Topologies of N⁶-adenosine methylation (m⁶A) in land plant mitochondria and their putative effects on organellar gene expression

Mitochondria serve as major sites of ATP production and play key roles in many other metabolic processes that are critical to the cell. As relicts of an ancient bacterial endosymbiont, mitochondria contain their own hereditary material (i.e. mtDNA, or mitogenome) and a machinery for protein biosynthesis. The expression of the mtDNA in plants is complex, particularly at the post-transcriptional level. Following transcription, the polycistronic pre-RNAs undergo extensive modifications, including trimming, splicing and editing, before being translated by organellar ribosomes. Our study focuses on N⁶-methylation of adenosine ribonucleotides (m⁶A-RNA) in plant mitochondria. m⁶A is a prevalent modification in nuclear-encoded mRNAs. The biological significance of this dynamic modification is under investigation, but it is widely accepted that m⁶A mediates structural switches that affect RNA stability and/or activity. Using m⁶A-pulldown/RNA-seq (m⁶A-RIP-seq) assays of Arabidopsis and cauliflower mitochondria, we provide information on the m⁶A-RNA landscapes in Arabidopsis thaliana and Brassica oleracea mitochondria. The results show that m⁶A targets different types of mitochondrial transcripts, including known genes, mtORFs, as well as non-coding (transcribed intergenic) RNA species. While ncRNAs undergo multiple m⁶A modifications, N⁶-methylation of adenosine residues with mRNAs seem preferably positioned near start codons and may modulate their translatability.