mRNA m6A regulates gene expression via H3K4me3 shift in 5’ UTR

Yuna Yang; Yuqing Huang; Tian Wang; Song Li; Jiafu Jiang; Sumei Chen; Fadi Chen; Likai Wang

doi:10.1186/s13059-025-03515-8

. 2025 Mar 12;26:54. doi: 10.1186/s13059-025-03515-8

mRNA m⁶A regulates gene expression via H3K4me3 shift in 5’ UTR

Yuna Yang ^1,², Yuqing Huang ^1,², Tian Wang ^1,², Song Li ^1,², Jiafu Jiang ^1,², Sumei Chen ^1,², Fadi Chen ^1,², Likai Wang ^1,^2,^✉

PMCID: PMC11900566 PMID: 40075435

Abstract

Background

N⁶-methyladenosine (m⁶A) is a prevalent and conserved RNA modification in eukaryotes. While its roles in the 3’ untranslated regions (3’ UTR) are well-studied, its role in the 5' UTR and its relationship with histone modifications remain underexplored.

Results

We demonstrate that m⁶A methylation in the 5’ UTR of mRNA triggers a downstream shift in H3K4me3 modification. This regulatory mechanism is conserved in Arabidopsis, rice, and chrysanthemum. The observed shift in H3K4me3 is genetically controlled by m⁶A modifiers and influences gene expression. MTA, the m⁶A methylase, preferentially binds to phosphorylated serine 5 (Ser5P)-CTD of RNA Pol II during transcription, leading to the displacement of ATX1, the H3K4me3 methylase. This dynamic binding of MTA and ATX1 to RNA Pol II ultimately results in the shift of H3K4me3 modification. Genetic evidence demonstrates that m⁶A in the 5' UTR controls H3K4me3 shift, thereby affecting SEDOHEPTULOSE-BISPHOSPHATASE expression and leaf senescence.

Conclusions

Our study provides new insights into the roles of m⁶A modification and its crosstalk with histone modification in 5’ UTRs, shedding light on the mechanism of m⁶A-mediated gene expression regulation.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13059-025-03515-8.

Background

N⁶-methyladenosine (m⁶A) is the most common, abundant, and conserved internal transcriptional modification in eukaryotes, and it is closely related to various biological functions, and plays vital roles in cells [1, 2]. m⁶A is a key regulatory mechanism that controls gene expression, and has been identified in many organisms, including mammals [1], yeast [3], and plants [4]. The “writer” complex installs the mRNA m⁶A modification, a conserved m⁶A methyltransferase complex including methyltransferases like 3 (METTL3), METTL14, Wilms’ tumor 1-associating protein (WTAP), MTA, FIP37 and FIONA1 [4–6]. In addition, the “reader” proteins, such as the YTH domain family, recognize the m⁶A modified transcripts and mediate RNA metabolism [7, 8]. The mRNA m⁶A modification is reversible and can be removed by “eraser” proteins, including fat-mass and obesity-associated protein (FTO), alkylated DNA repair protein alkB homolog 5 (ALKBH5) [9, 10], ALKBH9B [11] and ALKBH10B [12].

The roles and regulatory mechanisms of m⁶A modification in 3’ untranslated regions (3’ UTR) have been extensively studied. However, little attention has been given to 5′ UTR-specific m⁶A functions. Recent studies showed that m⁶A modifications in 5’ UTR regions are essential in regulating cortical development in mice, especially through mRNA transport and surveillance [13]. mRNAs containing m⁶A in their 5’ UTR can be translated cap-independently, by binding eukaryotic initiation factor 3 (eIF3) to recruit the 43S complex to initiate translation without the cap-binding factor [14]. 5′ UTR-specific m⁶A controls ribosome scanning and subsequent start codon selection to control global alternative translation during amino acid starvation [15]. In response to heat shock stress, m⁶A is preferentially deposited at the 5′UTR of newly transcribed mRNAs, resulting from stress-induced nuclear localization of m⁶A “reader” YTHDF2 [16]. All previous studies have focused on the roles of 5′ UTR-specific m⁶A in post-transcriptional regulation. However, whether these m⁶A modifications are involved in transcriptional regulation, especially in plant species, remains unknown.

Chromatin states regulate gene expression and responses to environmental stimuli during plant development. Various histone modification combinations, including histone methylation, acetylation, and phosphorylation, determine chromatin states. Recent studies have shown that m⁶A regulates chromatin accessibility and gene transcription through DNA demethylation [17], chromosome-associated regulatory RNAs (carRNAs) [18] and histone modification [19]. In addition, the m⁶A modification of mRNAs is mainly guided by the tri-methylation of histone H3 Lys-36 (H3K36me3) in animals [20]. The loss of H3K36me3 leads to a significant reduction in RNA m⁶A modification [20]. However, in plants, the histone modification H3K36me2, not H3K36me3, is a potential determinant of m⁶A modification [21]. Many other histone modification markers are associated with m⁶A methylation. For instance, H3K4me3 histone modifications are typically associated with active gene promoters. In mammalian research, it has been shown that the RNA m⁶A methyltransferase METTL3 binds to the transcription start site (TSS) in the vicinity of H3K4me3 histone marker peaks and facilitates m⁶A modification and enhances translation of the associated RNA transcript [22]. H3K27me3 histone modification, unlike H3K4me3, typically inhibits gene expression within heterochromatin. Although over-representation of the H3K27me3 histone modification near the m⁶A peaks was not detected [20], METTL3 depletion was observed to reduce the levels of H3K27me3 in animals [23]. However, the link between histone modifications and RNA m⁶A methylation remains unclear in plant.

In this work, by analyzing m⁶A methylation in different regions, we observed a consistent and conserved association between m⁶A methylation specifically in the 5’ UTR regions, referred to as m⁶A₅, and low levels of transcription across multiple plant species, including Arabidopsis, rice, and chrysanthemum. We identified that H3K4me3 modification was involved in the m⁶A-associated expression regulation of m⁶A₅. We found that MTA preferentially binding to the phosphorylated serine 5 (Ser5P)-CTD domain of RNA Pol II displaces the H3K4me3 methylase ATX1, resulting in H3K4me3 modification shifts at the transcription initiation and early elongation process. Through genetic validation, we observed that the shift of H3K4me3 modification is regulated by m⁶A modification in 5’ UTR regions, resulting in gene expression and leaf senescence regulation. Overall, this work establishes a mechanism by which m⁶A modification in 5’ UTR regions regulates H3K4me3 shift to finetune gene expression.

Results

m⁶A methylation, uniquely deposited in 5’ UTR regions, contributes to gene repression

The untranslated regions (UTRs) are recognized for their important role in regulating gene expression [24]. To explore the potential roles of 5’ UTRs in m⁶A-mediated transcriptional regulation, we re-analyzed the publicly available meRIP-seq and RNA-seq data in Arabidopsis from NCBI GEO GSE180768. We divided genes into five groups based on the location of m⁶A methylation in the gene transcripts in Arabidopsis, including genes with m⁶A methylation in 5’ UTR region (5’ UTR), genes with m⁶A methylation in both 5’ UTR and 3’ UTR region (m⁶A₅₃; Additional file 1: Fig. S1A), genes with m⁶A methylation only in 5’ UTR region (m⁶A₅; Additional file 1: Fig. S1B), genes with m⁶A methylation only in 3’ UTR region (m⁶A₃; Additional file 1: Fig. S1C), and genes with m⁶A methylation in 3’ UTR region (3’ UTR). Previous studies have consistently reported lower levels of m⁶A modification in the 5' UTR region compared to the 3' UTR region [25, 26]. To validate the presence of m⁶A modification signals within the 5' UTR regions, we conducted a re-analysis of publicly available meRIP-seq data obtained from Arabidopsis plants harboring mutations in FIP37, a known m⁶A writer protein. The dataset was retrieved from the NCBI GEO GSE174573. Our analysis revealed that ~ 94% of the 5’ UTR genes with m⁶A modification identified in Col-0 were absent in the meRIP-seq data of fip37 mutant (Additional file 1: Fig. S1D). This observation implies that the majority of genes with m⁶A modification in their 5’ UTR regions lost this modification in the mutants, indicating the reliability of these 5’ UTR genes with m⁶A modification. We then compared their expression with all m⁶A-methylated genes (m⁶A). We observed that m⁶A₅ genes showed the lowest expression, compared to genes in other groups (Fig. 1A), indicating that the 5’ UTR-specific m⁶A modification is associated with low transcription levels.

Fig. 1 — m⁶A methylation depositing in 5’ UTR regions represses gene expression. **A-C** Boxplot show the comparison of gene expression among genes with m⁶A modification at different regions in *Arabidopsis*, *Oryza sativa* and chrysanthemum. 5’ UTR: genes with m⁶A methylation in 5’ UTR region; m⁶A₅₃: genes with m⁶A methylation in both 5’ UTR and 3’ UTR region; m⁶A₅: genes with m⁶A methylation only in 5’ UTR region; m⁶A₃: genes with m⁶A methylation only in 3’ UTR region; 3’ UTR: genes with m⁶A methylation in 3’ UTR region; m⁶A: all genes with m⁶A methylation. Statistical analysis was conducted using the Student’s t-test. ** indicates P value < 0.01, * indicates P value < 0.05, and NS indicates no significance. D Relationship between m⁶A modification and target gene expression in *Arabidopsis*, *Oryza sativa* and chrysanthemum. Scatter plots showing the correlation between m⁶A fold enrichment and target mRNA abundance. E Comparison of H3K4me3-marked genes or H3K27me3-marked genes with genes marked by m⁶A in *Arabidopsis*. F Distribution of H3K4me3 or H3K27me3 reads in transcript segments with or without m⁶A modification. G Comparison of H3K4me3-marked genes or H3K27me3-marked genes with genes marked by m⁶A in *Oryza sativa*

To investigate whether m⁶A₅-associated low levels of transcription are evolutionarily conserved, we re-analyzed publicly available meRIP-seq and RNA-seq data from NCBI GEO GSE211828 in panicles of rice, a model crop plant, and performed meRIP-seq using a commercial anti-m⁶A antibody (202–003; Synaptic Systems) in leaf tissues of chrysanthemum, a segmental allopolyploid, self-incompatible and highly heterozygous plant [27] (Additional file 2: Table S1). We further divided the genes into five groups based on the location of m⁶A methylation in the gene transcripts in rice and chrysanthemum and then compared the group expression levels. Consistent with the results obtained in Arabidopsis, m⁶A₅ showed the lowest expression in rice and chrysanthemum (Fig. 1B and C). These results indicate that m⁶A methylation, uniquely deposited in 5’ UTR regions, is associated with lower gene expression.

