High-throughput m6A-seq reveals RNA m6A methylation patterns in the chloroplast and mitochondria transcriptomes of Arabidopsis thaliana

Abstract

This study is the first to comprehensively characterize m6A patterns in the Arabidopsis chloroplast and mitochondria transcriptomes based on our open accessible data deposited in NCBI's Gene Expression Omnibus with GEO Series accession number of GSE72706. Over 86% of the transcripts were methylated by m6A in the two organelles. Over 550 and 350 m6A sites were mapped, with ~5.6 to ~5.8 and ~4.6 to ~4.9 m6A sites per transcript, to the chloroplast and mitochondria genome, respectively. The overall m6A methylation extent in the two organelles was greatly higher than that in the nucleus. The m6A motif sequences in the transcriptome of two organelles were similar to the nuclear motifs, suggesting that selection of the m6A motifs for RNA methylation was conserved between the nucleus and organelle transcriptomes. The m6A patterns of rRNAs and tRNAs in the organelle were similar to those in the nucleus. However, the m6A patterns in coding RNAs were distinct between the nucleus and the organelle, suggesting that that regulation of the m6A methylation patterns may be different between the nuclei and the organelles. The extensively methylated transcripts in the two organelles were mainly associated with rRNA, ribosomal proteins, photosystem reaction proteins, tRNA, NADH dehydrogenase and redox. On average, 64% and 79% of the transcripts in the two organelles showed differential m6A methylation across three organs of the leaves, flowers and roots. The m6A methylation extent in the chloroplast was higher than that in the mitochondria. This study provides deep insights into the m6A methylation topology and differentiation in the plant organelle transcriptomes.

Fig 1. Screenshots from the IGV visualized program present two typical types of m⁶A topologies in the coding RNAs in the Arabidopsis mitochondria.

Extent of m⁶A methylation, sequencing depth, sequencing fragment alignment, and gene ID of the sequencing data can be clearly visualized by the IGV program [42]. The area in the screenshot indicated by the arrow, “Red leftwards arrow”, presents m⁶A methylation extent across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow”, presents sequencing fragment alignment across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow with tail”, presents gene ID information including gene ID, sequence reading direction, the intron and exon regions. (a) Type 1 (representative gene, ‘ATMG00920’, expressed for ‘a hypothetical protein’), the whole transcript without intron was highly methylated by m⁶A; (b) Type 2 (representative gene, ‘ATMG00160’, expressed for ‘cytochrome oxidase 2’), the exon was highly methylated but the intron was not methylated by m⁶A. Trace files of three organs, leaves (the upper), flowers (in the middle) and roots (the lower) were presented within a screenshot.

Fig 2. Number of the overlapped m⁶A transcripts in the two m⁶A-seq replicates.

(a) in the leaf chloroplast; (b) in the flower chloroplast; (c) in the root amyloplast; (d)in the leaf mitochondria; (e) in the flower mitochondria; and (f) in the root mitochondria.

Fig 3. Features of m⁶A methylation in two organelles.

(a) Proportion of the transcribed methylated genes containing different m⁶A sites in the chloroplast/amyloplast transcriptome; (b) proportion of the transcribed methylated genes containing different m⁶A sites in the mitochondria transcriptome; (c) the most common consensus motif (RRm⁶ACH) in the m⁶A peaks in the chloroplast/amyloplast transcriptome; and (d) the most common consensus motif (RRm⁶ACH) in the m⁶A peaks the mitochondria transcriptome.

Fig 4. Screenshots from the IGV visualized program present two typical types of m⁶A topologies in the coding RNAs in the Arabidopsis chloroplast/amyloplast.

Extent of m⁶A methylation, sequencing depth, sequencing fragment alignment, and gene ID of the sequencing data can be clearly visualized by the IGV program [42]. The area in the screenshot indicated by the arrow, “Red leftwards arrow”, presents m⁶A methylation extent across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow”, presents sequencing fragment alignment across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow with tail”, presents gene ID information including gene ID, sequence reading direction, the intron and exon regions. (a) Type 1 (representative gene, ‘ATCG00020’, expressed for ‘photosystem II reaction center protein A’), the whole transcript without intron was highly methylated by m⁶A; and (b) Type 2 (representative gene, ‘ATCG00130’, expressed for ‘ATPase, F0 complex, subunit B/B’), the exon was highly methylated but the intron was less methylated by m⁶A. Trace files of three organs, leaves (the upper), flowers (in the middle) and roots (the lower) were presented within a screenshot.

Fig 5. Screenshots from the IGV visualized program present m⁶A topologies in rRNA and tRNAs in the Arabidopsis chloroplast/amyloplast.

Extent of m⁶A methylation, sequencing depth, sequencing fragment alignment, and gene ID of the sequencing data can be clearly visualized by the IGV program [42]. The area in the screenshot indicated by the arrow, “Red leftwards arrow”, presents m⁶A methylation extent across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow”, presents sequencing fragment alignment across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow with tail”, presents gene ID information including gene ID, sequence reading direction, the intron and exon regions. (a) The whole rRNA was highly methylated by m⁶A, representative rRNA, ‘ATCG00920’; (b) The whole tRNA with intron was highly methylated, representative tRNA, ‘ATCG00100’; and (c) The whole tRNA without intron was highly methylated, representative tRNA, ‘ATCG00110’. The Trace files of three organs, leaves (the upper), flowers (in the middle) and roots (the lower) were presented within a screenshot.

Fig 6. Screenshots from the IGV visualized program present m⁶A topologies in rRNA and tRNAs in the Arabidopsis mitochondria.

Extent of m⁶A methylation, sequencing depth, sequencing fragment alignment, and gene ID of the sequencing data can be clearly visualized by the IGV program [42]. The area in the screenshot indicated by the arrow, “Red leftwards arrow”, presents m⁶A methylation extent across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow”, presents sequencing fragment alignment across the transcript. The area in the screenshot indicated by the arrow, “Black leftwards arrow with tail”, presents gene ID information including gene ID, sequence reading direction, the intron and exon regions. (a) The whole rRNA was highly methylated by m⁶A, representative rRNA, ‘ATMG01390’; (b) The whole tRNA was slightly methylated by m⁶A, representative tRNA, ‘ATMG00380’, expressed for tRNA-Asn. The Trace files of three organs, leaves (the upper), flowers (in the middle) and roots (the lower) were presented within a screenshot.