. To explore the potential functional impact of m⁶A methylation, we performed RNA pull-down assays and identified three virulent candidate m⁶A-binding proteins: Tuf (elongation factor Tu), prfA (peptide chain release factor A), and mgtA (magnesium transporter A). Microscale thermophoresis (MST) analysis also revealed that the three proteins exhibited significantly stronger binding affinities to m⁶A-modified RNA compared to their unmethylated counterparts, demonstrating their selective recognition of methylated transcripts. Further structural prediction using AlphaFold3 suggested specific amino acid residues mediating interactions with methylated adenines, offering mechanistic insights into m⁶A-protein interactions. Together, these findings firstly provided the landscape of m⁶A RNA methylation in mycoplasma and suggest that m⁶A may participate in post-transcriptional regulation by modulating RNA-protein interactions in mycoplasma genome, hinting that epitranscriptomic m⁶A regulation of mycoplasma mRNA may be associated with pathogenicity.