To analyze the relationship between m⁶A modification and gene expression in m⁶A₅ genes, we performed a correlation analysis between m⁶A peak fold-enrichment and target mRNA abundance. The negative correlations were conservatively observed in Arabidopsis, rice and chrysanthemum (Fig. 1D), indicating that m⁶A methylation uniquely depositing in 5’ UTR regions contributes to gene repression.

H3K4me3 is involved in the transcriptional regulation of m⁶A-modified genes

Chromatin remodeling plays an essential role in transcriptional regulation by altering the chromatin architecture to allow dynamic access to condensed DNA. Recent studies have shown that the chromatin state is regulated by m⁶A methylation of chromosome-associated regulatory RNA or by the methyltransferase METTL3/METTL14 [18, 28], indicating a direct link between m⁶A modification and the dynamic chromatin state. Histone H3 Lys 4 tri-methylation (H3K4me3) is an evolutionarily conserved epigenetic marker of transcriptionally active euchromatin [29], and H3K27me3 contributes to the repression of its associated genes [30]. To determine whether these two epigenetic histone markers correlate with levels of m⁶A modification in mRNA, we re-analyzed the H3K4me3 and H3K27me3 dataset from NCBI GEO GSE193251 [31]. We compared the genes associated with H3K4me3 and H3K27me3 with m⁶A-modified transcripts. As a control, we also included randomly generated genes with the same number of H3K4me3 targets. Our analysis revealed that approximately 60% of genes marked with H3K4me3 exhibited m⁶A modifications, accounting for 95% of the total m⁶A-modified genes (Fig. 1E, Additional file 1: Fig. S1E). In contrast, only 40% of randomly generated genes carried m⁶A modifications, accounting for 60% of the total m⁶A-modified genes (Fig. 1E). Interestingly, only 26% of genes marked with H3K27me3 overlapped with m⁶A-modified transcripts, representing 25% of the total m⁶A-modified genes. This proportion was lower than the overlapping proportion observed with randomly generated genes (Fig. 1E). To further validate the enrichment of both histone marks on m⁶A-modified genes, we examined the H3K4me3 and H3K27me3 profiles around the gene body of m⁶A-modified genes and compared them with genes without m⁶A modification. We observed a significant enrichment of H3K4me3 in the vicinity of m⁶A targets, while minimal enrichment was observed in genes lacking m⁶A modification (Fig. 1F). Additionally, we found a relatively higher level of H3K27me3 modification in genes without m⁶A modification compared to genes with m⁶A modification (Fig. 1F). Collectively, these findings suggest that H3K4me3 is involved in the transcriptional regulation of m⁶A-modified genes.

To investigate whether the co-enrichment of H3K4me3 and m⁶A modification exists in other plant species, we obtained H3K4me3 and H3K27me3 ChIP-seq data from the NCBI GEO GSE142570 [32] for rice panicles. Consistent with the findings in Arabidopsis, our analysis revealed that H3K4me3, but not H3K27me3, exhibited a specific enrichment in m⁶A-modified genes (Fig. 1G, Additional file 1: Fig. S1F and G). These results provide further support for the presence of co-enrichment between H3K4me3 and m⁶A modification in multiple plant species. The high percentage of m⁶A-modified genes among those marked with H3K4me3 and the specific enrichment of H3K4me3 in m⁶A-modified genes reinforce the association between H3K4me3 and the regulation of m⁶A-modified mRNA transcripts in a conservative manner across different plant species.

m⁶A depositing in 5’ UTR shifts H3K4me3 modification

To explore the combined roles of epigenetic histone markers and mRNA m⁶A methylation in transcriptional regulation, we examined the H3K4me3 profiles around the TSS region of different gene groups. In Arabidopsis, we observed low levels of H3K4me3 modification in m⁶A₅ genes (Fig. 2A), which is consistent with the low expression of these genes (Fig. 1A). To our surprise, we observed a difference in the location of H3K4me3 enrichment peaks, relative to the TSS, between genes with m⁶A modification in 5’ UTR regions (m⁶A₅, m⁶A₅₃ and 5’ UTR genes) and m⁶A₃ genes (Fig. 2A). To determine the exact length of this peak shift, we used 10-bp resolution intervals (10-bp bins) to partition the sequences downstream of the TSS and counted the number of bins between the TSS and H3K4me3-enriched peak summits in the m⁶A₃ and m⁶A₅ genes. We observed that the shift of the H3K4me3 peak on the m⁶A₅ genes was 60 bp (from 310 to 370 bp downstream of the TSS; Additional file 1: Fig. S2A) compared to that in the m⁶A₃ genes.

Fig. 2 — The relationship between H3K4me3 and m⁶A methylation. **A-C** The meta-gene profiles of the H3K4me3 generated along gene bodies in leaves of *Arabidopsis* (A), panicle (B) and leaves (C) of *Oryza sativa*. 5’ UTR: genes with m⁶A methylation in 5’ UTR region; m⁶A₅₃: genes with m⁶A methylation in both 5’ UTR and 3’ UTR region; m⁶A₅: genes with m⁶A methylation only in 5’ UTR region; m⁶A₃: genes with m⁶A methylation only in 3’ UTR region; 3’ UTR: genes with m⁶A methylation in 3’ UTR region; All genes: all the genes in corresponding genome that have both 5’ UTR and 3’ UTR; m⁶A: genes with m⁶A methylation. **D-E** Genome browser traces and ChIP-qPCR to show the m⁶A enrichment and H3K4me3 modification, respectively, for genes with m⁶A modification specifically in 5’ UTR regions (D) and 3’ UTR regions (E). About 700 bp regions were divided into 6 parts for ChIP-qPCR examination. F Correlation analysis to examine the relationship between m⁶A modification and the shift of H3K4me3 in the 5’ UTR regions of m⁶A₅ genes. We obtained the fold enrichment of each m⁶A peak and calculated the distance between the summit of H3K4me3 peak and the TSS. Subsequently, we calculated the Pearson correlation coefficient (PCC) to assess the correlation between these variables

To examine whether the H3K4me3 modification shifts at m⁶A₅ genes generally appeared in other species, we examined the H3K4me3 profiles around the TSS region of different gene groups in panicles of rice and identified that the H3K4me3 modification shifted 150 bp downstream of TSS when m⁶A methylation deposited in the 5’ UTR of mRNA, compared with these deposited in 3’ UTR (Fig. 2B, Additional file 1: Fig. S2B). To explore whether this downstream shift is observed in other rice tissues, we re-analyzed meRIP-seq data from NCBI GEO GSE211828 [33] and H3K4me3 ChIP-seq data from NCBI GEO GSE142570 in rice leaves [32] and observed a 140 bp downstream shift in m⁶A₅ genes (Fig. 2C and Fig. S2C). We confirmed that a downstream shift in H3K4me3 enrichment in m⁶A₅ occurred in the chrysanthemum leaf tissues (Fig. 2D-E and Additional file 1: Fig. S2D-H). These data suggest that H3K4me3 modification shifts in m⁶A₅ genes occur in different tissues and plant species.

To examine the relationship between m⁶A modification and the shift of H3K4me3 in the 5’ UTR regions of m⁶A₅ genes, we conducted a comparison of m⁶A enrichment with the downstream distance between the summit of the H3K4me3 peak and the TSS of target genes. The results demonstrated a positive correlation between higher m⁶A modification levels and larger downstream shifts of H3K4me3 enrichment in both Arabidopsis and rice (Fig. 2F), indicating that the shift of H3K4me3 modification in the 5' UTR regions of m⁶A₅ genes is indeed correlated with m⁶A modification.

The downstream shift of H3K4me3 is genetically affected by the m⁶A writer and eraser

The m⁶A₅₃ genes exhibit strong m⁶A modifications in the 3’ UTR regions, which may play dominant roles in gene expression regulation and overshadow the significance of 5’ UTR regions. Therefore, our focus was specifically on genes that possess m⁶A modification exclusively in their 5’ UTR regions (referred to as m⁶A₅ genes).

To investigate the impact of m⁶A demethylases on H3K4me3 modification shifts, we utilized the alkbh10b-2 mutant (Salk_107289C) [34]. This mutant contains a T-DNA insertion in the coding gene ALKBH10B, which is responsible for mRNA m⁶A erasure (Additional file 1: Fig. S3A). Consequently, the mutant of ALKBH10B exhibits a global increase in m⁶A modification (Additional file 1: Fig. S3B) [12, 34]. We performed ChIP-seq using an H3K4me3 antibody. In the alkbh10b−2 mutant, we observed a 100 bp downstream shift of H3K4me3 in the m⁶A₅ genes (Fig. 3A and B). This shift was more prominent than that observed in the Col-0 wild-type plants (Fig. 2A), providing evidence for the potential role of m⁶A demethylases in regulating the downstream shift of H3K4me3.

Fig. 3 — The downstream shift of H3K4me3 is genetically affected by the m⁶A writer and eraser. A The meta-gene profiles of the H3K4me3 generated along gene bodies in leaves of *Arabidopsis alkbh10b-2* mutant. 5’ UTR: genes with m⁶A methylation in 5’ UTR region; m⁶A₅₃: genes with m⁶A methylation in both 5’ UTR and 3’ UTR region; m⁶A₅: genes with m⁶A methylation only in 5’ UTR region; m⁶A₃: genes with m⁶A methylation only in 3’ UTR region; 3’ UTR: genes with m⁶A methylation in 3’ UTR region; All genes: all the genes in corresponding genome that have both 5’ UTR and 3’ UTR; m⁶A: genes with m⁶A methylation. B Screenshots showing normalized sequencing signals of H3K4me3, meRIP-seq and RNA-seq in both Col-0 and *alkbh10b* mutant over a representative gene. The binding levels or gene expression levels were indicated by reads per kilobase per million reads in sample (RPKM). The 5’ UTR regions with H3K4me3 shit and elevated m⁶A levels were highlighted with blue box. **C-D** Protoplast transient assay system to examine the m⁶A levels and H3K4me3 shift in *SBPASE* (C) and *AGO7* (D) genes. The protoplasts were transformed with *35S:AtMTA-GFP*, *35S:AtALKBH10B* or empty vectors. We assessed m⁶A modification enrichment (left panel) and H3K4me3 enrichment (right panel) on each gene. Trendlines were added to trace the H3K4me3 shift. Different letters were used to indicate statistically significance difference (P < 0.05, Student’s t-test). Diagram in (C) illustrates the primers used for H3K4me3 ChIP-qPCR. E Comparison of m⁶A modification in 5’ UTR regions among Col-0, *atx1-2* and *sdg2-1* plants. * indicates P value < 0.05. **F-G** Correlation analysis to examine the relationship between m⁶A modification and gene expression (F), and the relationship between m⁶A modification and H3K4me3 downstream shift (G) in m⁶A₅ genes in *atx1-2* and *sdg2-1*. The Pearson Correlation Coefficient (PCC) was used to assess the correlation between these variables

To validate the impact of ALKBH10B-mediated m⁶A modification on H3K4me3 shift, we performed ChIP-qPCR using an anti-H3K4me3 antibody and m⁶A-qPCR using an anti-m⁶A antibody in Arabidopsis protoplast cells that were transformed with 35S:AtALKBH10B-GFP. These results were compared with cells transformed with empty vectors. The data indicated decreased m⁶A modification levels in the 5’ UTR regions upon transformation with 35S:AtALKBH10B-GFP (Fig. 3C and D, Additional file 1: Fig. S3C), while H3K4me3 modification in the 5’ UTR regions closer to the TSS (Fig. 3C and D), consistent with the observations in the alkbh10b-2 mutant (Fig. 3A and B).

