The Critical Role of OsYTH10 in Promoting Early Flowering of Rice Under Long Sunlight

The Critical Role of OsYTH10 in Promoting Early Flowering of Rice Under Long Sunlight | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Critical Role of OsYTH10 in Promoting Early Flowering of Rice Under Long Sunlight Jun Yang, Zhihao Chen, Peng Wang, Mvuyeni Nyasulu, Qin Chen, Xiaopeng He, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6593659/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Theoretical and Applied Genetics → Version 1 posted 5 You are reading this latest preprint version Abstract RNA m6A modification plays a crucial role in plant growth and crop yield. Proteins that can recognize m6A modifications, known as m6A reader proteins, are essential for the regulatory functions of m6A in gene expression. Among mRNA modification, methylation of internal adenosine N6 positions (m6As) is the most prevalent. The functional impact of m6A modifications largely relies on reader proteins. In this study, we identified OsYTH10, a member of the rice YTH-domain family protein, as a key player in m6A binding. The m6A-binding activity of OsYTH10 is mediated by its YTH structural domain. Deletion of OsYTH10 function results in early flowering in rice. Through FA-CLIP and m6A-seq analysis, we discovered that OsYTH10 binds to m6A in the 3'UTR region of mRNA. Our findings reveal that OsYTH10 stabilizes the mRNAs of target genes OsDTH7 and OsGI , thereby regulating the normal flowering process in rice under prolonged sunlight conditions. This study sheds light on the critical role of OsYTH10 in m6A-mediated gene regulation and its impact on flowering time in rice. RNA m6A OsYTH10 Flowering Time Rice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Key message A crucial RNA N6 -methyladenosine (m6A) reader protein OsYTH10 in rice was identified to physically binds mRNA's 3'UTR via its YTH domain, stabilizing OsDTH7 and OsGI transcripts to accelerate flowering under long-day conditions. Introduction In recent years, epigenetic modifications have garnered significant attention for their crucial roles in plant physiology, with notable advancements in research on histone methylation, histone acetylation, and ubiquitination. However, there is a notable gap in the study of RNA epigenetic modifications. RNA methylation, particularly N6-methyladenosine (m6A), plays a vital post-transcriptional role in regulating gene expression in eukaryotes. This dynamic and reversible modification is orchestrated by a complex network of methyltransferases, de-methyltransferases, and m6A binding proteins (Liang et al. 2020; Shen et al. 2019; Tang et al. 2023; Wang and Zhao 2016; Wu et al. 2022). Methyltransferases serve as writers, de-methyltransferases as erasers, and m6A binding proteins as readers, collectively influencing various biological processes in response to environmental and internal cues (Lim and Pawson 2010). The dynamic reversibility of m6A modification allows eukaryotes to swiftly adapt to changing conditions, contributing to the regulation of diverse cellular processes. In animals, the RNA m6A methylation mystery has been well-characterized, and its biological role and applications have become increasingly clear. It has been definitively established that YT521-B homology (YTH) domain-containing reader proteins mediate mRNA recognition for precise regulation of RNA metabolism by m6A in mammalian cells (Xu et al. 2015; Yang et al. 2022; Zhou et al. 2021). The YT521-B homology (YTH) domain is a highly conserved structural domain of YTH family proteins in various species, containing a hydrophobic pocket critical for m6A recognition in the cytoplasm (Li et al. 2014). In mammals, there are two branches of YTH family proteins, YTHDFs and YTHDCs. For instance, YTHDF1 promotes RNA translation, YTHDF2 facilitates RNA decay, and YTHDF3 exhibits a dual function depending on its binding partner (Shi et al. 2017). These functionally distinct YTHDF family proteins (YTHDF1, YTHDF2, YTHDF3) can bind the same m6A-modified mRNAs through their YTH domains, redundantly mediating mRNA degradation and cellular differentiation (Zaccara and Jaffrey 2020). It has been observed that YTHDF1 and YTHDF3, but not YTHDF2, carry high levels of nutrient-sensing O-GlcNAc modifications, which attenuate the translation-promoting function of YTHDF1 and YTHDF3 by blocking their interactions with proteins associated with mRNA translation. This leads to the assembly, stability, and disassembly of stress granules, enabling better recovery from stress (Chen et al. 2023). In the YTHDC family, YTHDC1 mediates the export of methylated mRNA from the nucleus to the cytoplasm in Hela cells and is involved in m6A epigenetic regulation of FSP1, alleviating FSP1-dependent ferroptosis suppression. It also modulates autophagy by regulating the stability of SQSTM1 nuclear mRNA in diabetic keratinocytes (Liang et al. 2022; Roundtree et al. 2017; Yuan et al. 2023). YTHDC2 enhances the translation efficiency of its targets and decreases their mRNA abundance by selectively binding m6A at its consensus motif (Hsu et al. 2017). YTHDC2 is involved in rescuing lung tumorigenesis by suppressing cystine uptake and blocking the downstream antioxidant program (Ma et al. 2021). Coincidentally, research on m6A readers in plants is limited, and the currently studied reader proteins all belong to the YTH family, especially in Arabidopsis and rice. In Arabidopsis, the YTH family consists of 13 proteins containing highly conserved YTH domains at the C-terminus, referred to as the Evolutionarily Conserved C-Terminal Region (ECT) family (Bhat et al. 2018). AtECT1 was reported to sequester SA-induced m 6 A modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses (Lee et al. 2024). AtECT9 was involved in regulating plant immunity by interacting with AtECT1 (Wang et al. 2023). AtECT2, AtECT3, and AtECT4 show genetic redundancy in multiple biological processes, including plant developmental timing, morphogenesis, and organogenesis, suggesting that they cooperatively stabilize the bound m6A-modified mRNA to affect target gene expression (Arribas-Hernandez et al. 2021; Arribas-Hernandez et al. 2020; Song et al. 2023). In rice, there are 12 YTH proteins. It was shown that OsYTH03, OsYTH05, and OsYTH10 impact on the diterpenoid and brassinolide synthesis pathway by physically recognizing and binding to m6A-containing RNAs to redundantly modulate rice plant height (Cai et al. 2023). OsYTH07 physically interacts with OsEHD6 to enhance EHD6-YTH07 with strong-affinity to m6A targets and leads to the partial relocation of YTH07 from the cytoplasm to RNP granules through phase-separated condensation, thereby sequestering the OsCOL4 mRNA, reducing OsCOL4 protein accumulation, and promoting flowering via the EHD1 pathway (Cui et al. 2024). However, although the function of m6A readers in plants is rapidly being unveiled, the molecular mechanism underlying their regulatory roles remains poorly understood. Notably, we identified a YTH family protein, OsYTH10, that mediates the rice heading date in this study. Loss of function of OsYTH10 promotes early flowering in rice, while overexpression of OsYTH10 delays the rice heading date significantly. We found that OsYTH10 is localized in the nucleus and cytoplasm. The flowering-related genes OsGI and OsDTH7 have been identified as target genes of OsYTH10, and it has been demonstrated that OsYTH10 can specifically recognize and bind to m6A-containing mRNA in their 3'UTR. Additionally, loss of function of OsYTH10 can repress the expression levels of OsGI and OsDTH7 by accelerating their mRNA degradation, thereby affecting the rice heading date. Our results demonstrate that OsYTH10 plays a critical role in controlling m6A-dependent heading date regulation in rice. Materials and methods Plant materials and environment The wild-type (WT) rice variety used in this study was Nipponbare, a japonica variety. All plants were grown in the experimental field of Jiangxi Agricultural University, located in Jiangxi province, China (28°45′36″N, 115°22′58″E) during the