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Regulation of m6A RNA reader protein OsECT3 activity by lysine acetylation in the cold stress response in rice

Nature Plants volume 11, pages 1165–1180 (2025)Cite this article

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Abstract

N6-Methyladenosine (m6A) reader proteins, which recognize m6A to regulate RNA metabolism, are important for plant adaptation to the changing environment. It remains unknown how the activities of plant m6A reader proteins are regulated in plant responses to stress. Here we show that the rice m6A reader protein EVOLUTIONARILY CONSERVED C-TERMINAL REGION 3 (OsECT3), required for rice tolerance to cold, is post-translationally modified by lysine acetylation, which reduces its m6A-binding activity. Under cold conditions, OsECT3 acetylation is reduced by cold-induced histone deacetylase HDA705 and low ACLA2-sourced acetyl-CoA levels, resulting in an increase in OsECT3 m6A-binding activity, the accumulation of cold-response-related mRNAs and improved tolerance of rice to cold stress. These results unravel a regulatory mechanism of an m6A reader protein to dynamically control m6A RNA levels under stress and suggest a link between lysine acetylation, metabolism and m6A pathways.

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Fig. 1: The OsECT3 protein is acetylated in vivo mainly at the Lys471 residue.
Fig. 2: OsECT3 is an m6A reader, and Lys471 acetylation influences its m6A-binding activity in rice.
Fig. 3: OsECT3 acetylation is regulated by HDA705 and acetyl-CoA levels.
Fig. 4: OsECT3 m6A-binding activity and lysine deacetylation are required for cold tolerance in rice.
Fig. 5: HDA705 and acetyl-CoA levels mediate OsECT3 lysine deacetylation under cold stress.
Fig. 6: OsECT3 deacetylation improves its transcriptome-wide m6A-binding activity under cold stress.
Fig. 7: OsECT3 stabilizes cold-response-related genes under cold stress.
Fig. 8: Acetylation of OsECT3 affects mRNA stability of cold-response-related genes under cold stress.

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Data availability

The raw sequence data from m6A-seq and CLIP reported in this paper have been deposited in the Genome Sequence Archive at the National Genomics Data Center, Beijing Institute of Genomics, Chinese Academy of Sciences, under accession number PRJCA033583, which is publicly accessible at https://bigd.big.ac.cn/gsa. Source data are provided with this paper. All remaining data are available in the main text or in Supplementary Information.

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Acknowledgements

We thank T. Hu for the construction of transgenic plants. We thank X. Li for management and H. Song for help in confocal microscopy. We also thank the National Center for Protein Sciences at Peking University in Beijing, China, for assistance with sequencing, and G. Li and X. Zhang for help with the MGI2000 experiment. This work was supported by grants from the National Key R&D Program of China (nos 2024YFF1000302 and 2023ZD04073), the National Natural Science Foundation of China (nos 32070563, 22225704 and 32470307), the Fundamental Research Funds for the Central Universities (no. 2662023SKPY002) and the earmarked fund for China Agriculture Research System (no. CARS-01). The Fundamental Research Program of Hubei Province (2024AFE001).

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Author notes

  1. These authors contributed equally: Nini Ma, Peizhe Song.

Authors and Affiliations

  1. National Key Laboratory of Crop Genetic Improvement, Hubei Hongshan Laboratory, Huazhong Agricultural University, Wuhan, China

    Nini Ma, Ziyang Liu, Mengyue Ding, Xuan Ma, Qiutao Xu, Yaping Yue, Tangdi Luo, Dao-Xiu Zhou & Yu Zhao

  2. Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking-Tsinghua Center for Life Sciences, Beijing Advanced Center of RNA Biology (BEACON), Peking University, Beijing, China

    Peizhe Song, Zhihe Cai & Guifang Jia

  3. State Key Laboratory of Agricultural Microbiology, College of Informatics, Huazhong Agricultural University, Wuhan, China

    Yangjie Li

  4. Institute of Plant Science Paris-Saclay (IPS2), CNRS, INRAE, University of Paris-Saclay, Orsay, France

    Dao-Xiu Zhou

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  1. Nini Ma
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Contributions

Y.Z. conceived and designed the research, and D.-X.Z. supervised the research. N.M. performed the experiments and wrote the initial draft. P.S. performed the m6A-seq, FA-CLIP, m6A-IP–qPCR and FA-RIP–qPCR experiments. Z.C., Y.L. and P.S. performed the data analysis. Z.L., M.D. and T.L. participated in the experiments. X.M., Q.X. and Y.Y. joined in the transgenic plant regeneration. Y.Z. and D.-X.Z. analysed the data. Y.Z., D.-X.Z. and G.J. revised the paper with input from all authors.

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Correspondence to Guifang Jia or Yu Zhao.

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Nature Plants thanks Hunseung Kang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Relative expression levels of YTH domain-containing genes in rice.