Next, we employed protoplast transient assay system to examine whether MTA-mediated m⁶A modification regulates the shift of H3K4me3. We observed increased m⁶A modification levels in the 5’ UTR regions upon transformation with 35S:AtMTA-GFP (Fig. 3C and D), whereas H3K4me3 modification in the 5’ UTR regions more distant from the TSS compared to cells transformed with empty vectors (Fig. 3C and D), indicating that the downstream shift of H3K4me3 is affected by the m⁶A writer and eraser.

m⁶A-mediated gene expression depends on H3K4me3 modification in 5’ UTR regions

To investigate whether H3K4me3 modification regulates m⁶A levels in the 5’ UTR regions, we performed meRIP-seq analysis using leaves from atx1-2 (Salk_149002C) [35] and sdg2-1 (Salk_021008) [36] mutants (Fig. S3) that carry T-DNA insertions in the SET DOMAIN GROUP (SDG) genes, which have been previously shown to be essential for H3K4 trimethylation. We quantified the m⁶A enrichment in the 5’ UTR regions of m⁶A₅ genes. The results revealed higher m⁶A modification levels in the atx1-2 and sdg2-1 mutants compared to the Col-0 control (Fig. 3E), indicating a negative effect of H3K4me3 modification on m⁶A levels.

Next, we examined the gene expression patterns in both mutants, and compared them with the changes in m⁶A modifications. Notably, in the sdg2-1 mutant, we did not detect a significant correlation between the changes in m⁶A and the variations in gene expression (Fig. 3F). Moreover, in the atx1-2 mutant, we only observed a weak negative correlation (Fig. 3F), which was significantly lower than that observed in Col-0, the control strain with normal H3K4me3 modification (Fig. 1D). We then examined the correlation between the changes of m⁶A and H3K4me3 modification shifts, we found no significant correlation in atx1-2 and sdg2 mutants (Fig. 3G), which was significantly lower than that observed in Col-0 (Fig. 2F). These findings suggest that m⁶A modification alone is insufficient to regulate gene expression in the absence of H3K4me3 modification in the 5’ UTR regions, further indicating that the presence of H3K4me3 modification is crucial for the regulatory effects of m⁶A in the 5' UTR regions.

MTA and ATX1 interact with CTD of RNA Pol II in vitro and in vivo

Previous studies have reported that ATX1, the H3K4me3 writer containing a SET domain, binds to the CTD domain of RNA Pol II [37]. Similarly, MTA, the writer of m⁶A, has been shown to interact with RNA Pol II in Arabidopsis [38]. Based on these reports, we hypothesized that MTA may compete with ATX1 for binding to RNA Pol II during transcription initiation, leading to a downstream shift in H3K4me3 modification. To test this hypothesis, we performed in vitro and in vivo protein–protein interaction assays, including split-luciferase complementation (Split-LUC) imaging and pulldown assays. In the Split-LUC assay, we cloned the coding sequences of each gene/domain fused with either the C-terminal or N-terminal half of LUC. These constructs were then transformed into Agrobacterium strain GV3101 and infiltrated into Nicotiana benthamiana leaves. We observed luciferase activity when co-infiltrating AtSET-CLUC and AtCTD-NLUC constructs, indicating interaction between the two proteins (Fig. 4A). Interestingly, we also detected luciferase activity when co-infiltrating the AtMTA-CLUC and AtCTD-NLUC constructs, and AtSET-CLUC and AtMTA-NLUC constructs (Fig. 4A). To further confirm these interactions, we conducted a pull-down assay. In this assay, we cloned AtSET and AtMTA fused with GST and AtCTD fused with a His tag, respectively. We observed bands of the expected molecular weight for GST-AtMTA and GST-AtSET in the pull-down assay using anti-His antibody (Fig. 4B). These interacts were also detected when using the coding sequences from chrysanthemum (Fig. 4C), suggesting that the interactions between ATX1, MTA, and the CTD of RNA Pol II are conserved across plant species.

Fig. 4 — MTA and ATX1 interact with CTD of RNA Pol II. A Split-luciferase complementation (Split-LUC) assays examining the interaction between AtSET and AtCTD (left panel), AtMTA and AtCTD (middle panel), and AtSET and AtMTA (right panel). Split-LUC assays were conducted by the infiltration of *Agrobacterium* carrying indicated constructs into *N. benthamiana* leaves. Images of plants sprayed with 500 μM luciferin and placed in the dark for 5 min are shown. B Pull-down assays to examine the interaction between AtSET or AtMTA and AtCTD. Increasing concentrations of GST-AtSET was respectively incubated with a fixed concentration of GST-AtMTA. GST-fusion proteins and GST were detected with an anti-GST antibody. Input means total protein lysate without immunoprecipitation. C Split-LUC assays examining the interaction between CmSET and CmCTD (left panel), CmMTA and CmCTD (middle panel), and CmSET and CmMTA (right panel). Split-LUC assays were conducted by the infiltration of *Agrobacterium* carrying indicated constructs into *N. benthamiana* leaves. Images of plants sprayed with 500 μM luciferin and placed in the dark for 5 min are shown. D Split-LUC assays examining the competition between AtMTA and AtSET (upper panel), and CmMTA and CmSET (lower panel). Split-LUC assays were conducted by the infiltration of *Agrobacterium* carrying indicated constructs into *N. benthamiana* leaves. Images of plants sprayed with 500 μM luciferin and placed in the dark for 5 min are shown. Quantitation of luciferase intensity from three biological replications are shown in plots (error bars are SD). Different letters were used to indicate statistically significance difference (P < 0.05, Student’s t-test)

MTA competes ATX1 to interact with CTD of RNA Pol II in vitro and in vivo

To explore the competitive binding between ATX1 and MTA to the CTD of RNA Pol II, we co-infiltrated Nicotiana benthamiana leaves with AtMTA-CLUC and AtCTD-NLUC constructs, along with varying amounts of AtSET or an empty vector (EV) control. As the concentration of AtSET increased, the luciferase activity, indicative of the interaction between AtMTA and AtCTD, significantly decreased (Fig. 4D, upper panel). Similar results were observed when using genes from chrysanthemum (Fig. 4D, lower panel). To further validate these findings, we conducted a pull-down assay. The amount of GST-AtSET pulled down by His-AtCTD decreased when 1 × AtMTA was added, while the amount of GST-AtMTA pulled down by His-AtCTD was not affected by the addition of 1 × AtSET (Fig. 4B). This indicates that AtMTA can compete with AtSET for binding to the AtCTD of RNA Pol II. Moreover, as the concentration of GST-AtSET increased, the amount of GST-AtSET pulled down by His-AtCTD also increased, while the amount of GST-AtMTA pulled down decreased (Fig. 4B). We attempted to employ a truncated version of AtSET as a negative control. Initially, we divided AtSET into three segments (Additional file 1: Fig. S4A) and assessed their individual interactions with AtCTD and ATMTA. Our findings revealed that, excluding the AtSET3 and AtMTA group, all other group combinations exhibited interactions (Additional file 1: Fig. S4B and C). Subsequently, we conducted a pull-down assay by escalating the amount of GST, which served as a negative control (Additional file 1: Fig. S4D). Results indicated that the amount of GST-MTA pulled down by His-AtCTD remained unaffected by increasing GST (Additional file 1: Fig. S4D). These results collectively suggest that ATX1 and MTA compete for binding to the CTD of RNA Pol II.

MTA competes ATX1 to interact with Ser5P-CTD of RNA Pol II in vitro and in vivo

Phosphorylation of serine 5 (Ser5P) of the CTD is known to be a marker for transcription initiation and early elongation, while serine 2 (Ser2P) phosphorylation is associated with transcription elongation [39]. Previous studies have shown that the SET domain of AtATX1 preferentially binds to the Ser5P form of the CTD of RNA Pol II [37]. This led us to investigate whether MTA interacts with phosphorylated CTD. We performed pull-down assays using antibodies specific to Ser5P and Ser2P for capturing Ser5P-CTD and Ser2P-CTD, respectively. Our results revealed that AtMTA interacts with both Ser2P-CTD and Ser5P-CTD peptides, with a higher affinity observed for the Ser5P-CTD peptide (Fig. 5A). Similar findings were obtained with the MTA from chrysanthemum (Fig. 5B). However, CmSET exhibits equal interaction strength with both Ser2P-CTD and Ser5P-CTD (Fig. 5C).

Fig. 5 — Binding of the SET or MTA to different phosphorylated forms of the CTD of Pol II. **A-C** Binding of AtMTA (A), CmMTA (B), CmSET (C) or GST to bead-bound peptides containing four consensus CTD heptad repeats (YSPTSPS) with phosphorylated at Ser5P or Ser2P were examined. The amount of GST or GST fused protein bound to the peptides on the beads was determined by immunoblot analysis with antibody to GST. D Pull down assay to examine the competition between AtMTA and AtSET for binding to Ser5P-CTD of RNA Pol II. Increasing concentrations of GST-AtSET was respectively incubated with a fixed concentration of GST-AtMTA. GST-fusion proteins were detected with an anti-GST antibody. Input means total protein lysate without immunoprecipitation. E Protoplast transient assay system to examine the competition between AtMTA and AtSET for binding to Ser5P-CTD of RNA Pol II. Protoplast cells obtained from *atx1-2* mutant and WT plants were used for *35S:AtMTA-GFP* transformation. Total proteins extracted from protoplast cells were immunoprecipitated with anti-Ser5P-CTD and analyzed by western blot with antibody to GFP. F Model for the m⁶A-mediated H3K4me3 shift on gene expression regulation. In cases where there is no m⁶A modification in the 5' UTR regions, histone methyltransferase ATX1 promotes H3K4me3 modification, leading to high level expression of genes. However, when the m⁶A writer MTA binds to the CTD domain of RNA Pol II, it competes with ATX1, resulting in reduced H3K4me3 modification during transcriptional initiation and early elongation stages. As the transcription elongation progresses and MTA dissociates from RNA Pol II, ATX1 is recruited by RNA Pol II to perform H3K4me3 modification on target genes. This downstream shift of H3K4me3 is associated with a decrease in gene expression levels

To investigate whether ATX1 competes MTA for binding to Ser5P-CTD, we performed a pull-down assay using an anti-Ser5P antibody. Our findings revealed that GST-AtSET was predominantly outcompeted by GST-MTA (Fig. 5D, 1st lane vs. 3rd lane). Conversely, GST-MTA was only slightly competed by GST-SET (Fig. 5D, 2nd lane vs. 3rd lane), even though a higher amount of GST-SET was utilized in the pull-down assay (Fig. 5D, input). Additionally, as the concentration of GST-AtSET increased, the amount of GST-ATSET pulled down by anti-Ser5P exhibited a slight increase, while the amount of GST-AtMTA pulled down showed a slight decrease (Fig. 5D). These results indicate that AtMTA exhibits a higher binding affinity to the Ser5P-CTD of RNA Pol II compared to AtATX1.