summer. RT-qPCR and western blotting Total RNA was extracted using Trizol (Aidlab, China), and the first-strand complementary DNA was reverse transcribed using ToloScript All-in-one RT EasyMix for qPCR (TOLOBIO, China). RT-qPCR was performed using TB Green Premix Ex Taq II (Takara) on a CFX96 Real-Time PCR Detection System (Bio-Rad), and the RT-qPCR primers used in this study are listed in supplemental table S1. Total protein extracts from genotypic plants were separated on the 12% SDS-PAGE gel, and then transferred onto nitrocellulose membranes (GE Healthcare). proteins were detected using antibodies anti-ACTIN (Abmart), anti-GST (Abcam) and anti-MYC (Abcam). Eletrophoretic mobility shift analysis (EMSA) The recombinant GST, GST-OSYTH10 were expressed with pGEX-6P-3 containing a GST tag, and purified using an Escherichia coli (BL21-RIL) expression system. The GST protein was used the negative control. Two conserved sites, Trp431 and Trp444, for YTH function were discovered by aligning sequence of YTH domains in OsYTH10 and AtECT2. Then the sites both Trp431 and Trp444 were mutated into Ala using the Fast Mutagenesis System kit (TransGene Biotech, China) for GST-OsYTH10 mutants. EMSA assays were performed using kit as described previously (Wei et al. 2018). The synthesized FAM 5’ end-labeled RNA fragment listed in supplemental table1 was used as the RNA probe, while the corresponding unlabeled fragment was used as the competitor probes. mRNA decay assay The procedure was conducted as the previously described method (Duan et al. 2017; Tang et al. 2022). 7-day-old WT and osyth10-1 seedlings were cultured with liquid half‐strength MS medium. Then the seedlings were treated with a final concentration of 200 µM actinomycin D in the medium. After infiltration for 30 min, seedlings were harvested as time 0 controls, respectively. and subsequent samples were harvested every 3 h. To determine remaining mRNA levels using RT‐qPCR, 18S rRNA was used as the internal control. The primers used in this assay are listed in supplemental table S1. RIP assay and RNA-protein pull-down assay in vitro RIP assay with OsYTH10-4×Myc line was performed according to a previously described method (Tang et al. 2022). Briefly, 35-day-old seedlings were harvested and cross-linked by using 1% formaldehyde for 10 min. RNA-protein complexes were immunoprecipitated by incubating with antibody Myc binding to protein G beads (Sigma) at 4°C for 4 h. Finally, the cross-linking was reversed, and RNA was purified by Trizol (Aidlab, China) for qPCR analysis. Primers used for RIP assay are listed in supplemental Table S1. RNA pull-down assay was referred to a previously described method (Yang et al. 2023). In brief, biotin 5’ end-labelled RNA fragment from OsDTH7 3’UTR region with m6A modification was synthetized and denatured to be immobilized onto streptavidin magnetic beads. The assay was carried out with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific). 80 picomole (pM) of biotin labeled-RNA and 5 mg of soluble protein were used for once. The eluted RNA-proteins complexes were analyzed by western blotting with GST antibody (Abmart). RNA m6A sequencing RNA m 6 A-seq was conducted following the established protocols. Total RNA was isolated and purified using Trizol reagent following the manufacturer's procedure with the panicles of 30-day-old WT plants. Poly (A) RNA was purified from 50μg total RNA, and was fragmented into small pieces using Magnesium RNA Fragmentation Module. The cleaved RNA fragments were incubated for 2h at 4℃ with m 6 A-specific antibody in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to cDNA by SuperScript™ II Reverse Transcriptase, which was next used to synthesis U-labeled second-stranded DNAs with E. coli DNA polymerase I, RNase H and dUTP Solution. An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and then the ligated products were amplified. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 platform. Formaldehyde-crosslinking and RNA Immunoprecipitation (FA-CLIP) To explore the region where OsYTH10 binds to the mRNA of flowering related genes, FA-CLIP was used with OE-OsYTH10 plants. FA-CLIP was performed with minor modifications based on previously described (Wei et al. 2018; Wu et al. 2020). First, 3g of rice leaves are ground into powder in liquid nitrogen and transferred to a pre-cooled lysis buffer (containing 150 mM KCl, 50 mM HEPES (PH7.5), 2 mM EDTA, 2% Nonidet P-40, 40 U/mL Ribolock RNase Inhibitor (Thermo), 1 × cocktail protease inhibitor (Roche)). After complete dissolution, 1% formaldehyde is added and incubated at 4°C for 10 min, then the crosslinking is terminated by 2 M glycine and further incubated for 40 min at 4°C. The sample is centrifuged, and the supernatant is collected after filtering. 2 μL/mL of DNaseⅠ (Promega) is added to digest genomic DNA for 5 min at 37℃, and 50 μL of sample was transferred out as input. Then, 7 μg Myc antibody is added and incubated with the sample, followed by the addition of protein G beads and overnight incubation. After that, the supernatant is removed, and the beads are washed with NT2 buffer (containing 300 mM KCl, 50 mM HEPES (PH7.5), 0.05% Nonidet P-40, 200 U/mL Ribolock RNase Inhibitor, 1 × cocktail protease inhibitor). 10 U/μL RNase T1 (NEB) is added to digest RNA to about 30 nucleotides in size. After washing with high salt buffer, the beads are treated with 400 μL 1 × T4 PNK buffer and 5 μL T4 PNK (NEB) for 1 h at 37℃. The pre-treated protease K solution (containing 400 μL 20 mg/mL protease K (Roche), 160 μL protease K buffer) by incubation at 37℃ for 20 min is then added for digestion at 37℃ for 20 min with a rate of 1000g per 3 min, and subsequently, Trizol and chloroform are used to extract the supernatant. Finally, the RNA from input and IP sample was used to send to the biological company for sequencing and analysis. The spatiotemporal expression of OsYTH10 To investigate the subcellular localization of OsYTH10, the full-length cDNA sequences of OsYTH10 was amplified and fused into the eGFP vector pD1301s-eGFP driven by the double 35S promoter. The recombinant constructs were transformed into rice protoplasts, which were prepared as described previously. After transformation, the protoplasts were incubated at 28℃ for 14-16 h, and the fluorescence signals were detected using a confocal microscope (Zeiss LSM 900). All primers used in this experiment are listed in supplemental table S1. Besides, 30-day-old WT plants were used for nuclear-cytoplasmic fractionation according to the previous description. As quality controls for the fractionation, tubulin protein was detected with tubulin antibody and used as cytoplasmic markers, and histone H3 was detected with histone H3 antibody and used as nuclear markers. Sequence alignment The amino acid sequences of OsYTH10 from Oryza sativa and AtECT2 from Arabidopsis thaliana were obtained from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html), and a multiple sequence alignment of YTH domains was performed using ClustalW with default parameter. Results OsYTH10 is an m6A reader dependent on W431 and W444 The m6A reader protein AtECT2 can specifically recognize and bind to mRNA 3’UTRs containing one of three types of m6A-modified motifs in Arabidopsis: UGUAA, RRACH (R=A/G, H=A/C/U), and URUAY (Y=C/U). It was presented that the two conserved tryptophan residues (W404 and W417) are crucial for AtECT8 to recognize and bind m6A modifications. By aligning the YTH domain sequences in OsYTH10 and AtECT2, it was revealed that the conserved W431 and W444 in OsYTH10 are likely important sites affecting its function as an m6A reader protein (Fig. 1a). To confirm the significance of these sites in OsYTH10 for recognizing and binding m6A modifications, we expressed and purified GST protein, GST-tagged OsYTH10 protein, and three non-binding variants (GST-OsYTH10 m1 , GST-OsYTH10 m2 , and GST-OsYTH10 m3 ) with corresponding mutations (W431A, W444A, and W431A/W444A) (Fig. S1). Additionally, FAM 5’-end-labeled RNA probes with or without m6A modification were synthesized to assess the m6A-binding ability