RT-qPCR analysis of YTH domain family gene expression levels in 14-day-old wild-type rice seedlings. Actin was used as a reference gene. Data represent the means ± s.d. (three separate experiments per sample). The significance was calculated using one-way ANOVA with Tukey’s multiple comparison tests. Different lowercase letters over the bars indicate significant differences (p < 0.05).

Source data

Extended Data Fig. 2 Sequence alignment of the YTH domains from different species.

Os, Oryza sativa; At, Arabidopsis thaliana; Hs, Homo sapiens. Tryptophan (W), which forms the methyl-interacting aromatic cage, is highlighted in cyan. Lysine (K), the acetylation site shown in Fig. 1a, is highlighted in magenta.

Extended Data Fig. 3 Production and detection of OsECT3 transgenic plants.

a, Production of rice OsECT3 CRISPR-Cas9 mutants. The positions of single guide RNA (sgRNA) in the gene (indicated by black arrows) and decoded sequence mutations of the OsECT3 gene are shown. b, c, Detection of relative mRNA and protein levels in WT and COM-E3-F (OsECT3pro::OsECT3-Flag/osect3) plants by RT-qPCR (b) and immunoblotting with anti-Flag antibody (c), respectively. d, Determination of relative OsECT3 mRNA levels in WT and COM-E3-G (OsECT3pro::OsECT3-GFP/osect3), COM-E3K471R (OsECT3pro::OsECT3K471R-GFP/osect3), and COM-E3K471Q (OsECT3pro::OsECT3K471Q-GFP/osect3) plants by RT-qPCR. e-g, Detection of GFP-targeted OsECT3 (e), OsECT3(K471R/Q) (f, g) protein levels using anti-GFP antibodies in the respective complementary transgenic plants. For all data, bars indicate the mean ± s.d. of three replicates (one-way ANOVA with Tukey’s multiple comparison tests, the significance level was p < 0.05). RT-qPCR used ACTIN as a reference gene, each sample are three biological replicates. Immunoblot analyses show the indicated protein expression of the transgenes in T2 seedlings 14 days after germination. Four independent lines are shown for each construct, anti-actin used as a control.

Source data

Extended Data Fig. 4 Analysis of OsECT3/OsECT33WA RNA-binding activities and production of OsECT33WA transgenic plants.

a-b, Tests of OsECT3- (a) and OsECT33WA- (b) binding activities to RNA probes containing the RRACH motif. For the RNA-EMSA, 4 nmol of the RNA probe were used. RNA probe sequence is 5′-FAM-UCUUUGGXCUGACUUGGACUCUUUA-3′ (X = A/m6A). Protein concentrations were indicated. Two repetitions were performed. c-d, Detection of OsECT3 mRNA and protein levels in WT and COM-E33WA (OsECT3pro::OsECT33WA-Flag/osect3) plants by RT-qPCR (c) and immunoblotting with anti-Flag antibody (d). Bars indicate the mean ± s.d. of three replicates (one-way ANOVA with Tukey’s multiple comparison tests, the significance level was p < 0.05).

Source data

Extended Data Fig. 5 Acetylation of the lysine-471 residue of OsECT3 affects its m6A binding activity, but not its subcellular localization, protein level or stability.

a, Computer modeling of m6A (colored in magenta) binding affinity of OsECT3(K471Q) and OsECT3(K471R) (colored in orange) proteins. b, OsECT3 and OsECT3(K471R/Q) mutants localized in the cytoplasm under normal and cold conditions. Confocal images showing cytoplasmic localization of GFP in root tips of OsECT3pro::OsECT3-GFP/osect3 and OsECT3pro::OsECT3K471R/Q-GFP/osect3. Scale bar = 5 μm. c, Analysis of OsECT3 protein levels in 14-day-old seedlings in complementation plants with WT OsECT3 or its mutant variants. OsECT3 was detected using an anti-GFP antibody, with anti-actin used as a control. d, Recombinant OsECT3 and its mutant proteins (OsECT3(K471R/Q)) were incubated with rice cell extracts for semi-in vitro protein degradation assays. The degradation rate of OsECT3 and OsECT3(K471R/Q) were not significantly different. CBB (Coomassie Blue) staining was used as a loading control. Two independent experiments were performed in b-d.

Source data

Extended Data Fig. 6 Phenotypes of osect3 mutants in response to heat, salt, and PEG6000 stress.

Phenotypes of 14-day-old wild-type, osect3 seedlings before and after 42 °C treatment (a), 20% PEG6000 treatment (b), 180 mM NaCl treatment (c) for 3 days and subsequent recovery for 14 days. Bars = 4 cm. Mean ± s.d. of 5 biological replicates (n = 36 plants for each replicate) are shown in a-c. P values from two-tailed t-test.