To confirm these findings in an in vivo context, we generated GFP-fusion constructs of the AtMTA and introduced it into the protoplast derived from atx1-2 mutant (Salk_149002C) [35] (Additional file 1: Fig. S3A) and WT plants. Compared to the cells without AtATX1, the amount of AtMTA-GFP co-immunoprecipitated by anti-Ser5P in cells with AtATX1 (the WT cells) decreased (Fig. 5E), consistent with the results obtained from the pull-down assay. Collectively, these results suggest that during the transcription initiation and early elongation stage of m⁶A₅ genes, Ser5P-CTD of RNA Pol II preferentially interacts with MTA for m⁶A synthesis, while ATX1 is displaced during this process, resulting in a downstream shift of H3K4me3 modification (Fig. 5F).

ALKBH10B competes with MTA for binding to the Ser5P-CTD of RNA Pol II both in vitro and in vivo

Given the opposite effects of MTA and ALKBH10B on the shift of H3K4me3 (Fig. 3), we next investigated the role ALKBH10B in the MTA-CTD-ATX1 complex. Firstly, we performed Split-LUC assays, which revealed that ALKBH10B interacts with CTD of RNA Pol II in both Arabidopsis and chrysanthemum (Fig. 6A). Subsequently, pull-down assays were performed to confirm their interactions (Fig. 6B). Based on our observations, we hypothesized that ALKBH10B may compete with MTA for binding to RNA Pol II during transcription initiation. To test this hypothesis, split-LUC assays were conducted, and the results demonstrated that as the concentration of AtALKBH10B increased, the interaction between AtMTA and AtCTD significantly decreased in both Arabidopsis (Fig. 5C) and chrysanthemum (Fig. 5D). Furthermore, the pull down-assays indicated that with an increase in the concentration of GST-AtALKBH10B, the amount of GST-AtALKBH10B pulled down by His-AtCTD also increased, while the amount of GST-AtMTA pulled down decreased (Fig. 6B). Collectively, these results strongly suggest a competitive binding relationship between ALKBH10B and MTA for the CTD of RNA Pol II.

Fig. 6 — ALKBH10B competes with MTA for binding to the Ser5P-CTD of RNA Pol II. A Split-LUC assays examining the interaction between AtALKBH10B and AtCTD (upper panel), and CmALKBH10B and CmCTD (lower panel). Split-LUC assays were conducted by the infiltration of *Agrobacterium* carrying indicated constructs into *N. benthamiana* leaves. Images of plants sprayed with 500 μM luciferin and placed in the dark for 5 min are shown. B Pull-down assays to examine the interaction between AtALKBH10B or AtMTA and AtCTD. Increasing concentrations of GST-AtALKBH10B was respectively incubated with a fixed concentration of GST-AtMTA. **C-D** Split-LUC assays examining the competition between AtALKBH10B and AtSET (C), and CmALKBH10B and CmSET (D). Split-LUC assays were conducted by the infiltration of *Agrobacterium* carrying indicated constructs into *N. benthamiana* leaves. Images of plants sprayed with 500 μM luciferin and placed in the dark for 5 min are shown (upper panel). Quantitation of luciferase intensity from more than five biological replications are shown in plots (error bars are SD, lower panel). Different letters were used to indicate statistically significance difference (P < 0.05, Student’s t-test). E Binding of AtALKBH10B or GST to bead-bound peptides containing four consensus CTD heptad repeats (YSPTSPS) with phosphorylated at Ser5P or Ser2P were examined. The amount of GST or GST-AtALKBH10B protein bound to the peptides on the beads was determined by immunoblot analysis with antibody to GST. F Pull down assay to examine the competition between AtALKBH10B and AtMTA for binding to Ser5P-CTD of RNA Pol II. Increasing concentrations of GST-AtALKBH10B was respectively incubated with a fixed concentration of GST-AtMTA. G Protoplast transient assay system to examine the competition between AtMTA and AtALKBH10B for binding to Ser5P-CTD of RNA Pol II. Protoplast cells obtained from *alkbh10b-2* mutant and WT plants were used for 35S:AtMTA-GFP transformation. Total proteins extracted from protoplast cells were immunoprecipitated with anti-Ser5P-CTD and analyzed by western blot with antibody to GFP

We then examined whether the competition happened on the Ser5P-CTD of RNA Pol II. Pull down assays were conducted, revealing that AtALKBH10B interacts with both Ser2P-CTD and Ser5P-CTD peptides (Fig. 6E). Furthermore, we observed that GST-AtMTA was competitively displaced by GST-ALKBH10B when pulled down by Ser5P-CTD (Fig. 6F). As the concentration of GST-AtALKBH10B increased, the amount of GST-ATALKBH10B pulled down by anti-Ser5P exhibited a slight increase, while the amount of GST-AtMTA pulled down decreased (Fig. 6F). These results indicate that ALKBH10B could compete MTA at Ser5P-CTD of RNA Pol II. To further support this, a protoplast transient assay was performed, demonstrating that the amount of AtMTA-GFP co-immunoprecipitated by anti-Ser5P in cells with AtALKBH10B (Col-0) decreased compared to cells without AtALKBH10B (Fig. 6G), consistent with the results obtained from the pull-down assay. Collectively, these results strongly suggest that ALKBH10B plays a role in determining the occupancy of MTA at transcriptional initiation.

m⁶A-mediated H3K4me3 shift in the 5’ UTR is involved in leaf senescence

To gain deeper insights into the function of the m⁶A₅ genes, we performed a Gene Ontology (GO) analysis and found their enrichment in regulation of histone methylation, development, cell death, and metabolic processes (Fig. 7A). Previous studies have demonstrated that alkbh10b-1 (SALK_004215C; Additional file 1: Fig. S5) and alkbh10b-2 (SALK_107289C) mutants exhibit delayed flowering and suppressed vegetative growth [12]. Additionally, both mutants display a delayed leaf senescence phenotype (Fig. 7B-D). To unravel the mechanisms underlying m⁶A-mediated leaf senescence, we re-evaluated the m⁶A₅ genes and observed that the SEDOHEPTULOSE-BISPHOSPHATASE (SBPASE) gene is predominantly expressed in leaf tissues across various plant species (Fig. 7E). Moreover, mutations in the SBPASE gene result in a notable delay in developmental leaf senescence and flowering time [40], similar to the phenotype observed in alkbh10b mutants (Fig. 7B-D). Notably, we observed lower expression levels of SBPASE in alkbh10b plants compared to WT plants (Fig. 7F). Furthermore, we re-analyzed the H3K4me3 ChIPseq data (GSE67776) obtained from green leaves at 42 days and senescent leaves at 57 days [41]. Our analysis revealed an upstream shift in H3K4me3 during leaf senescence that resulted in increased SBPASE expression (Fig. 7G), which is consistent with our qRT-PCR results using green and senescent leaves harvested at 30 and 50 days, respectively (Fig. 7F). To confirm this regulatory mechanism, we performed m⁶A-qPCR on green and senescent leaves of both Col-0 and alkbh10b plants. The results showed down-regulation of m⁶A modification on the 5’UTR region of SBPASE during senescence in both Col-0 and alkbh10b, with alkbh10b exhibiting higher levels of m⁶A modification (Fig. 7H). Furthermore, we examined H3K4me3 modification downstream of the TSS region of SBPASE and observed an upstream shift during senescence, whereas no significant shift was observed in alkbh10b (Fig. 7I). These findings collectively indicate that SBPASE regulates leaf senescence controlled by m⁶A-mediated H3K4me3 shift in 5’ UTR.

Fig. 7 — m⁶A-mediated H3K4me3 shift in the 5’ UTR is involved in leaf senescence. A Gene ontology (GO) biological processes enrichment analyses of genes with m⁶A modification only in 5’ UTR regions. B The senescence phenotype of 50 d old Col-0, *alkbh10b-1* and *alkbh10b-2* plants. C The senescence phenotype of leaves. The leaves were detached and arranged according to their age. D Total chlorophyll contents in leaves from Col-0, *alkbh10b-1* and *alkbh10b-2* plants were measured. Three biological replicates were performed. Error bars represent SD. Different letters indicate a statistically significant difference with P ≤ 0.05 by Student's t-test. E The expression analysis of *SBPASE* from different plant species was performed using GERDH database (https://dphdatabase.com/). Left panel showing the phylogenetic tree of SPBASE protein from different plant species; right panel showing the expression of *SPBASE* gene in root, stem, leaf, bud and flower across various plant species. F Quantitative PCR detection of *SBPASE* expression in Col-0 and *alkbh10b-2* plants at 30 d and 50 d. ** indicates p value < 0.01 by Student's t-test. G Genome browser traces of H3K4me3 ChIP-seq data and RNA-seq data from *SBPASE* gene during leaf senescence in *Arabidopsis*. Red box highlights the different enrichment of H3K4me3. H m⁶A RNA immunoprecipitation quantitative PCR (m⁶A-IP-qPCR) detection of m⁶A modification enrichment on *SBPASE* gene in Col-0 and *alkbh10b-2* plants at 30 d and 50 d. Total RNA was fragmented and immunoprecipitated with anti-m⁶A antibody. Both input control and m⁶A-IP samples were subjected to qPCR analysis. * indicates p value < 0.05 by Student's t-test. I ChIP-qPCR detection of H3K4me3 enrichment on *SBPASE* gene in Col-0 and *alkbh10b-2* plants at 30 d and 50 d. The sonicated chromatin was immunoprecipitated using anti-H3K4me3 antibody, DNA was eluted and amplified by primers (P1-P7) indicated in the figure. Trendlines were added to trace the H3K4me3 shift. * indicates p value < 0.05 by Student's t-test between 30 and 50 days for each primer in Col-0 or *alkbh10b* plants

In summary, all these results suggested that the deposition of m⁶A in the 5’ UTR alters H3K4me3 modification, finely tuning the expression of target genes and contributing to leaf senescence.