of YTH10 and its mutated forms using EMSA assays. The EMSA results confirmed the direct binding of OsYTH10 to m6A-modified RNA probes (Fig. 1b). Importantly, none of the three mutated OsYTH10 proteins were able to bind to the m6A-modified probe in vitro (Fig. 1c). Furthermore, 5’-end biotin-labeled RNA fragments, identical to the probe sequence used in EMSA assays, were designed and synthesized to further validate the recognition and binding of OsYTH10 to the m6A site through RNA-protein pull-down assays in vitro. GST-OsYTH10 specifically bound to the RNA fragments, while GST-OsYTH10ms and GST proteins did not (Fig. 1d), indicating that OsYTH10 binding to m6A-modified RNA is dependent on both of indispensable W431 and W444. OsYTH10 negatively regulates heading date in rice In the initial study, we used m6A-seq to identify differentially expressed genes in response to cadmium stress in rice, with YTH10 emerging as a key gene in the m6A-mediated regulatory network under this stress (Cheng et al. 2021). To investigate the function of OsYTH10 in rice, we generated two OsYTH10 mutants ( osyth10 - 1 and osyth10 - 2 ) using the CRISPR/Cas9 system, resulting in premature termination of OsYTH10 translation (Fig. 2a). Additionally, we developed OsYTH10 overexpressing plants (OE- OsYTH10 -1 and OE- OsYTH10 -2) by expressing 4 x Myc-tagged OsYTH10 under the 35S promoter. Subsequent qRT-PCR analysis confirmed significant upregulation of OsYTH10 expression in the overexpressing plants compared to wild-type (WT) seedlings (Fig. 2b). Western blot analysis with MYC antibody further validated the expression of OsYTH10-4 x Myc in the overexpressing plants (Fig. 2c). Surprisingly, phenotypic analysis did not reveal significant differences between OsYTH10 mutants and WT seedlings under cadmium stress (Fig. S2), indicating OsYTH10 mutants are insensitive to cadmium. However, both osyth10 - 1 and osyth10 - 2 knockout mutants exhibited similar early heading phenotypes compared to WT plants under natural subtropical climatic conditions with short-day photoperiods in Nanchang, Jiangxi province (Fig. 2d). The osyth10 - 1 and osyth10 - 2 mutants initiated heading at 68 and 69 days after germination (DAG), respectively, representing 6-day and 5-day advancements compared to WT plants (Fig. 2e). Conversely, OsYTH10 overexpressing lines (OE- OsYTH10 -1 and OE- OsYTH10 -2) displayed delayed heading times of 83 and 82 DAG, corresponding to 9-day and 8-day delays relative to WT plants (Fig. 2d and e). These results demonstrate that OsYTH10 functions as a negative regulator of heading date in rice, with its overexpression significantly postponing the heading transition. Furthermore, m6A dot blot analysis revealed the lowest m6A levels in the mutant and the highest levels in YTH10 overexpressing plants, suggesting that YTH10 positively regulates m6A levels in rice (Fig. S3). m6A cumulative percent analysis also showed that m6A modified RNA was enriched in OE- OsYTH10 -1 using RIP with anti-IgG and anti-Myc (Fig. S4). Expression pattern and subcellular relocation of OsYTH10 To investigate the new function of OsYTH10 , we initially examined its spatial and temporal transcription expression patterns in various tissues and at different growth stages in rice using RT-qPCR assays. The expression analysis of OsYTH10 revealed relatively high expression levels in the root, stem, leaf, sheath, and panicles (Fig. 3a). The subcellular localization of OsYTH10 in rice protoplasts was also investigated. The results showed GFP fluorescent signals in the cytoplasm and nucleus, overlapping with the mCherry fluorescent signals of the nuclear marker protein NLS-mCherry (Fig. 3b). Furthermore, nuclear-cytoplasmic fractionation assays confirmed the distribution of OsYTH10 protein in both the cytoplasm and nucleus, as shown by western blot analysis (Fig. S5). These results provide insights into the subcellular localization of OsYTH10. OsYTH10 regulates the expression of OsDTH7 and OsGI To investigate the regulatory mechanism of OsYTH10 on the rice heading stage, we initiated m6A-seq analysis using 30-day-old WT plants. The results revealed that m6A modified peaks were predominantly concentrated in the 3'UTR region (Fig. 4a), with the UGUAA motif showing the highest enrichment in this region (Fig 4b and c). Subsequent analysis of differentially expressed genes in the m6A-seq data identified 6 genes associated with late flowering, namely OsDTH7 , OsCRCT , OsGI , OsRE1 , OsHBF1 , and OsHBF2 , based on the phenotypic observations of OsYTH10 mutants and overexpressing plants. m6A-IP-qPCR results confirmed the presence of m6A modifications in these genes (Fig 4d). OsYTH10 physically binds to the m6A modification of 3’UTR in OsDTH7 and OsGI mRNA To identify the regulatory target genes of OsYTH10 in vivo , we utilized OE- OsYTH10 -2 plants to perform RIP qPCR assays aimed at identifying candidate target genes that interact with OsYTH10 in vivo . The results indicated that only OsDTH7 and OsGI were significantly enriched in the IP sample with the Myc antibody, suggesting a direct interaction between OsYTH10 and their 3'UTR mRNA (Fig. 5a). In vitro , FAM 5’ end-labeled RNA probes with or without m6A modification based on the 3’UTR sequences of OsDTH7 and OsGI were designed for ESMA assays to validate the RIP-qPCR results. The EMSA results showed OsYTH10's ability to bind to the m6A-modified 3'UTR in OsDTH7 and OsGI (Fig. 5b-I). Furthermore, to confirm whether OsYTH10 recognizes and binds to OsDTH7 mRNA in vivo , FA-CLIP assay was conducted using the leaves of 30-day-old OE- OsYTH10 -2 plants and anti-Myc magnetic beads. Data analysis revealed high enrichment of OsDTH7 and OsGI mRNA in the immunoprecipitation compared to the input, indicating binding of OsYTH10 to OsDTH7 and OsGI mRNA. No enrichment was observed in the mRNA level of a non-related control gene. Additionally, FA-CLIP-seq analysis unveiled that OsYTH10's binding sites on OsDTH7 and OsGI transcripts contain m6A modifications, as analyzed using integrative genomics viewer (IGV) on the sequenced data (Fig. 6a), consistent with the previous m6A-seq result. OsYTH10 inhibits decay of OsDTH7 and OsGI mRNA We performed mRNA lifetime assays on OsDTH7 and OsGI in vivo in both WT and OsYTH10 mutants treated with actinomycin D to examine the impact of mRNA m6A readers on the stability or degradation of Arabidopsis mRNA. The findings revealed that the mRNA degradation rates of OsDTH7 and OsGI were increased in osyth10-1 and osyth10-2 mutants compared to WT (Fig. 6b and c), suggesting that OsYTH10 mutation accelerates the decay of OsDTH7 and OsGI mRNA. Discussion m6A is the most abundant chemical modification in eukaryotic organisms and plays a crucial role in regulating plant and animal cells. Proteins known as m6A readers are essential for mediating the functions of m6A. While the m6A reader in Arabidopsis has been extensively studied, its role in rice remains largely unexplored. Our study reveals that OsYTH10 in rice regulates flowering by recognizing m6A in the 3'UTR region of mRNA, thereby maintaining the stability of mRNA for key flowering-related genes like OsGI , OsDTH7 , and OsCRCT (Fig. 2). Meanwhile, We utilized FA-CLIP to investigate OsYTH10. The FA-CLIP-seq analysis of the rice transcriptome revealed that OsYTH10 predominantly binds to the 3'UTR region of mRNA (Fig. 6). The primary binding motif of OsYTH10 was identified as UGUA, consistent with the AtECT2 target motif in Arabidopsis. Furthermore, our m6A-seq results indicated that m6A modifications in rice were predominantly located in the mRNA 3'UTR region with the major motif being UGUA (Fig. 6). These findings suggest that OsYTH10 may recognize m6A-modified UGUA motifs in the mRNA 3'UTR region to perform its reading function. To confirm this hypothesis, we designed two RNA probes, UGUAA and UGU(m6A)A, and conducted EMSA experiments with GST-OsYTH10, demonstrating that OsYTH10 could bind to these motifs (Fig. 1b and c). Additionally, Biotin-RNA Pulldown experiments showed that UGU(m6A)A successfully pulled down OsYTH10, while UGUAA did not (Fig. 