Transcriptome-wide high-throughput deep m(6)A-seq reveals unique differential m(6)A methylation patterns between three organs in Arabidopsis thaliana

Abstract

Background: m(6)A is a ubiquitous RNA modification in eukaryotes. Transcriptome-wide m(6)A patterns in Arabidopsis have been assayed recently. However, differential m(6)A patterns between organs have not been well characterized.

Results: Over two-third of the transcripts in Arabidopsis are modified by m(6)A. In contrast to a recent observation of m(6)A enrichment in 5' mRNA, we find that m(6)A is distributed predominantly near stop codons. Interestingly, 85 % of the modified transcripts show high m(6)A methylation extent compared to their transcript level. The 290 highly methylated transcripts are mainly associated with transporters, stress responses, redox, regulation factors, and some non-coding RNAs. On average, the proportion of transcripts showing differential methylation between two plant organs is higher than that showing differential transcript levels. The transcripts with extensively higher m(6)A methylation in an organ are associated with the unique biological processes of this organ, suggesting that m(6)A may be another important contributor to organ differentiation in Arabidopsis. Highly expressed genes are relatively less methylated and vice versa, and different RNAs have distinct m(6)A patterns, which hint at mRNA fate. Intriguingly, most of the transposable element transcripts maintained a fragmented form with a relatively low transcript level and high m(6)A methylation in the cells.

Conclusions: This is the first study to comprehensively analyze m(6)A patterns in a variety of RNAs, the relationship between transcript level and m(6)A methylation extent, and differential m(6)A patterns across organs in Arabidopsis.

Fig. 1

Number of overlapped m⁶A transcripts in the two m⁶A-seq replicates of three organs of leaves (a), flowers (b), and roots (c) in Arabidposis. The analysis indicated that over 83 % of m⁶A transcripts were overlapped in the two m⁶A-seq replicates

Fig. 2

Number of the overlapped transcripts in the two (a, b) m⁶A-seq replicates

Fig. 3

The general m⁶A patterns in Arabidopsis. a Proportion of the modified transcripts containing different m⁶A sites. b Sequence logo representing the most common consensus motif (RRm⁶ACH) in the m⁶A peaks in Arabidopsis

Fig. 4

Schematic screen shots of two typical types of m⁶A topologies in mRNA in Arabidopsis. a Type 1 (representative gene, ‘AT2G01910’; trace files of two organs (flowers and roots) were presented), one or two predominant peaks in 3′UTR or at stop codon with several much lower signals in the codon regions. The peaks in 3′UTR or at stop codon were two to tens of folds of the signals in the codon regions. Most of the messenger RNA (over 70 %) presented this type of m⁶A topology. b Type 2 (representative gene, ‘At3g29030’; trace files of three organs, leaves (the upper), flowers (in the middle), and roots (the lower) were presented). Several m⁶A sites distributing in the transcripts with low m⁶A signals (in the middle and lower parts of the figure, representative of flowers and roots) or without peaks in 3′UTR or at stop codon (in the upper part, representative of leaves)

Fig. 5

The overall m⁶A distributing pattern from 5′ (left) to 3′ (right) in the m⁶A-seq datasets and the overall transcriptional pattern from 5′ (left) to 3′ (right) in the mRNA-seq datasets in the three organs of Arabidopsis. The patterns with the caption of ‘CK’ in the image was deduced from the mRNA-seq datasets. The other threes were from the m⁶A-seq dataset. The number ‘0’ on the left refers to TSS. The numbers from 49 to 59 represents stop codon or the proximate 3′UTR. As shown in this figure, only one dominant peak of m⁶A enrichment was detected around 3′UTR or stop codon in the overall dataset of the Arabidopsis transcriptome in this study

Fig. 6

Schematic screen shots of the m⁶A patterns in rRNA, tRNA and sn(o)RNA. a Numerous m⁶A sites distributed in a rRNA transcript (reprehensive ‘AT2g01010’). b A single m⁶A in a rRNA transcript (representative ‘AT2g01020’). c Low m⁶A methylation in most of tRNA (approximately 90 %, reprehensive ‘AT1g06480’). d sn(o) RNA was highly methylated by a single m⁶A site (representative ‘AT3G56705’)

Fig. 7

GO analysis of the biological process (a) and molecular functions (b) for the 290 transcripts extensively methylated by m⁶A in the Arabidopsis transcriptome. Proportions in the figures indicated the level of the hits for each classification in the blast. As the majority of mRNA was methylated by m⁶A, methylation occurs on a functionally distinct subset of transcripts. However, most of the gene transcripts extensively methylated by m⁶A were related to the proteins that they were a direct transporter for energy molecules, for example, ATP or GTP, or accomplishment of their biological functions was dependent on these energy chaperones

Fig. 8

Heatmap by the gene alphabetic order presenting the overall differential patterns of both gene transcript and m⁶A methylation level in the transcripts in Arabidopsis. a Overview of the differential patterns of m⁶A methylation between leaves and flowers. b Overview of the differential patterns of m⁶A methylation between leaves and roots. c Overview of the differential patterns of m⁶A methylation between flowers and roots. d Overview of the differential patterns of gene transcript level between leaves and flowers. e Overview of the differential patterns of gene transcript level between leaves and roots. f Overview of the differential patterns of gene transcript level between flowers and roots. The patterns in all of the six comparisons (a to f) above were based on the alphabetic order of the gene ID representing Chromosomes 1 to 5 (up to down)

Fig. 9

GO analysis of molecular functions of transcripts presenting a higher extent of m⁶A methylation in leaves (a), flowers (b), or roots (c) than other two organs in Arabidopsis

Fig. 10

The transcriptional pattern (a) and m⁶A patterns (b) in the TE transcripts. The ‘fragmented form’ was observed in most of the TE transcript (the representative gene, AT5G35835, and trace files of two organs (leaves and flowers))