Discussion

Previous studies have primarily focused on m⁶A modifications in the vicinity of stop codons and 3’ untranslated regions (UTRs), rather than the 5’ UTRs [25, 42]. The molecular functions of 3’ UTR-specific m⁶A modifications have been extensively investigated. They have been found to be associated with alternative polyadenylation of transcripts [43, 44], and the regulation of gene expression [45]. In spermatocytes and round spermatids, proper erasure of m⁶A in the 3’ UTR is crucial for correct splicing and stability of mRNA molecules with long 3’ UTR [46]. On the other hand, 5’ UTR-specific m⁶A modifications have received less attention but have been shown to play essential roles in regulating cortical development [13], and cellular stress responses [14, 15] at post-transcriptional level. However, the roles of 5’ UTR-specific m⁶A modifications in transcriptional regulation have received limited attention. In this study, we found that m⁶A methylation deposited in the 5’ UTR regions potentially contributes to gene repression, and this phenomenon is conserved in Arabidopsis, rice and chrysanthemum (Fig. 1). Furthermore, we made a notable discovery that gene expression is regulated by a downstream shift of H3K4me3, controlled by m⁶A modification in the 5’ UTR regions, which is implicated in the regulation of leaf senescence. Nevertheless, we cannot overlook the potential impact of m⁶A on mRNA stability, translational efficiency, alternative splicing, and other factors, leading to varied expression dynamics of genes/proteins beyond m⁶A₅, thereby influencing leaf senescence. Furthermore, the precise functional significance of the H3K4me3 shift remains to be fully elucidated. Additional evidence is necessary to address the unanswered questions concerning the functions and underlying mechanisms of H3K4me3 shift in the regulation of gene expression in future studies.

To date, there have been limited studies on the shift of histone modification in genes with m⁶A methylation. One study reported that when the transcriptional status of a gene changes from silenced to activated, the first nucleosome downstream of TSS shifts in the downstream direction [47], indicating that nucleosome positioning plays an important role in gene expression regulation. In addition, a recent report showed that incorporation of N1-methylpseudouridylation into mRNA causes + 1 ribosomal frameshifting in vitro and in vivo [48], indicating the effect of mRNA modification in translational regulation. In our study, we observed a shift in H3K4me3, mostly less than 150 bp, which is not sufficient to bypass a nucleosome, suggesting a repositioning of the nucleosome.

Furthermore, it has been reported that the m⁶A writer protein METTL14 interacts with H3K36me3 on chromatin and guides N6-methylation on nascent RNAs, leading to a higher level of N6-methylation in long exons [20]. A recent study demonstrated that super-enhancer RNA m⁶A (seRNA m⁶A) promotes local chromatin accessibility and facilitates the transcription of oncogenes. This effect is mediated by the seRNA m⁶A-YTHDC2 module, which recruits the H3K4 methyltransferase MLL1 to promote H3K4me3 modification [19]. These findings suggest a connection between mRNA m⁶A modification and histone modifications. Profiling of histone modifications upon m⁶A loss via METTL14 knockout revealed increased levels of H3K4me3, H3K27me3 and H3K27ac in cell-proliferation-related genes [49]. This finding can be interpreted in light of our results, which indicate that MTA and ATX1 compete to interact with RNA Pol II. Loss of H3K4me3 has been reported to have minimal effects on transcriptional initiation but leads to a widespread decrease in transcriptional output, an increase in RNA Pol II pausing and slower elongation [50]. In our study, we found that MTA binding to the CTD domain of RNA Pol II competes with ATX1, resulting in lower H3K4me3 modification at the transcription initiation and early elongation process. When MTA dissociates from RNA Pol II during transcription elongation, RNA Pol II recruits ATX1 for H3K4me3 modification of target genes, leading to a downstream shift of H3K4me3 modification and lower level of gene expression (Fig. 5F). The MTA binding to RNA Pol II results in H3K4me3 loss, which may lead to increased RNA Pol II pausing and slower early elongation, consequently resulting in decreased transcriptional output. In addition, it is important to consider the possibility that m⁶A modification may impact mRNA stability, leading to reduced transcription levels. Furthermore, investigating whether the enzymatic activity of MTA or ALKBH10B is necessary for the regulation of gene expression through H3K4me3 shift would be an intriguing aspect to explore in future studies.

Conclusions

In conclusion, our findings indicate that m⁶A-mediated shifts in H3K4me3 contributing to gene repression and leaf senescence. These findings provide valuable insights into the mechanism underlying m⁶A-mediated gene expression regulation in the 5' UTR regions, opening up promising avenues for further research in this field.

Methods

Plant materials

The cut flower chrysanthemum cultivar, ‘Hualing’, was obtained from Chrysanthemum Germplasm Resource Conservation Center in Nanjing Agricultural University (Nanjing, China). The alkbh10b-1 (Salk_004215C) Arabidopsis seeds were kindly donated by Dr. Jungnam Cho, and the atx1-2 (Salk_149002C) and alkbh10b-2 (Salk_107289C) mutant lines were obtained from AraShare (www.arashare.cn).

Assay for natural and dark-induced leaf senescence

To investigate age-dependent leaf senescence, we selected the third and fourth rosette leaves of individual Arabidopsis plants. These leaves were used for various analyses, including chlorophyll content measurement, gene expression analysis, and examination of m⁶A and H3K4me3 modifications.

For the induction of dark-induced leaf senescence, the selected leaves were excised and placed in petri dishes containing wet paper. The petri dishes were wrapped with double-layer aluminum foil and incubated at a temperature of 22 °C throughout the experiment.

Measurements of chlorophyll content

The detached leaves were incubated in 80% acetone (v/v) in darkness for 24 h. Then the absorbance of the solution was measured at 645 and 657 nm using a spectrophotometer. The chlorophyll content was calculated using the formula: (20.2 × A₆₄₅ + 8.02 × A₆₅₇)/g fresh weight.

m⁶A-immunoprecipitation and sequencing (m⁶A-seq) and analysis

mRNAs were fragmented into 100-nucleotide fragments and incubated with 0.5 mg/ml anti-m⁶A polyclonal antibody (202–003; Synaptic Systems). Bound mRNA was eluted and used to construct the libraries. Sequencing was performed on the Illumina Hiseq 2500 platform.

Clean reads were aligned to reference genomes using bowtie2 [51]. The peak-caller MACS2 [52] was used for peak detection with FDR value was less than 0.01 and fold enrichment more than 1.5.

RNA-seq processing and analysis

Total RNA was used to prepare RNA-seq libraries using TruSeq RNA Library Prep Kit (Illumina). Multiplexed libraries were sequenced on an Illumina HiSeq 2500. Clean reads were aligned to the reference genome using HISAT2 [53]. DEGs were identified using Cufflinks [54], and detected with |log2(fold change)| and RPKM values larger than 1. The expression analysis of SBPASE was performed using GERDH database [55].

ChIP-seq processing and analysis

Chromatin immunoprecipitation was performed according to published protocol [56]. Clean reads were mapped to the reference genome using bowtie2 [51]. Peaks significantly enriched in ChIP-seq tags were identified using MACS2 [52].

Chromatin-immunoprecipitated DNA was amplified by primers corresponding to genes of interest. Primers used for ChIP-qPCR are listed in Additional file 2: Table S2.

Western blot

To analyze the proteins, we performed SDS–PAGE followed by electroblotted onto a nitrocellulose membrane. The membrane was probed with specific primary antibodies and then secondary antibodies. The signals were visualized using the SuperSignal West Pico PLUS kit (ThermoFisher).

Pull-down assay

The fusion proteins were incubated together and subsequently washed using a pull-down buffer. The precipitated magnetic beads were collected and then resuspended in a protein extraction buffer. The proteins were then separated by SDS-PAGE and detected using specific antibodies.

Immunoprecipitation assays

The harvested cells were incubated in co-IP buffer on ice for 30 min, allowing the proteins to be solubilized in the co-IP buffer. After following centrifugation, the cleared extract was then combined with anti-Ser5P-CTD antibody and incubated overnight at 4 °C. The magnetic beads were collected and resuspended in a protein extraction buffer. The proteins were then separated by SDS-PAGE and detected using a specific antibody.

N. benthamiana transient expression assay

The Agrobacterium strains were injected into N. benthamiana plants. 2 days later, the leaves of the plants were sprayed with 500 μM luciferin (Promega) and examined using a CCD imaging apparatus (Tanon, Shanghai, China).

Supplementary Information

13059_2025_3515_MOESM1_ESM.pdf^{(890.9KB, pdf)}

Additional file 1: Fig. S1. m⁶A methylation in Arabidopsis and Oryza sativa. Fig. S2. m⁶A methylation deposits in the 5’ UTR of mRNA shifted H3K4me3 histone modification. Fig. S3. Genotyping of T-DNA insertion mutants and m⁶A levels in different plants. Fig. S4. The negative control of MTA and ATX1 interact with CTD of RNA Pol II. Fig. S5. The diagram to show the T-DNA insertion for alkbh10b-1

13059_2025_3515_MOESM2_ESM.pdf^{(142.8KB, pdf)}

Additional file 2: Table S1. Summary of reads and mapped rates in different samples. Table S2. Primers used in this paper

13059_2025_3515_MOESM3_ESM.pdf^{(341.4KB, pdf)}

Additional file 3: Uncropped images of Western blots in Fig. 4, Fig. 5, Fig. 6 and Fig. S4

Additional file 4: Review history.

Acknowledgements

We thank Dr. Jungnam Cho (Chinese Academy of Sciences, Shanghai, China) for kindly providing the alkbh10b-1 (Salk_004215C) Arabidopsis seeds. This work was supported by the high-performance computing platform of Bioinformatics Center, Nanjing Agricultural University.

Peer review information

Shuangxia Jin and Wenjing She were the primary editors of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. The peer-review history is available in the online version of this article.

Authors’ contributions

Y.Y. performed most of the experiments and analyzed the data, with the help from Y.H., T.W. and S.L.. J.J., S.C., F.C., and L.W. conceived and designed the research. L.W. supervised the project and wrote the paper. All authors read and approved the final manuscript.