1d), indicating that OsYTH10 binds to UGUA motifs by recognizing m6A modifications. Previous studies have shown that editing OsYTH10 in Dongjin led to changes in rice plant height. In this study, we observed that knockdown of OsYTH10 in NIP caused rice to flower earlier under long-day conditions, while overexpression of OsYTH10 resulted in late flowering, indicating a crucial role of OsYTH10 in regulating rice flowering. The function of m6A modification primarily relies on its reader proteins. Research on human YTH family proteins has demonstrated their involvement in pre-mRNA splicing, nuclear export, mRNA degradation, and mRNA translation efficiency (Hsu et al. 2017; Roundtree and He 2016; Shi et al. 2017; Wang et al. 2014). In Arabidopsis, AtECT2 is known to maintain mRNA stability (Wei et al. 2018), while in rice, YTH07 binds to mRNAs modified by m6A and reduces their translation efficiency (Cui et al. 2024). Flowering time in rice is controlled by numerous genes, so we focused on the genes associated with rice flowering time among the target genes of OsYTH10. We analyzed the mRNA abundance of these genes in osyth10-1 versus WT and found that the abundance of these genes in osyth10-1 was lower than in WT. Given the high homology between OsYTH10 and AtECT2, we inferred that OsYTH10 has the capability of maintaining mRNA stability. Treating rice seedlings with actinomycin D showed that OsDTH7 and OsGI degraded more rapidly in the osyth10-1 strain compared to WT (Fig. 6b and c). RIP-qPCR confirmed that OsYTH10 can bind to the mRNAs of OsDTH7 and OsGI . These findings suggest that OsYTH10 maintains mRNA stability of OsDTH7 and OsGI by interacting with their transcripts, and that deletion of OsYTH10 accelerates the degradation of OsDTH7 and OsGI , resulting in an early flowering phenotype. Conclusions This study investigates the functional role of OsYTH10, an m6A-binding protein, in regulating rice flowering under prolonged light exposure. Genetic analyses demonstrate that OsYTH10 serves as a key modulator of flowering time by stabilizing transcripts of OsDTH7 and OsGI . Given that precise control of flowering timing directly impacts yield potential, OsYTH10 emerges as a promising molecular target for breeding rice varieties adapted to regions with extended daylight cycles, offering a strategic avenue to improve grain production. Declarations Funding This study was supported by Biological Breeding-National Science and Technology Maior Project (20302022ZD040010202), the National Natural Science Foundation of China (32160485), and the Jiangxi Province Double Thousand Plan by Science and Technology Innovation High-Talent Project (JXSQ2023201057). Competing interests The authors declare no competing interests. Author Contributions Jianmin Bian and Haohua He conceived the project and designed the experiments. Jun Yang, Zhihao Chen and Peng Wang executed the experiments. Jun Yang, Mvuyeni Nyasulu, Qin Cheng, Xiaopeng He, Jie Xu, Junru Fu, Dahu Zhou, Linjuan Ouyang and Jianmin Bian analyzed the data. Jun Yang, Mvuyeni Nyasulu, Peng Wang and Jianmin Bian wrote the manuscript. Data availability All data supporting the conclusions of this article are provided within the article (and its additional files), and the raw sequence data for this article are deposited in the NCBI (BioProject accession no. PRJNA1247134). Data generated in this study are available from the corresponding author upon reasonable request. References Arribas-Hernandez L, Rennie S, Schon M, Porcelli C, Enugutti B, Andersson R, Nodine MD, Brodersen P (2021) The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation. Elife 10. Arribas-Hernandez L, Simonini S, Hansen MH, Paredes EB, Bressendorff S, Dong Y, Ostergaard L, Brodersen P (2020) Recurrent requirement for the m(6)A-ECT2/ECT3/ECT4 axis in the control of cell proliferation during plant organogenesis. Development 147. Bhat SS, Bielewicz D, Jarmolowski A, Szweykowska-Kulinska Z (2018) N(6)-methyladenosine (m(6)A): Revisiting the Old with Focus on New, an Arabidopsis thaliana Centered Review. Genes (Basel) 9. Cai L, Cui S, Jin T, Huang X, Hou H, Hao B, Xu Z, Cai L, Hu Y, Yang X, Zhou L, Yu T, Tian Y, Liu X, Chen L, Liu S, Jiang L, Zhou S, Wan J (2023) The N(6)-methyladenosine binding proteins YTH03/05/10 coordinately regulate rice plant height. Plant Sci 329:111546. Chen Y, Wan R, Zou Z, Lao L, Shao G, Zheng Y, Tang L, Yuan Y, Ge Y, He C, Lin S (2023) O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins. Nat Cell Biol 25:1676-1690. Cheng Q, Wang P, Wu G, Wang Y, Tan J, Li C, Zhang X, Liu S, Huang S, Huang T, Yang M, He H, Bian J (2021) Coordination of m(6)A mRNA methylation and gene transcriptome in rice response to cadmium stress. Rice (N Y) 14:62. Cui S, Song P, Wang C, Chen S, Hao B, Xu Z, Cai L, Chen X, Zhu S, Gan X, Dong H, Hu Y, Zhou L, Hou H, Tian Y, Liu X, Chen L, Liu S, Jiang L, Wang H, Jia G, Zhou S, Wan J (2024) The RNA binding protein EHD6 recruits the m(6)A reader YTH07 and sequesters OsCOL4 mRNA into phase-separated ribonucleoprotein condensates to promote rice flowering. Mol Plant 17(6):935-954. Duan HC, Wei LH, Zhang C, Wang Y, Chen L, Lu Z, Chen PR, He C, Jia G (2017) ALKBH10B Is an RNA N(6)-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition. Plant Cell 29:2995-3011. Hsu PJ, Zhu Y, Ma H, Guo Y, Shi X, Liu Y, Qi M, Lu Z, Shi H, Wang J, Cheng Y, Luo G, Dai Q, Liu M, Guo X, Sha J, Shen B, He C (2017) Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis. Cell Res 27:1115-1127. Lee KP, Liu K, Kim EY, Medina-Puche L, Dong H, Di M, Singh RM, Li M, Qi S, Meng Z, Cho J, Zhang H, Lozano-Duran R, Kim C (2024) The m6A reader ECT1 drives mRNA sequestration to dampen salicylic acid-dependent stress responses in Arabidopsis. Plant Cell 36:746-763. Li F, Zhao D, Wu J, Shi Y (2014) Structure of the YTH domain of human YTHDF2 in complex with an m(6)A mononucleotide reveals an aromatic cage for m(6)A recognition. Cell Res 24:1490-1492. Liang D, Lin WJ, Ren M, Qiu J, Yang C, Wang X, Li N, Zeng T, Sun K, You L, Yan L, Wang W (2022) m(6)A reader YTHDC1 modulates autophagy by targeting SQSTM1 in diabetic skin. Autophagy 18:1318-1337. Liang Z, Riaz A, Chachar S, Ding Y, Du H, Gu X (2020) Epigenetic Modifications of mRNA and DNA in Plants. Mol Plant 13:14-30. Lim WA, Pawson T (2010) Phosphotyrosine signaling: evolving a new cellular communication system. Cell 142:661-667. Ma L, Chen T, Zhang X, Miao Y, Tian X, Yu K, Xu X, Niu Y, Guo S, Zhang C, Qiu S, Qiao Y, Fang W, Du L, Yu Y, Wang J (2021) The m(6)A reader YTHDC2 inhibits lung adenocarcinoma tumorigenesis by suppressing SLC7A11-dependent antioxidant function. Redox Biol 38:101801. Roundtree IA, He C (2016) RNA epigenetics--chemical messages for posttranscriptional gene regulation. Curr Opin Chem Biol 30:46-51. Roundtree IA, Luo GZ, Zhang Z, Wang X, Zhou T, Cui Y, Sha J, Huang X, Guerrero L, Xie P, He E, Shen B, He C (2017) YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs. Elife 6:e31311. Shen L, Liang Z, Wong CE, Yu H (2019) Messenger RNA Modifications in Plants. Trends Plant Sci 24:328-341. Shi H, Wang X, Lu Z, Zhao BS, Ma H, Hsu PJ, Liu C, He C (2017) YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA. Cell Res 27:315-328. Song P, Wei L, Chen Z, Cai Z, Lu Q, Wang C, Tian E, Jia G (2023) m(6)A readers ECT2/ECT3/ECT4 enhance mRNA stability through direct recruitment of the poly(A) binding proteins in Arabidopsis. Genome biology 24:103. Tang J, Chen S, Jia G (2023) Detection, regulation, and functions of RNA N(6)-methyladenosine modification in plants. Plant Commun 4:100546. Tang J, Yang J, Lu Q, Tang Q, Chen S, Jia G (2022) The RNA N(6) -methyladenosine demethylase ALKBH9B modulates ABA responses in Arabidopsis. J Integr Plant Biol 64:2361-2373. Wang H, Niu R, Zhou Y, Tang Z, Xu G, Zhou G (2023) ECT9 condensates with ECT1 and regulates plant immunity. Front Plant Sci 14:1140840. Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505:117-120. Wang Y, Zhao JC (2016) Update: Mechanisms Underlying N(6)-Methyladenosine Modification of Eukaryotic mRNA. Trends Genet 32:763-773. Wei LH, Song P, Wang Y, Lu Z, Tang Q, Yu Q, Xiao Y, Zhang X, Duan HC, Jia G (2018) The m(6)A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis. Plant Cell 30:968-985. Wu J, Peled-Zehavi H, Galili G (2020) The m(6) A reader ECT2 post-transcriptionally regulates proteasome activity in Arabidopsis. New Phytol 228:151-162. Wu M, Nie F, Liu H, Zhang T, Li M, Song X, Chen W (2022) The evolution of N(6)-methyladenosine regulators in plants. Methods 203:268-275. Xu C, Liu K, Ahmed H, Loppnau P, Schapira M, Min J (2015) Structural Basis for the Discriminative Recognition of N6-Methyladenosine RNA by the Human YT521-B Homology Domain Family of Proteins. J Biol Chem 290:24902-24913. Yang H, Chiang C, Luo Q, Chen C, Huang J, Zhu L, Zheng D (2022) YT521-B homology domain family proteins as N6-methyladenosine readers in tumors. Front Genet 13:934223. Yang J, He R, Qu Z, Gu J, Jiang L, Zhan X, Gao Y, Adelson DL, Li S, Wang ZY, Zhu Y, Wang D (2023) Long noncoding RNA ARTA controls ABA response through MYB7 nuclear trafficking in Arabidopsis. Dev Cell 58:1206-1217 e1204. Yuan S, Xi S, Weng H, Guo MM, Zhang JH, Yu ZP, Zhang H, Yu Z, Xing Z, Liu MY, Ming DJ, Sah RK, Zhou Y, Li G, Zeng T, Hong X, Li Y, Zeng XT, Hu H (2023) YTHDC1 as a tumor progression suppressor through modulating FSP1-dependent ferroptosis suppression in lung cancer. Cell Death Differ 30:2477-2490. Zaccara S, Jaffrey SR (2020) A Unified Model for the Function of YTHDF Proteins in Regulating m(6)A-Modified mRNA. Cell 181:1582-1595 e1518. Zhou B, Liu C, Xu L, Yuan Y, Zhao J, Zhao W, Chen Y, Qiu J, Meng M, Zheng Y, Wang D, Gao X, Li X, Zhao Q, Wei X, Wu D, Zhang H, Hu C, Zhuo X, Zheng M, Wang H, Lu Y, Ma X (2021) N(6) -Methyladenosine Reader Protein YT521-B Homology Domain-Containing 2 Suppresses Liver Steatosis by Regulation of mRNA Stability of Lipogenic Genes. Hepatology 73:91-103. Supplementary Files SupplementaryMaterial.docx Supplementary Information (SI) Fig. S1. Induction of GST-OsYTH10 and its mutants. Fig. S2. Phenotypic analysis of OsYTH10 mutants under Cadmium (Cd) stress. Fig. S3. Dot blot analysis of m6A levels of mRNA extracted from WT, osyth10 - 1 , osyth10 - 2 , OE- OsYTH10 -1, and OE- OsYTH10 -2 seedlings. Fig. S4. In vivo IP showed that m6A modifications were enriched in OsYTH10-4*Myc-bound RNA compared to IgG-bound RNA. Fig. S5. Immunoblot analyses showing the nuclear and cytoplasmic distributions of OsYTH10-4 × Myc protein in OE-OsYTH10-1 plants. Table S1. Primers used in this study. Cite Share Download PDF Status: Published Journal Publication published 24 Sep, 2025 Read the published version in Theoretical and Applied Genetics → Version 1 posted Editorial decision: Major revisions 04 Jun, 2025 Reviewers agreed at journal 08 May, 2025 Reviewers invited by journal 08 May, 2025 Editor assigned by journal 05 May, 2025 First submitted to journal 05 May, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6593659","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":453708471,"identity":"5af85aed-16df-4f13-a6ec-f38ebf57cee2","order_by":0,"name":"Jun Yang","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Yang","suffix":""},{"id":453708472,"identity":"47abd9f7-09ab-4a1a-865d-8c9146914df3","order_by":1,"name":"Zhihao Chen","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Zhihao","middleName":"","lastName":"Chen","suffix":""},{"id":453708473,"identity":"6f867548-8ac3-4346-bf92-37e2d9ce5a98","order_by":2,"name":"Peng Wang","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Wang","suffix":""},{"id":453708474,"identity":"8a9a5d79-5cfb-49f2-a140-2fa9b4c3a255","order_by":3,"name":"Mvuyeni Nyasulu","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Mvuyeni","middleName":"","lastName":"Nyasulu","suffix":""},{"id":453708475,"identity":"d1c79c01-c0b1-4bea-9340-3789d67ac3b4","order_by":4,"name":"Qin Chen","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qin","middleName":"","lastName":"Chen","suffix":""},{"id":453708476,"identity":"0c73e8f3-4ac2-44b4-995c-a0721199d02c","order_by":5,"name":"Xiaopeng He","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiaopeng","middleName":"","lastName":"He","suffix":""},{"id":453708477,"identity":"b4e2d467-5a02-4e65-a55b-22d7f4b32fe6","order_by":6,"name":"Jie Xu","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Xu","suffix":""},{"id":453708478,"identity":"e16353f5-c1a5-4033-b50c-407dff058785","order_by":7,"name":"Junru Fu","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Junru","middleName":"","lastName":"Fu","suffix":""},{"id":453708479,"identity":"7a900e3b-8318-4682-ac09-500736ac7a82","order_by":8,"name":"Dahu Zhou","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dahu","middleName":"","lastName":"Zhou","suffix":""},{"id":453708480,"identity":"5d218a01-89f2-4895-a82b-8ad5267fb3b2","order_by":9,"name":"Linjuan Ouyang","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Linjuan","middleName":"","lastName":"Ouyang","suffix":""},{"id":453708481,"identity":"79dff427-c8f2-4739-8aa7-07b883f509f8","order_by":10,"name":"Haohua He","email":"","orcid":"","institution":"Jiangxi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Haohua","middleName":"","lastName":"He","suffix":""},{"id":453708482,"identity":"d64c3dac-925d-4963-b859-a8673ebc9cd5","order_by":11,"name":"Bian Jianmin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYBACxgYehgMMDDY8/MzMBx+QoiVNRrKdLdmASHt4QMRhG4PzPGYCRGlgnnb24IGfO9J4jA8zmDEw1NhEE3bY7LyEg71nbHjMDjOkPWA4lpbbQFhLjsEB3rY0kJbjBowNh4nTcvBv22Ee42bGNgmitRzmBWoxYGZmI1ZLXsJhWaDDJA6zMRskEOMXw9m5hz++bbOx5+8///HBhxobIrSgqEggpBwE5IlRNApGwSgYBSMcAADjDj+bLoWSHgAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3703-379X","institution":"Jiangxi Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Bian","middleName":"","lastName":"Jianmin","suffix":""}],"badges":[],"createdAt":"2025-05-05 10:41:01","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6593659/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6593659/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00122-025-05041-4","type":"published","date":"2025-09-24T15:57:11+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82583041,"identity":"12bbd143-a04b-4d81-a4e2-d83e5b4afb41","added_by":"auto","created_at":"2025-05-13 06:48:04","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":252474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDetection of the OsYTH10’s ability to bind m6A modifications.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a). Alignment of YTH domains from OsYTH10 and AtECT2. The vital sites W431 and W444 are marked with ‘*’. (b). EMSA of GST-OsYTH10 binding with RNA probe containing m6A-modified UGUAA motif but not with unmethylated probe. UGU(m6A)A RNA probe: 5′-UGGCCGUUCAUCUAAAAUGU(m6A)AGCUUUUUUGGCUUU*G*U-3′, competitor probe: 5′-UGGCCGUUCAUCUAAAAUGUAAGCUUUUUUGGCUUU*G*U-3′, * Indicates thiol-protected bases used in the experiment. (c). EMSA assay showing abolished binding affinity of GST-OsYTH10 (containing GST-OsYTH10\u003csup\u003em1\u003c/sup\u003e, GST-OsYTH10\u003csup\u003em2\u003c/sup\u003e, and GST-OsYTH10\u003csup\u003em3\u003c/sup\u003e) binding the UGU(m6A)A RNA probe. (d). Detection of GST-OsYTH10 binding RNA probe containing m6A-modified UGUAA motif but not with unmethylated probe in vitro by RNA pull-down assay. GST protein was used as a negative control.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/e0b90f9c15aa0ceddbc3fe25.png"},{"id":82584763,"identity":"92fe9e95-ccbd-4f79-a237-2ffe53c6f6b1","added_by":"auto","created_at":"2025-05-13 06:56:04","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":318965,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic identification of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsYTH10\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant and overexpressing plants.