Source data

Extended Data Fig. 7 HDA705 transgenic plant production and stress-induced expression analysis.

a, Generation of hda705 knockout mutants using CRISPR/Cas9 technology. Two positions (black arrow indicated) of the designed sgRNA and the decoded mutations of the HDA705 gene are shown. b, Detection of HDA705 in hda705 complementation plants (HDA705pro::HDA705-GFP/hda705) by immunoblotting with anti-GFP, and anti-Actin used as a control. Two independent experiments were performed. c-d, Relative expression levels of HDA705 in wild-type seedlings under 20% PEG6000 (c) and 180 mM NaCl (d) treatments. Actin was used as the internal control gene. Data are presented as mean ± s.d. of three replicates. p values indicate significant differences compared to normal at each time point, as determined by two-tailed, unpaired t-test.

Source data

Extended Data Fig. 8 HDA705 deacetylase activity towards OsECT3 in the HDA705 overexpression plants under cold stress.

a, Detection of relative HDA705 mRNA levels in WT and HDA705 overexpression (OE-HDA705) plants under normal condition. b, Relative expression levels of HDA705 under cold conditions for 0, 6, 12, and 24 h in the OE-HDA705 plants. ACTIN was used as a reference gene, data are presented as mean ± s.d. of three replicates. The significance was calculated using one-way ANOVA with Tukey’s multiple comparison tests in a-b. c, Protein levels of HDA705 treated by cold for 0, 12 h in the OE-HDA705 plants. HDA705-Flag protein was detected with anti-Flag antibody and anti-Actin antibody was used as a loading control. d, Detection of deacetylation level of OsECT3 treated by cold for 0, 12 h in the OE-HDA705 plants. OsECT3 protein was immunoprecipitated with anti-OsECT3 antibody, followed by immunoblotting with anti-OsECT3 or anti-ac-lys antibodies. e, Tests of HDA705 deacetylase activity in 14-day-old WT seedlings under normal and cold conditions. HDA705 protein was isolated by immunoprecipitation using anti-HDA705. HDA705 activities were shown relative to normal conditions (set at 100 %). Error bars are mean ± s.d. from three biological replicates. Significant difference was calculated by the two-tailed, unpaired t-test, the significance level was p < 0.05. Two replicates are shown.

Source data

Extended Data Fig. 9 OsECT3 binds to mRNA 3’UTR regions and m6A levels quantification under normal and cold conditions.

a, Go enrichment analysis of known genes bond by OsECT3 under cold stress. b, Motifs identified by HOMER software based on the OsECT3 binding and m6A sites under normal and cold stress conditions. c, Distribution of the distance of OsECT3 binding and m6A sites under cold stress conditions compared to those under normal conditions. d, Boxplot showing m6A levels of 1,701 common OsECT3- and m6A-targeted genes under both conditions. Results were calibrated with m6A spike-ins to diminish the difference in efficiency during immunoprecipitation in m6A-seq. The medians (horizontal lines), interquartile ranges (boxes), whiskers (± 1.5× interquartile range) and outliers of the data are shown (n = 1,701 common OsECT3- and m6A-targeted genes from two biological replicates). P-values were calculated using two-sided Wilcoxon test. e, LC-MS/MS quantification of the m6A/A ratio in polyadenylated RNA isolated from 14-day-old WT seedlings under both conditions. Data are presented as mean ± s.d., n = 6 independent experiments with 2 technical replicates each. ns, not significant by unpaired two-tailed t-test. f, Venn diagrams showing the overlap of OsECT3 and OsECT3(K471Q/R) binding sites detected by FA-CLIP with m6A peaks detected by m6A-seq under cold condition. g, Go enrichment analysis of genes bond by OsECT3, but not by OsECT3(K471Q) under cold stress.

Extended Data Fig. 10 mRNA m6A enrichment and stability of 4 OsETC3 target genes under normal and cold conditions.

a. Relative mRNA m6A levels of the 4 cold-repsonsive mark genes by m6A-IP-qPCR in WT, osect3, COM-E3-G, COM-E33WA, COM-E3K471R, and COM-E3K471Q seedlings under normal and cold conditions. External m6A spike-in was used for calibration. Data are presented as mean ± SE, n = 3 independent experiments. The significance was calculated using one-way ANOVA with Tukey’s multiple comparison tests. b-c, The mRNA half-lives of the 4 genes in WT and osect3 seedlings under normal conditions (b) and COM-E3K471Q, COM-E3K471R seedlings under cold conditions (c). Data are presented as mean ± s.d., n = 2 independent experiments, each with 2 technical replicates.

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Supplementary information

Supplementary Information (download PDF )

Supplementary Tables 1 and 2.

Reporting Summary (download PDF )

Supplementary Dataset 1 (download XLSX )

Statistics of OsECT3 and m6A targets under normal conditions.

Supplementary Dataset 2 (download XLSX )

Statistics of OsECT3 and m6A targets under cold conditions.

Supplementary Dataset 3 (download XLSX )

Statistics of OsECT3/OsECT3(K471Q), OsECT3(K471R) and m6A targets under cold conditions.

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Ma, N., Song, P., Liu, Z. et al. Regulation of m6A RNA reader protein OsECT3 activity by lysine acetylation in the cold stress response in rice. Nat. Plants 11, 1165–1180 (2025). https://doi.org/10.1038/s41477-025-02013-w

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