Funding

This work was supported by grants from the National Key Research and Development Program of China (2022YFF1003104), the Fundamental Research Funds for the Central Universities (KJJQ2025009, RENCAI2023007, RENCAI2024015, KJJQ2024011), the National Natural Science Foundation of China (32371955) and the interdisciplinary project of College of Horticulture, Nanjing Agricultural University (YYJC202202).

Data availability

All data supporting the findings of this study are available in the article and its supplementary figures and tables. The raw meRIP-, ChIP- and RNA-sequencing data reported in this paper are available at the Genome Sequence Archive in National Genomics Data Center, Chinese Academy of Sciences, with accession number CRA009857 [57].

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

1.Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, et al. Topology of the human and mouse m⁶A RNA methylomes revealed by m⁶A-seq. Nature. 2012;485:201–6. [DOI] [PubMed] [Google Scholar]
2.Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3’ UTRs and near stop codons. Cell. 2012;149:1635–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
3.Clancy MJ, Shambaugh ME, Timpte CS, Bokar JA. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene. Nucleic Acids Res. 2002;30:4509–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Liang Z, Riaz A, Chachar S, Ding Y, Du H, Gu X. Epigenetic modifications of mRNA and DNA in plants. Mol Plant. 2020;13:14–30. [DOI] [PubMed] [Google Scholar]
5.Ping X-L, Sun B-F, Wang L, Xiao W, Yang X, Wang W-J, Adhikari S, Shi Y, Lv Y, Chen Y-S, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24:177–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
6.Xu T, Wu X, Wong CE, Fan S, Zhang Y, Zhang S, Liang Z, Yu H, Shen L. FIONA1-mediated m⁶A modification regulates the floral transition in arabidopsis. Adv Sci. 2022;9:2103628. [DOI] [PMC free article] [PubMed] [Google Scholar]
7.Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505:117–20. [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene expression regulation. Cell. 2017;169:1187–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
9.Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG, He C. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7:885–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vågbø CB, Shi Y, Wang WL, Song SH, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013;49:18–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Fan W, Wang L, Lei Z, Li H, Chu J, Yan M, Wang Y, Wang H, Yang J, Cho J. m6A RNA demethylase AtALKBH9B promotes mobilization of a heat-activated long terminal repeat retrotransposon in Arabidopsis. Sci Adv. 2023;9:eadf3292. [DOI] [PMC free article] [PubMed]
12.Duan HC, Wei LH, Zhang C, Wang Y, Chen L, Lu Z, Chen PR, He C, Jia G. ALKBH10B Is an RNA N(6)-Methyladenosine demethylase affecting arabidopsis floral transition. Plant Cell. 2017;29:2995–3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Zhang LB, Qiu TT, Yang WWJ. Bioinformatic analysis reveals the distinct role of 5′UTR-specific m6A RNA modification in mice developing cerebral cortices. Dev Neurosci. 2022;44:67–79. [DOI] [PubMed] [Google Scholar]
14.Meyer KD, Patil DP, Zhou J, Zinoviev A, Skabkin MA, Elemento O, Pestova TV, Qian SB, Jaffrey SR. 5’ UTR m(6)A promotes cap-independent translation. Cell. 2015;163:999–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Zhou J, Wan J, Shu XE, Mao Y, Liu XM, Yuan X, Zhang X, Hess ME, Brüning JC, Qian SB. N6-Methyladenosine guides mRNA alternative translation during integrated stress response. Mol Cell. 2018;69:636–647.e637. [DOI] [PMC free article] [PubMed] [Google Scholar]
16.Zhou J, Wan J, Gao X, Zhang X, Jaffrey SR, Qian SB. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature. 2015;526:591–4. [DOI] [PMC free article] [PubMed] [Google Scholar]
17.Deng S, Zhang J, Su J, Zuo Z, Zeng L, Liu K, Zheng Y, Huang X, Bai R, Zhuang L, et al. RNA m⁶A regulates transcription via DNA demethylation and chromatin accessibility. Nat Genet. 2022;54:1427–37. [DOI] [PubMed] [Google Scholar]
18.Liu J, Dou X, Chen C, Chen C, Liu C, Xu MM, Zhao S, Shen B, Gao Y, Han D, He C. N (6)-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science. 2020;367:580–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Li R, Zhao H, Huang X, Zhang J, Bai R, Zhuang L, Wen S, Wu S, Zhou Q, Li M, et al. Super-enhancer RNA m(6)A promotes local chromatin accessibility and oncogene transcription in pancreatic ductal adenocarcinoma. Nat Genet. 2023;55:2224–34. [DOI] [PubMed] [Google Scholar]
20.Huang H, Weng H, Zhou K, Wu T, Zhao BS, Sun M, Chen Z, Deng X, Xiao G, Auer F, et al. Histone H3 trimethylation at lysine 36 guides m⁶A RNA modification co-transcriptionally. Nature. 2019;567:414–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Shim S, Lee HG, Lee H, Seo PJ. H3K36me2 is highly correlated with m(6) A modifications in plants. J Integr Plant Biol. 2020;62:1455–60. [DOI] [PubMed] [Google Scholar]
22.Barbieri I, Tzelepis K, Pandolfini L, Shi J, Millán-Zambrano G, Robson SC, Aspris D, Migliori V, Bannister AJ, Han N, et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature. 2017;552:126–31. [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Chen J, Zhang YC, Huang C, Shen H, Sun B, Cheng X, Zhang YJ, Yang YG, Shu Q, Yang Y, Li X. m(6)A regulates neurogenesis and neuronal development by modulating histone methyltransferase Ezh2. Genom Proteom Bioinf. 2019;17:154–68. [DOI] [PMC free article] [PubMed] [Google Scholar]
24.Mignone F, Gissi C, Liuni S, Pesole G. Untranslated regions of mRNAs. Genome Biol. 2002;3(reviews0004):0001. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Luo G-Z, MacQueen A, Zheng G, Duan H, Dore LC, Lu Z, Liu J, Chen K, Jia G, Bergelson J, He C. Unique features of the m⁶A methylome in Arabidopsis thaliana. Nat Commun. 2014;5:5630. [DOI] [PMC free article] [PubMed] [Google Scholar]
26.Cheng Q, Wang P, Wu G, Wang Y, Tan J, Li C, Zhang X, Liu S, Huang S, Huang T, et al. Coordination of m(6)A mRNA methylation and gene transcriptome in rice response to cadmium stress. Rice (N Y). 2021;14:62. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Song A, Su J, Wang H, Zhang Z, Zhang X, Van de Peer Y, Chen F, Fang W, Guan Z, Zhang F, et al. Analyses of a chromosome-scale genome assembly reveal the origin and evolution of cultivated chrysanthemum. Nat Commun. 2023;14:2021. [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Li Y, Xia L, Tan K, Ye X, Zuo Z, Li M, Xiao R, Wang Z, Liu X, Deng M, et al. N6-Methyladenosine co-transcriptionally directs the demethylation of histone H3K9me2. Nat Genet. 2020;52:870–7. [DOI] [PubMed] [Google Scholar]
29.Pinon V, Yao X, Dong A, Shen WH. SDG2-mediated H3K4me3 is crucial for chromatin condensation and mitotic division during male gametogenesis in arabidopsis. Plant Physiol. 2017;174:1205–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
30.Zhang X, Clarenz O, Cokus S, Bernatavichute YV, Pellegrini M, Goodrich J, Jacobsen SE. Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol. 2007;5:e129. [DOI] [PMC free article] [PubMed]
31.Qi PL, Zhou HR, Zhao QQ, Feng C, Ning YQ, Su YN, Cai XW, Yuan DY, Zhang ZC, Su XM, et al. Characterization of an autonomous pathway complex that promotes flowering in Arabidopsis. Nucleic Acids Res. 2022;50:7380–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.Zhao L, Xie L, Zhang Q, Ouyang W, Deng L, Guan P, Ma M, Li Y, Zhang Y, Xiao Q, et al. Integrative analysis of reference epigenomes in 20 rice varieties. Nat Commun. 2020;11:2658. [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Wang L, Yang C, Shan Q, Zhao M, Yu J, Li YF. Transcriptome-wide profiling of mRNA N(6)-methyladenosine modification in rice panicles and flag leaves. Genomics. 2023;115: 110542. [DOI] [PubMed] [Google Scholar]
34.Shoaib Y, Hu J, Manduzio S, Kang H. Alpha-ketoglutarate-dependent dioxygenase homolog 10B, an N6-methyladenosine mRNA demethylase, plays a role in salt stress and abscisic acid responses in Arabidopsis thaliana. Physiol Plant. 2021;173:1078–89. [DOI] [PubMed] [Google Scholar]
35.Wang X, Wang D, Xu W, Kong L, Ye X, Zhuang Q, Fan D, Luo K. Histone methyltransferase ATX1 dynamically regulates fiber secondary cell wall biosynthesis in Arabidopsis inflorescence stem. Nucleic Acids Res. 2021;49:190–205. [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Berr A, McCallum EJ, Ménard R, Meyer D, Fuchs J, Dong A, Shen WH. Arabidopsis SET DOMAIN GROUP2 is required for H3K4 trimethylation and is crucial for both sporophyte and gametophyte development. Plant Cell. 2010;22:3232–48. [DOI] [PMC free article] [PubMed] [Google Scholar]
37.Ding Y, Avramova Z, Fromm M. Two distinct roles of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) at promoters and within transcribed regions of ATX1-regulated genes. Plant Cell. 2011;23:350–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
38.Bhat SS, Bielewicz D, Gulanicz T, Bodi Z, Yu X, Anderson SJ, Szewc L, Bajczyk M, Dolata J, Grzelak N, et al. mRNA adenosine methylase (MTA) deposits m⁶A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2020;117:21785. [DOI] [PMC free article] [PubMed] [Google Scholar]
39.Phatnani HP, Greenleaf AL. Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 2006;20:2922–36. [DOI] [PubMed] [Google Scholar]
40.Liu XL, Yu HD, Guan Y, Li JK, Guo FQ. Carbonylation and loss-of-function analyses of SBPase reveal its metabolic interface role in oxidative stress, carbon assimilation, and multiple aspects of growth and development in Arabidopsis. Mol Plant. 2012;5:1082–99. [DOI] [PubMed] [Google Scholar]
41.Brusslan JA, Bonora G, Rus-Canterbury AM, Tariq F, Jaroszewicz A, Pellegrini M. A genome-wide chronological study of gene expression and two histone modifications, H3K4me3 and H3K9ac, during developmental leaf senescence. Plant Physiol. 2015;168:1246–61. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Zhang H, Shi X, Huang T, Zhao X, Chen W, Gu N, Zhang R. Dynamic landscape and evolution of m⁶A methylation in human. Nucleic Acids Res. 2020;48:6251–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Ke S, Alemu EA, Mertens C, Gantman EC, Fak JJ, Mele A, Haripal B, Zucker-Scharff I, Moore MJ, Park CY, et al. A majority of m⁶A residues are in the last exons, allowing the potential for 3’ UTR regulation. Genes Dev. 2015;29:2037–53. [DOI] [PMC free article] [PubMed] [Google Scholar]
44.Yue Y, Liu J, Cui X, Cao J, Luo G, Zhang Z, Cheng T, Gao M, Shu X, Ma H, et al. VIRMA mediates preferential m(6)A mRNA methylation in 3’UTR and near stop codon and associates with alternative polyadenylation. Cell Discov. 2018;4:10. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Bushkin GG, Pincus D, Morgan JT, Richardson K, Lewis C, Chan SH, Bartel DP, Fink GR. m⁶A modification of a 3′UTR site reduces RME1 mRNA levels to promote meiosis. Nat Commun. 2019;10:3414. [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Tang C, Klukovich R, Peng H, Wang Z, Yu T, Zhang Y, Zheng H, Klungland A, Yan W. ALKBH5-dependent m6A demethylation controls splicing and stability of long 3’-UTR mRNAs in male germ cells. Proc Natl Acad Sci U S A. 2018;115:E325–e333. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Chen J, Li E, Zhang X, Dong X, Lei L, Song W, Zhao H, Lai J. Genome-wide nucleosome occupancy and organization modulates the plasticity of gene transcriptional status in maize. Mol Plant. 2017;10:962–74. [DOI] [PubMed] [Google Scholar]
48.Mulroney TE, Pöyry T, Yam-Puc JC, Rust M, Harvey RF, Kalmar L, Horner E, Booth L, Ferreira AP, Stoneley M, et al. N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Nature. 2024;625:189–94. [DOI] [PMC free article] [PubMed] [Google Scholar]
49.Wang Y, Li Y, Yue M, Wang J, Kumar S, Wechsler-Reya RJ, Zhang Z, Ogawa Y, Kellis M, Duester G, Zhao JC. N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications. Nat Neurosci. 2018;21:195–206. [DOI] [PMC free article] [PubMed] [Google Scholar]
50.Wang H, Fan Z, Shliaha PV, Miele M, Hendrickson RC, Jiang X, Helin . H3K4me3 regulates RNA polymerase II promoter-proximal pause-release. Nature. 2023;615:339–348. [DOI] [PMC free article] [PubMed]
51.Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
52.Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008;9:R137. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907–15. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–78. [DOI] [PMC free article] [PubMed] [Google Scholar]
55.Cheng H, Zhang H, Song J, Jiang J, Chen S, Chen F, Wang L. GERDH: an interactive multi-omics database for cross-species data mining in horticultural crops. Plant J. 2023;116(4):1018–29. [DOI] [PubMed] [Google Scholar]
56.Wang L, Zhang Z, Zhang F, Shao Z, Zhao B, Huang A, Tran J, Hernandez FV, Qiao H. EIN2-directed histone acetylation requires EIN3-mediated positive feedback regulation in response to ethylene. Plant Cell. 2021;33:322–37. [DOI] [PMC free article] [PubMed] [Google Scholar]
57.Yang Y, Huang Y, Wang T, Li S, Jiang J, Chen S, Chen F, Wang L. mRNA m⁶A regulates gene expression via H3K4me3 shift in 5’ UTR. meRIP-, ChIP- and RNA-sequencing data on Genome Sequence Archive in National Genomics Data Center. 2025. https://ngdc.cncb.ac.cn/gsa/browse/CRA009857. Accessed Jan 17.