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a). Schematic representation of the \u003cem\u003eOsYTH10\u003c/em\u003e gene structure in mutants (\u003cem\u003eosyth10-1 \u003c/em\u003eand \u003cem\u003eosyth10-2\u003c/em\u003e) with the corresponding nucleotide sequences at the mutation sites compared with that in WT. UTRs, exons and introns are indicated by blank rectangles, orange rectangles and black lines, respectively. (b) and (c). Detection of the transcript levels (b) and protein levels (c) of \u003cem\u003eOsYTH10 \u003c/em\u003ein WT, OE-\u003cem\u003eOsYTH10\u003c/em\u003e-1 and OE-\u003cem\u003eOsYTH10\u003c/em\u003e-2 plants. OsYTH10-4×Myc fusion protein was detected as a band by the anti-Myc antibody in \u003cem\u003eOsYTH10\u003c/em\u003e over-expressing transgenic plants, and \u003cem\u003eOsActin1\u003c/em\u003e was shown as a loading control. Error bars represent SD from three bio logical replicates, and significant differences in expression fold change are indicated by asterisks (**p \u0026lt; 0.01). (d). \u003cem\u003eOsYTH10\u003c/em\u003emutation promote flowering in rice. Representative 75-day-old plants of mutants compared to wild-type (WT) grown under long-day (LD) photoperiod conditions. Bar, 30cm. (e). Heading date measurements of WT, \u003cem\u003eosyth10-1\u003c/em\u003e, \u003cem\u003eosyth10-2\u003c/em\u003e, OE-\u003cem\u003eOsYTH10\u003c/em\u003e-1, and OE-\u003cem\u003eOsYTH10\u003c/em\u003e-2 plants. Values are shown as means ± standard deviation from \u003cem\u003en\u003c/em\u003e =30 individual plants (**p \u0026lt; 0.01).\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/81b84573936f1b64c8aaf595.png"},{"id":82583042,"identity":"46af359b-8554-47b6-9ba4-05a4882e63e2","added_by":"auto","created_at":"2025-05-13 06:48:04","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":103400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpatiotemporal expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsYTH10\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a). Quantitative analysis of \u003cem\u003eOsYTH10\u003c/em\u003eexpression in root, culm, leaf, sheath and panicle of WT. The \u003cem\u003eOsActin1\u003c/em\u003egene was used as a loading control. Values are means ± standard deviation from n=3 independent biological replicates. (b). Subcellular localization of OsYTH10-eGFP fusion protein in rice protoplast. Bar, 20 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/e3318cc3c42793ab3dd80950.png"},{"id":82585136,"identity":"92522f19-a193-411f-9d28-2a9fcd07a29f","added_by":"auto","created_at":"2025-05-13 07:04:04","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":137217,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsYTH10 binds to mRNA 3’UTR regions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a). Metagene profile illustrating the region distribution of OsYTH10- and m6A-binding sites across the indicated mRNA segments in rice. 5′ UTR, 5′ untranslated region; CDS, coding sequence; 3′ UTR, 3′ untranslated region. (b). Distribution of m6A peaks in rice genome. (c). Motif identified by HOMER software based on the OsYTH10- and m6A-binding site. (d). qRT-PCR analysis showing that \u003cem\u003eOsYTH10\u003c/em\u003eknockout decreases the transcript levels of \u003cem\u003eOsDTH7, OsOsGI, OsCRCT, OsRE1, OsHBF1\u003c/em\u003e and \u003cem\u003eOsHBF2\u003c/em\u003e. (e). m6A-IP was validated with WT, the transcript levels of \u003cem\u003eOsDTH7, OsOsGI, OsCRCT, OsRE1, OsHBF1\u003c/em\u003e and \u003cem\u003eOsHBF2\u003c/em\u003e in \u003cem\u003evivo\u003c/em\u003ein the immunoprecipitates were determined by qRT-PCR. Data are represented as means ±SE, \u003cem\u003en\u003c/em\u003e = 2 biological replicates 3 technical replicates.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/7d32b5d3ab2a1e5c07d48009.png"},{"id":82583045,"identity":"519a45c6-e965-4ce0-881e-adf094fa765b","added_by":"auto","created_at":"2025-05-13 06:48:04","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":311400,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of OsYTH10 binding to target gene mRNA in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003evivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a). RIP assay with OE-\u003cem\u003eOsYTH10\u003c/em\u003e-1 transgenic (35S: OsYTH10-4×Myc) plants was validated, and the levels of \u003cem\u003eOsDTH7, OsGI, OsCRCT, OsOsRE1, OsHBF1\u003c/em\u003e, and \u003cem\u003eOsHBF2\u003c/em\u003e in \u003cem\u003evivo\u003c/em\u003e in the immunoprecipitates were determined by qRT-PCR. IgG was used as the negative control. Error bars represent SD from three biological replicates, and asterisks represent significance differences determined by Student’s t test (**p \u0026lt; 0.01). (b)-(i). Verify the binding of GST-OsYTH10, OsYTH10\u003csup\u003em1\u003c/sup\u003e, OsYTH10\u003csup\u003em2\u003c/sup\u003e, OsYTH10\u003csup\u003em3\u003c/sup\u003e to DTH7 and OsGI probes with and without m6A-modified UGUAA motif using EMSA assays. Each lane was loaded with varying concentrations (100 x, 200 x, 500 x) of competitor probe and a consistent amount of protein.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/e916a9bafbb901d6c254f7c3.png"},{"id":82583044,"identity":"51ae49c8-28cf-4308-9d67-bc0635b23aa2","added_by":"auto","created_at":"2025-05-13 06:48:04","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":151345,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOsYTH10 enhances the mRNA stability of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsDTH7\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eOsGI\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a). Integrative genomics viewer (IGV) showing the m6A-seq and FA-CLIP sequencing results on \u003cem\u003eOsDTH7\u003c/em\u003eand \u003cem\u003eOsGI\u003c/em\u003e transcripts. FA-CLIP, formaldehyde crosslinking and immunoprecipitation. (b) and (c). The RNA half-lives of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003etranscripts in 14-day-old WT, \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e, and \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e2\u003c/em\u003eseedlings treated for different time with 200 µM actinomycin. (d). 18S was used as internal control. Data are presented as means ± SE, n=2 independent experiments, each with 3 technical replicates.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/fafe0c56510f83b26c6619d4.png"},{"id":92430631,"identity":"4f58167a-a00e-4ca1-866e-a6a6c296ca38","added_by":"auto","created_at":"2025-09-29 16:07:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2046844,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/49986db7-9a55-4f43-8dbc-371b37883a4e.pdf"},{"id":82583048,"identity":"695b589e-d565-41eb-858b-06abe3c4a8de","added_by":"auto","created_at":"2025-05-13 06:48:04","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1368979,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Information (SI)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S1.\u003c/strong\u003e Induction of GST-OsYTH10 and its mutants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S2.\u003c/strong\u003e Phenotypic analysis of\u003cem\u003e OsYTH10\u003c/em\u003emutants under Cadmium (Cd) stress.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S3. \u003c/strong\u003eDot blot analysis of m6A levels of mRNA extracted from WT, \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e, \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e2\u003c/em\u003e, OE-\u003cem\u003eOsYTH10\u003c/em\u003e-1, and OE-\u003cem\u003eOsYTH10\u003c/em\u003e-2 seedlings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S4. \u003c/strong\u003eIn vivo IP showed that m6A modifications were enriched in OsYTH10-4*Myc-bound RNA compared to IgG-bound RNA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. S5. \u003c/strong\u003eImmunoblot analyses showing the nuclear and cytoplasmic distributions of OsYTH10-4 × Myc protein in OE-OsYTH10-1 plants.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1.\u003c/strong\u003e Primers used in this study.\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-6593659/v1/3ebe9b2e4e47a051aa675678.docx"}],"financialInterests":"","formattedTitle":"The Critical Role of OsYTH10 in Promoting Early Flowering of Rice Under Long Sunlight","fulltext":[{"header":"Key message","content":"\u003cp\u003eA crucial RNA N6 -methyladenosine (m6A) reader protein OsYTH10 in rice was identified to physically binds mRNA\u0026apos;s 3\u0026apos;UTR via its YTH domain, stabilizing \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e transcripts to accelerate flowering under long-day conditions.