Transcriptome-wide N⁶-methyladenosine profiling of rice callus and leaf reveals the presence of tissue-specific competitors involved in selective mRNA modification

Abstract

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification present in mRNAs of all higher eukaryotes. With the development of MeRIP-seq technique, in-depth identification of mRNAs with m(6)A modification becomes feasible. Here we present a transcriptome-wide m(6)A modification profiling effort for rice transcriptomes of differentiated callus and leaf, which yields 8,138 and 14,253 m(6)A-modified genes, respectively. The m(6)A peak (m(6)A-modified nucleotide position on mRNAs) distribution exhibits preference toward both translation termination and initiation sites. The m(6)A peak enrichment is negatively correlated with gene expression and weakly positively correlated with certain gene features, such as exon length and number. By comparing m(6)A-modified genes between the 2 samples, we define 1,792 and 6,508 tissue-specific m(6)A-modified genes (TSMGs) in callus and leaf, respectively. Among which, 626 and 5,509 TSMGs are actively expressed in both tissues but are selectively m(6)A-modified (SMGs) only in one of the 2 tissues. Further analyses reveal characteristics of SMGs: (1) Most SMGs are differentially expressed between callus and leaf. (2) Two conserved RNA-binding motifs, predicted to be recognized by PUM and RNP4F, are significantly over-represented in SMGs. (3) GO enrichment analysis shows that SMGs in callus mainly participate in transcription regulator/factor activity whereas SMGs in leaf are mainly involved in plastid and thylakoid. Our results suggest the presence of tissue-specific competitors involved in SMGs. These findings provide a resource for plant RNA epitranscriptomic studies and further enlarge our knowledge on the function of RNA m(6)A modification.

Keywords: BPTM, bases per 10 millions of reads; MeRIP-seq; N6-methyladenosine; RNA epitranscriptomic; RNA m6A modification; RPKM, reads per kilo base per million mapped reads; SMG, selective methylated gene; TSMG, tissue specific methylated gene; rice.

Figure 1.

Distribution of m⁶A modification peaks along mRNA and chromosome (A) Transcriptome-wide distribution of m⁶A peaks. Bar graph shows the fraction of m⁶A peaks in each of the 5 non-overlapping transcript segments. (B) Distribution of m⁶A peaks across the length of mRNAs. 5′UTRs, CDS, and 3′UTRs of rice mRNAs are individually binned into regions spanning 1% of their total length, and the percentage of m⁶A peaks that fall within each bin is determined. (C)Distribution of m⁶A peaks along chromosomes of callus and leaf tissues. From top to bottom, 3 horizontal bars represent chromosomes, where the m⁶A peak density in callus, leaf, TE gene and expressed gene (taking callus as samples) in 500-kb windows are displayed with scales as indicated. Diamonds in different colors (light blue->green-> brown->purple) represent numbers of peaks or genes ascending.

Figure 2.

Characteristics and distribution of m⁶A modification peaks among rice genes (A) Percentage of m⁶A methylated genes with different m⁶A peak number. (B) Correlations between m⁶A enrichment value and the length of CDS, 5′UTR, 3′UTR, intron, gene, and mRNA, as well as exon number. The color bars represent the Pearson Correlation Coefficient (PCC) values. (C–D) Correlations between m⁶A peak enrichment and mRNA abundance. Peak enrichment value (the ratio of MeRIP sample reads to non-IP sample reads within the area of peaks, each normalized to the number of reads within the sample) relative to transcript abundance measured by RPKM (reads per kilo base per million mapped reads) values in the control are also plotted.

Figure 3

Examples of selected methylated genes and GO enriched functional classes for SMGs (A) Two examples of selectively methylated genes (SMGs). The peaks (gray vertical bars) of SMGs in callus (in the left panel; LOC_Os07g12510.1) and leaf (the right panel; LOC_Os09g10760.1) and BPTM (base per 10 millions of reads) values for the 2 genes are shown. (B) GO analysis of commonly methylated genes (CM) and selectively methylated genes in callus (CS) and leaf (LS). Diamonds in different colors represent GO term enrichment values, and gray color means the term is not significantly enriched. *MB, membrane-bound.