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

13059_2025_3515_MOESM1_ESM.pdf^{(890.9KB, pdf)}

13059_2025_3515_MOESM2_ESM.pdf^{(142.8KB, pdf)}

Additional file 2: Table S1. Summary of reads and mapped rates in different samples. Table S2. Primers used in this paper

13059_2025_3515_MOESM3_ESM.pdf^{(341.4KB, pdf)}

Additional file 3: Uncropped images of Western blots in Fig. 4, Fig. 5, Fig. 6 and Fig. S4

Data Availability Statement

[CR1] 1.Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, et al. Topology of the human and mouse m⁶A RNA methylomes revealed by m⁶A-seq. Nature. 2012;485:201–6. [DOI] [PubMed] [Google Scholar]

[CR2] 2.Meyer KD, Saletore Y, Zumbo P, Elemento O, Mason CE, Jaffrey SR. Comprehensive analysis of mRNA methylation reveals enrichment in 3’ UTRs and near stop codons. Cell. 2012;149:1635–46. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR3] 3.Clancy MJ, Shambaugh ME, Timpte CS, Bokar JA. Induction of sporulation in Saccharomyces cerevisiae leads to the formation of N6-methyladenosine in mRNA: a potential mechanism for the activity of the IME4 gene. Nucleic Acids Res. 2002;30:4509–18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR4] 4.Liang Z, Riaz A, Chachar S, Ding Y, Du H, Gu X. Epigenetic modifications of mRNA and DNA in plants. Mol Plant. 2020;13:14–30. [DOI] [PubMed] [Google Scholar]

[CR5] 5.Ping X-L, Sun B-F, Wang L, Xiao W, Yang X, Wang W-J, Adhikari S, Shi Y, Lv Y, Chen Y-S, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24:177–89. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR6] 6.Xu T, Wu X, Wong CE, Fan S, Zhang Y, Zhang S, Liang Z, Yu H, Shen L. FIONA1-mediated m⁶A modification regulates the floral transition in arabidopsis. Adv Sci. 2022;9:2103628. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505:117–20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR8] 8.Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene expression regulation. Cell. 2017;169:1187–200. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR9] 9.Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, Yi C, Lindahl T, Pan T, Yang YG, He C. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7:885–7. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, Vågbø CB, Shi Y, Wang WL, Song SH, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013;49:18–29. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR11] 11.Fan W, Wang L, Lei Z, Li H, Chu J, Yan M, Wang Y, Wang H, Yang J, Cho J. m6A RNA demethylase AtALKBH9B promotes mobilization of a heat-activated long terminal repeat retrotransposon in Arabidopsis. Sci Adv. 2023;9:eadf3292. [DOI] [PMC free article] [PubMed]

[CR12] 12.Duan HC, Wei LH, Zhang C, Wang Y, Chen L, Lu Z, Chen PR, He C, Jia G. ALKBH10B Is an RNA N(6)-Methyladenosine demethylase affecting arabidopsis floral transition. Plant Cell. 2017;29:2995–3011. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Zhang LB, Qiu TT, Yang WWJ. Bioinformatic analysis reveals the distinct role of 5′UTR-specific m6A RNA modification in mice developing cerebral cortices. Dev Neurosci. 2022;44:67–79. [DOI] [PubMed] [Google Scholar]

[CR14] 14.Meyer KD, Patil DP, Zhou J, Zinoviev A, Skabkin MA, Elemento O, Pestova TV, Qian SB, Jaffrey SR. 5’ UTR m(6)A promotes cap-independent translation. Cell. 2015;163:999–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR15] 15.Zhou J, Wan J, Shu XE, Mao Y, Liu XM, Yuan X, Zhang X, Hess ME, Brüning JC, Qian SB. N6-Methyladenosine guides mRNA alternative translation during integrated stress response. Mol Cell. 2018;69:636–647.e637. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR16] 16.Zhou J, Wan J, Gao X, Zhang X, Jaffrey SR, Qian SB. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature. 2015;526:591–4. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR17] 17.Deng S, Zhang J, Su J, Zuo Z, Zeng L, Liu K, Zheng Y, Huang X, Bai R, Zhuang L, et al. RNA m⁶A regulates transcription via DNA demethylation and chromatin accessibility. Nat Genet. 2022;54:1427–37. [DOI] [PubMed] [Google Scholar]

[CR18] 18.Liu J, Dou X, Chen C, Chen C, Liu C, Xu MM, Zhao S, Shen B, Gao Y, Han D, He C. N (6)-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription. Science. 2020;367:580–6. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR19] 19.Li R, Zhao H, Huang X, Zhang J, Bai R, Zhuang L, Wen S, Wu S, Zhou Q, Li M, et al. Super-enhancer RNA m(6)A promotes local chromatin accessibility and oncogene transcription in pancreatic ductal adenocarcinoma. Nat Genet. 2023;55:2224–34. [DOI] [PubMed] [Google Scholar]

[CR20] 20.Huang H, Weng H, Zhou K, Wu T, Zhao BS, Sun M, Chen Z, Deng X, Xiao G, Auer F, et al. Histone H3 trimethylation at lysine 36 guides m⁶A RNA modification co-transcriptionally. Nature. 2019;567:414–9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Shim S, Lee HG, Lee H, Seo PJ. H3K36me2 is highly correlated with m(6) A modifications in plants. J Integr Plant Biol. 2020;62:1455–60. [DOI] [PubMed] [Google Scholar]

[CR22] 22.Barbieri I, Tzelepis K, Pandolfini L, Shi J, Millán-Zambrano G, Robson SC, Aspris D, Migliori V, Bannister AJ, Han N, et al. Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control. Nature. 2017;552:126–31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR23] 23.Chen J, Zhang YC, Huang C, Shen H, Sun B, Cheng X, Zhang YJ, Yang YG, Shu Q, Yang Y, Li X. m(6)A regulates neurogenesis and neuronal development by modulating histone methyltransferase Ezh2. Genom Proteom Bioinf. 2019;17:154–68. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR24] 24.Mignone F, Gissi C, Liuni S, Pesole G. Untranslated regions of mRNAs. Genome Biol. 2002;3(reviews0004):0001. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR25] 25.Luo G-Z, MacQueen A, Zheng G, Duan H, Dore LC, Lu Z, Liu J, Chen K, Jia G, Bergelson J, He C. Unique features of the m⁶A methylome in Arabidopsis thaliana. Nat Commun. 2014;5:5630. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Cheng Q, Wang P, Wu G, Wang Y, Tan J, Li C, Zhang X, Liu S, Huang S, Huang T, et al. Coordination of m(6)A mRNA methylation and gene transcriptome in rice response to cadmium stress. Rice (N Y). 2021;14:62. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Song A, Su J, Wang H, Zhang Z, Zhang X, Van de Peer Y, Chen F, Fang W, Guan Z, Zhang F, et al. Analyses of a chromosome-scale genome assembly reveal the origin and evolution of cultivated chrysanthemum. Nat Commun. 2023;14:2021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.Li Y, Xia L, Tan K, Ye X, Zuo Z, Li M, Xiao R, Wang Z, Liu X, Deng M, et al. N6-Methyladenosine co-transcriptionally directs the demethylation of histone H3K9me2. Nat Genet. 2020;52:870–7. [DOI] [PubMed] [Google Scholar]