\u0026nbsp;\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eIn recent years, epigenetic modifications have garnered significant attention for their crucial roles in plant physiology, with notable advancements in research on histone methylation, histone acetylation, and ubiquitination. However, there is a notable gap in the study of RNA epigenetic modifications. RNA methylation, particularly N6-methyladenosine (m6A), plays a vital post-transcriptional role in regulating gene expression in eukaryotes. This dynamic and reversible modification is orchestrated by a complex network of methyltransferases, de-methyltransferases, and m6A binding proteins (Liang et al. 2020; Shen et al. 2019; Tang et al. 2023; Wang and Zhao 2016; Wu et al. 2022). Methyltransferases serve as writers, de-methyltransferases as erasers, and m6A binding proteins as readers, collectively influencing various biological processes in response to environmental and internal cues (Lim and Pawson 2010). The dynamic reversibility of m6A modification allows eukaryotes to swiftly adapt to changing conditions, contributing to the regulation of diverse cellular processes.\u003c/p\u003e\n\u003cp\u003eIn animals, the RNA m6A methylation mystery has been well-characterized, and its biological role and applications have become increasingly clear. It has been definitively established that YT521-B homology (YTH) domain-containing reader proteins mediate mRNA recognition for precise regulation of RNA metabolism by m6A in mammalian cells (Xu et al. 2015; Yang et al. 2022; Zhou et al. 2021). The YT521-B homology (YTH) domain is a highly conserved structural domain of YTH family proteins in various species, containing a hydrophobic pocket critical for m6A recognition in the cytoplasm (Li et al. 2014).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn mammals, there are two branches of YTH family proteins, YTHDFs and YTHDCs. For instance, YTHDF1 promotes RNA translation, YTHDF2 facilitates RNA decay, and YTHDF3 exhibits a dual function depending on its binding partner (Shi et al. 2017). These functionally distinct YTHDF family proteins (YTHDF1, YTHDF2, YTHDF3) can bind the same m6A-modified mRNAs through their YTH domains, redundantly mediating mRNA degradation and cellular differentiation (Zaccara and Jaffrey 2020).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIt has been observed that YTHDF1 and YTHDF3, but not YTHDF2, carry high levels of nutrient-sensing O-GlcNAc modifications, which attenuate the translation-promoting function of YTHDF1 and YTHDF3 by blocking their interactions with proteins associated with mRNA translation. This leads to the assembly, stability, and disassembly of stress granules, enabling better recovery from stress (Chen et al. 2023).\u003c/p\u003e\n\u003cp\u003eIn the YTHDC family, YTHDC1 mediates the export of methylated mRNA from the nucleus to the cytoplasm in Hela cells and is involved in m6A epigenetic regulation of FSP1, alleviating FSP1-dependent ferroptosis suppression. It also modulates autophagy by regulating the stability of SQSTM1 nuclear mRNA in diabetic keratinocytes (Liang et al. 2022; Roundtree et al. 2017; Yuan et al. 2023). YTHDC2 enhances the translation efficiency of its targets and decreases their mRNA abundance by selectively binding m6A at its consensus motif (Hsu et al. 2017). YTHDC2 is involved in rescuing lung tumorigenesis by suppressing cystine uptake and blocking the downstream antioxidant program (Ma et al. 2021).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCoincidentally, research on m6A readers in plants is limited, and the currently studied reader proteins all belong to the YTH family, especially in Arabidopsis and rice. In Arabidopsis, the YTH family consists of 13 proteins containing highly conserved YTH domains at the C-terminus, referred to as the Evolutionarily Conserved C-Terminal Region (ECT) family (Bhat et al. 2018). AtECT1 was reported to sequester SA-induced m\u003csup\u003e6\u003c/sup\u003eA modification-prone mRNAs through its conserved aromatic cage to facilitate their decay in cytosolic condensates, thereby dampening SA-mediated stress responses (Lee et al. 2024). AtECT9 was involved in regulating plant immunity by interacting with AtECT1 (Wang et al. 2023). AtECT2, AtECT3, and AtECT4 show genetic redundancy in multiple biological processes, including plant developmental timing, morphogenesis, and organogenesis, suggesting that they cooperatively stabilize the bound m6A-modified mRNA to affect target gene expression (Arribas-Hernandez et al. 2021; Arribas-Hernandez et al. 2020; Song et al. 2023). In rice, there are 12 YTH proteins. It was shown that OsYTH03, OsYTH05, and OsYTH10 impact on the diterpenoid and brassinolide synthesis pathway by physically recognizing and binding to m6A-containing RNAs to redundantly modulate rice plant height (Cai et al. 2023). OsYTH07 physically interacts with OsEHD6 to enhance EHD6-YTH07 with strong-affinity to m6A targets and leads to the partial relocation of YTH07 from the cytoplasm to RNP granules through phase-separated condensation, thereby sequestering the \u003cem\u003eOsCOL4\u003c/em\u003e mRNA, reducing OsCOL4 protein accumulation, and promoting flowering via the EHD1 pathway (Cui et al. 2024). However, although the function of m6A readers in plants is rapidly being unveiled, the molecular mechanism underlying their regulatory roles remains poorly understood.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNotably, we identified a YTH family protein, OsYTH10, that mediates the rice heading date in this study. Loss of function of \u003cem\u003eOsYTH10\u003c/em\u003e promotes early flowering in rice, while overexpression of \u003cem\u003eOsYTH10\u003c/em\u003e delays the rice heading date significantly. We found that OsYTH10 is localized in the nucleus and cytoplasm. The flowering-related genes \u003cem\u003eOsGI\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOsDTH7\u003c/em\u003e have been identified as target genes of OsYTH10, and it has been demonstrated that OsYTH10 can specifically recognize and bind to m6A-containing mRNA in their 3\u0026apos;UTR. Additionally, loss of function of \u003cem\u003eOsYTH10\u003c/em\u003e can repress the expression levels of \u003cem\u003eOsGI\u0026nbsp;\u003c/em\u003eand \u003cem\u003eOsDTH7\u003c/em\u003e by accelerating their mRNA degradation, thereby affecting the rice heading date. Our results demonstrate that \u003cem\u003eOsYTH10\u003c/em\u003e plays a critical role in controlling m6A-dependent heading date regulation in rice.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and environment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe wild-type (WT) rice variety used in this study was Nipponbare, a japonica variety. All plants were grown in the experimental field of Jiangxi Agricultural University, located in Jiangxi province, China (28\u0026deg;45\u0026prime;36\u0026Prime;N, 115\u0026deg;22\u0026prime;58\u0026Prime;E) during the summer.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRT-qPCR and western blotting\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using Trizol (Aidlab, China), and the first-strand complementary DNA was reverse transcribed using ToloScript All-in-one RT EasyMix for qPCR (TOLOBIO, China). RT-qPCR was performed using TB Green Premix Ex Taq II (Takara) on a CFX96 Real-Time PCR Detection System (Bio-Rad), and the RT-qPCR primers used in this study are listed in supplemental table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTotal protein extracts from genotypic plants were separated on the 12% SDS-PAGE gel, and then transferred onto nitrocellulose membranes (GE Healthcare). proteins were detected using antibodies anti-ACTIN (Abmart), anti-GST (Abcam) and anti-MYC (Abcam).