[CR29] 29.Pinon V, Yao X, Dong A, Shen WH. SDG2-mediated H3K4me3 is crucial for chromatin condensation and mitotic division during male gametogenesis in arabidopsis. Plant Physiol. 2017;174:1205–15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Zhang X, Clarenz O, Cokus S, Bernatavichute YV, Pellegrini M, Goodrich J, Jacobsen SE. Whole-genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol. 2007;5:e129. [DOI] [PMC free article] [PubMed]

[CR31] 31.Qi PL, Zhou HR, Zhao QQ, Feng C, Ning YQ, Su YN, Cai XW, Yuan DY, Zhang ZC, Su XM, et al. Characterization of an autonomous pathway complex that promotes flowering in Arabidopsis. Nucleic Acids Res. 2022;50:7380–95. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Zhao L, Xie L, Zhang Q, Ouyang W, Deng L, Guan P, Ma M, Li Y, Zhang Y, Xiao Q, et al. Integrative analysis of reference epigenomes in 20 rice varieties. Nat Commun. 2020;11:2658. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Wang L, Yang C, Shan Q, Zhao M, Yu J, Li YF. Transcriptome-wide profiling of mRNA N(6)-methyladenosine modification in rice panicles and flag leaves. Genomics. 2023;115: 110542. [DOI] [PubMed] [Google Scholar]

[CR34] 34.Shoaib Y, Hu J, Manduzio S, Kang H. Alpha-ketoglutarate-dependent dioxygenase homolog 10B, an N6-methyladenosine mRNA demethylase, plays a role in salt stress and abscisic acid responses in Arabidopsis thaliana. Physiol Plant. 2021;173:1078–89. [DOI] [PubMed] [Google Scholar]

[CR35] 35.Wang X, Wang D, Xu W, Kong L, Ye X, Zhuang Q, Fan D, Luo K. Histone methyltransferase ATX1 dynamically regulates fiber secondary cell wall biosynthesis in Arabidopsis inflorescence stem. Nucleic Acids Res. 2021;49:190–205. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Berr A, McCallum EJ, Ménard R, Meyer D, Fuchs J, Dong A, Shen WH. Arabidopsis SET DOMAIN GROUP2 is required for H3K4 trimethylation and is crucial for both sporophyte and gametophyte development. Plant Cell. 2010;22:3232–48. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Ding Y, Avramova Z, Fromm M. Two distinct roles of ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) at promoters and within transcribed regions of ATX1-regulated genes. Plant Cell. 2011;23:350–63. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Bhat SS, Bielewicz D, Gulanicz T, Bodi Z, Yu X, Anderson SJ, Szewc L, Bajczyk M, Dolata J, Grzelak N, et al. mRNA adenosine methylase (MTA) deposits m⁶A on pri-miRNAs to modulate miRNA biogenesis in Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2020;117:21785. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR39] 39.Phatnani HP, Greenleaf AL. Phosphorylation and functions of the RNA polymerase II CTD. Genes Dev. 2006;20:2922–36. [DOI] [PubMed] [Google Scholar]

[CR40] 40.Liu XL, Yu HD, Guan Y, Li JK, Guo FQ. Carbonylation and loss-of-function analyses of SBPase reveal its metabolic interface role in oxidative stress, carbon assimilation, and multiple aspects of growth and development in Arabidopsis. Mol Plant. 2012;5:1082–99. [DOI] [PubMed] [Google Scholar]

[CR41] 41.Brusslan JA, Bonora G, Rus-Canterbury AM, Tariq F, Jaroszewicz A, Pellegrini M. A genome-wide chronological study of gene expression and two histone modifications, H3K4me3 and H3K9ac, during developmental leaf senescence. Plant Physiol. 2015;168:1246–61. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR42] 42.Zhang H, Shi X, Huang T, Zhao X, Chen W, Gu N, Zhang R. Dynamic landscape and evolution of m⁶A methylation in human. Nucleic Acids Res. 2020;48:6251–64. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR43] 43.Ke S, Alemu EA, Mertens C, Gantman EC, Fak JJ, Mele A, Haripal B, Zucker-Scharff I, Moore MJ, Park CY, et al. A majority of m⁶A residues are in the last exons, allowing the potential for 3’ UTR regulation. Genes Dev. 2015;29:2037–53. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR44] 44.Yue Y, Liu J, Cui X, Cao J, Luo G, Zhang Z, Cheng T, Gao M, Shu X, Ma H, et al. VIRMA mediates preferential m(6)A mRNA methylation in 3’UTR and near stop codon and associates with alternative polyadenylation. Cell Discov. 2018;4:10. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR45] 45.Bushkin GG, Pincus D, Morgan JT, Richardson K, Lewis C, Chan SH, Bartel DP, Fink GR. m⁶A modification of a 3′UTR site reduces RME1 mRNA levels to promote meiosis. Nat Commun. 2019;10:3414. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR46] 46.Tang C, Klukovich R, Peng H, Wang Z, Yu T, Zhang Y, Zheng H, Klungland A, Yan W. ALKBH5-dependent m6A demethylation controls splicing and stability of long 3’-UTR mRNAs in male germ cells. Proc Natl Acad Sci U S A. 2018;115:E325–e333. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR47] 47.Chen J, Li E, Zhang X, Dong X, Lei L, Song W, Zhao H, Lai J. Genome-wide nucleosome occupancy and organization modulates the plasticity of gene transcriptional status in maize. Mol Plant. 2017;10:962–74. [DOI] [PubMed] [Google Scholar]

[CR48] 48.Mulroney TE, Pöyry T, Yam-Puc JC, Rust M, Harvey RF, Kalmar L, Horner E, Booth L, Ferreira AP, Stoneley M, et al. N1-methylpseudouridylation of mRNA causes +1 ribosomal frameshifting. Nature. 2024;625:189–94. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR49] 49.Wang Y, Li Y, Yue M, Wang J, Kumar S, Wechsler-Reya RJ, Zhang Z, Ogawa Y, Kellis M, Duester G, Zhao JC. N6-methyladenosine RNA modification regulates embryonic neural stem cell self-renewal through histone modifications. Nat Neurosci. 2018;21:195–206. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR50] 50.Wang H, Fan Z, Shliaha PV, Miele M, Hendrickson RC, Jiang X, Helin . H3K4me3 regulates RNA polymerase II promoter-proximal pause-release. Nature. 2023;615:339–348. [DOI] [PMC free article] [PubMed]

[CR51] 51.Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR52] 52.Zhang Y, Liu T, Meyer CA, Eeckhoute J, Johnson DS, Bernstein BE, Nusbaum C, Myers RM, Brown M, Li W, Liu XS. Model-based analysis of ChIP-Seq (MACS). Genome Biol. 2008;9:R137. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR53] 53.Kim D, Paggi JM, Park C, Bennett C, Salzberg SL. Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol. 2019;37:907–15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR54] 54.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–78. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR55] 55.Cheng H, Zhang H, Song J, Jiang J, Chen S, Chen F, Wang L. GERDH: an interactive multi-omics database for cross-species data mining in horticultural crops. Plant J. 2023;116(4):1018–29. [DOI] [PubMed] [Google Scholar]

[CR56] 56.Wang L, Zhang Z, Zhang F, Shao Z, Zhao B, Huang A, Tran J, Hernandez FV, Qiao H. EIN2-directed histone acetylation requires EIN3-mediated positive feedback regulation in response to ethylene. Plant Cell. 2021;33:322–37. [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR57] 57.Yang Y, Huang Y, Wang T, Li S, Jiang J, Chen S, Chen F, Wang L. mRNA m⁶A regulates gene expression via H3K4me3 shift in 5’ UTR. meRIP-, ChIP- and RNA-sequencing data on Genome Sequence Archive in National Genomics Data Center. 2025. https://ngdc.cncb.ac.cn/gsa/browse/CRA009857. Accessed Jan 17.

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mRNA m6A regulates gene expression via H3K4me3 shift in 5’ UTR

Yuna Yang

Yuqing Huang

Tian Wang

Song Li

Jiafu Jiang

Sumei Chen

Fadi Chen

Likai Wang

Abstract

Background

Results

Conclusions

Supplementary Information

Background

Results

m6A methylation, uniquely deposited in 5’ UTR regions, contributes to gene repression

Fig. 1.

H3K4me3 is involved in the transcriptional regulation of m6A-modified genes

m6A depositing in 5’ UTR shifts H3K4me3 modification

Fig. 2.

The downstream shift of H3K4me3 is genetically affected by the m6A writer and eraser

Fig. 3.

m6A-mediated gene expression depends on H3K4me3 modification in 5’ UTR regions

MTA and ATX1 interact with CTD of RNA Pol II in vitro and in vivo

Fig. 4.

MTA competes ATX1 to interact with CTD of RNA Pol II in vitro and in vivo

MTA competes ATX1 to interact with Ser5P-CTD of RNA Pol II in vitro and in vivo

Fig. 5.

ALKBH10B competes with MTA for binding to the Ser5P-CTD of RNA Pol II both in vitro and in vivo

Fig. 6.

m6A-mediated H3K4me3 shift in the 5’ UTR is involved in leaf senescence

Fig. 7.

Discussion

Conclusions

Methods

Plant materials

Assay for natural and dark-induced leaf senescence

Measurements of chlorophyll content

m6A-immunoprecipitation and sequencing (m6A-seq) and analysis

RNA-seq processing and analysis

ChIP-seq processing and analysis

Western blot

Pull-down assay

Immunoprecipitation assays

N. benthamiana transient expression assay

Supplementary Information

Acknowledgements

Peer review information

Authors’ contributions

Funding

Data availability

Declarations

Ethics approval and consent to participate

Consent for publication

Competing interests

Footnotes

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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Links to NCBI Databases

mRNA m⁶A regulates gene expression via H3K4me3 shift in 5’ UTR

m⁶A methylation, uniquely deposited in 5’ UTR regions, contributes to gene repression

H3K4me3 is involved in the transcriptional regulation of m⁶A-modified genes

m⁶A depositing in 5’ UTR shifts H3K4me3 modification

The downstream shift of H3K4me3 is genetically affected by the m⁶A writer and eraser

m⁶A-mediated gene expression depends on H3K4me3 modification in 5’ UTR regions

m⁶A-mediated H3K4me3 shift in the 5’ UTR is involved in leaf senescence

m⁶A-immunoprecipitation and sequencing (m⁶A-seq) and analysis