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEletrophoretic mobility shift analysis (EMSA)\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe recombinant GST, GST-OSYTH10 were expressed with pGEX-6P-3 containing a GST tag, and purified using an \u003cem\u003eEscherichia\u003c/em\u003e \u003cem\u003ecoli\u003c/em\u003e (BL21-RIL) expression system. The GST protein was used the negative control. Two conserved sites, Trp431 and Trp444, for YTH function were discovered by aligning sequence of YTH domains in OsYTH10 and AtECT2. Then the sites both Trp431 and Trp444 were mutated into Ala using the Fast Mutagenesis System kit (TransGene Biotech, China) for GST-OsYTH10 mutants. EMSA assays were performed using kit as described previously (Wei et al. 2018). The synthesized FAM 5\u0026rsquo; end-labeled RNA fragment listed in supplemental table1 was used as the RNA probe, while the corresponding unlabeled fragment was used as the competitor probes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emRNA decay assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe procedure was conducted as the previously described method (Duan et al. 2017; Tang et al. 2022). 7-day-old WT and \u003cem\u003eosyth10-1\u003c/em\u003e seedlings were cultured with liquid half‐strength MS medium. Then the seedlings were treated with a final concentration of 200 \u0026micro;M actinomycin D in the medium. After infiltration for 30 min, seedlings were harvested as time 0 controls, respectively. and subsequent samples were harvested every 3 h. To determine remaining mRNA levels using RT‐qPCR, 18S rRNA was used as the internal control. The primers used in this assay are listed in supplemental table S1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRIP assay and RNA-protein pull-down assay \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRIP assay with OsYTH10-4\u0026times;Myc line was performed according to a previously described method (Tang et al. 2022). Briefly, 35-day-old seedlings were harvested and cross-linked by using 1% formaldehyde for 10 min. RNA-protein complexes were immunoprecipitated by incubating with antibody Myc binding to protein G beads (Sigma) at 4\u0026deg;C for 4 h. Finally, the cross-linking was reversed, and RNA was purified by Trizol (Aidlab, China) for qPCR analysis. Primers used for RIP assay are listed in supplemental Table S1.\u003c/p\u003e\n\u003cp\u003eRNA pull-down assay was referred to a previously described method (Yang et al. 2023). In brief, biotin 5\u0026rsquo; end-labelled RNA fragment from \u003cem\u003eOsDTH7\u003c/em\u003e 3\u0026rsquo;UTR region with m6A modification was synthetized and denatured to be immobilized onto streptavidin magnetic beads. The assay was carried out with Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Scientific). 80 picomole (pM) of biotin labeled-RNA and 5 mg of soluble protein were used for once. The eluted RNA-proteins complexes were analyzed by western blotting with GST antibody (Abmart).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA m6A sequencing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRNA m\u003csup\u003e6\u003c/sup\u003eA-seq was conducted following the established protocols. Total RNA was isolated and purified using Trizol reagent following the manufacturer\u0026apos;s procedure with the panicles of 30-day-old WT plants. Poly (A) RNA was purified from 50\u0026mu;g total RNA, and was fragmented into small pieces using Magnesium RNA Fragmentation Module. The cleaved RNA fragments were incubated for 2h at 4℃\u0026nbsp;with m\u003csup\u003e6\u003c/sup\u003eA-specific antibody in IP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630). The IP RNA was reverse-transcribed to cDNA by SuperScript\u0026trade;\u0026nbsp;II Reverse Transcriptase, which was next used to synthesis U-labeled second-stranded DNAs with \u003cem\u003eE. coli\u003c/em\u003e DNA polymerase I, RNase H and dUTP Solution. An A-base was added to the blunt ends of each strand, preparing for ligation to the indexed adapters. Dual-index adapters were ligated to the fragments, and then the ligated products were amplified. At last, we performed paired-end sequencing (PE150) on an Illumina Novaseq\u0026trade;\u0026nbsp;6000 platform.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFormaldehyde-crosslinking and RNA Immunoprecipitation (FA-CLIP)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo explore the region where OsYTH10 binds to the mRNA of flowering related genes, FA-CLIP was used with OE-OsYTH10 plants. FA-CLIP was performed with minor modifications based on previously described (Wei et al. 2018; Wu et al. 2020). First, 3g of rice leaves are ground into powder in liquid nitrogen and transferred to a pre-cooled lysis buffer (containing 150 mM KCl, 50 mM HEPES (PH7.5), 2 mM EDTA, 2% Nonidet P-40, 40 U/mL Ribolock RNase Inhibitor (Thermo), 1 \u0026times; cocktail protease inhibitor (Roche)). After complete dissolution, 1% formaldehyde is added and incubated at 4\u0026deg;C for 10 min, then the crosslinking is terminated by 2 M glycine and further incubated for 40 min at 4\u0026deg;C. The sample is centrifuged, and the supernatant is collected after filtering. 2 \u0026mu;L/mL of DNaseⅠ (Promega) is added to digest genomic DNA for 5 min at 37℃, and 50 \u0026mu;L of sample was transferred out as input. Then, 7 \u0026mu;g Myc antibody is added and incubated with the sample, followed by the addition of protein G beads and overnight incubation. After that, the supernatant is removed, and the beads are washed with NT2 buffer (containing 300 mM KCl, 50 mM HEPES (PH7.5), 0.05% Nonidet P-40, 200 U/mL Ribolock RNase Inhibitor, 1 \u0026times; cocktail protease inhibitor). 10 U/\u0026mu;L RNase T1 (NEB) is added to digest RNA to about 30 nucleotides in size. After washing with high salt buffer, the beads are treated with 400 \u0026mu;L 1 \u0026times; T4 PNK buffer and 5 \u0026mu;L T4 PNK (NEB) for 1 h at 37℃. The pre-treated protease K solution (containing 400 \u0026mu;L 20 mg/mL protease K (Roche), 160 \u0026mu;L protease K buffer) by incubation at 37℃ for 20 min is then added for digestion at 37℃ for 20 min with a rate of 1000g per 3 min, and subsequently, Trizol and chloroform are used to extract the supernatant. Finally, the RNA from input and IP sample was used to send to the biological company for sequencing and analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe spatiotemporal expression of \u003cem\u003eOsYTH10\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the subcellular localization of OsYTH10, the full-length cDNA sequences of \u003cem\u003eOsYTH10\u003c/em\u003e was amplified and fused into the eGFP vector pD1301s-eGFP driven by the double 35S promoter. The recombinant constructs were transformed into rice protoplasts, which were prepared as described previously. After transformation, the protoplasts were incubated at 28℃\u0026nbsp;for 14-16 h, and the fluorescence signals were detected using a confocal microscope (Zeiss LSM 900). All primers used in this experiment are listed in supplemental table S1.\u003c/p\u003e\n\u003cp\u003eBesides, 30-day-old WT plants were used for nuclear-cytoplasmic fractionation according to the previous description. As quality controls for the fractionation, tubulin protein was detected with tubulin antibody and used as cytoplasmic markers, and histone H3 was detected with histone H3 antibody and used as nuclear markers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSequence alignment\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amino acid sequences of OsYTH10 from Oryza sativa and AtECT2 from Arabidopsis thaliana were obtained from the Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html), and a multiple sequence alignment of YTH domains was performed using ClustalW with default parameter.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eOsYTH10 is an m6A reader dependent on W431 and W444\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe m6A reader protein AtECT2 can specifically recognize and bind to mRNA 3\u0026rsquo;UTRs containing one of three types of m6A-modified motifs in Arabidopsis: UGUAA, RRACH (R=A/G, H=A/C/U), and URUAY (Y=C/U). It was presented that the two conserved tryptophan residues (W404 and W417) are crucial for AtECT8 to recognize and bind m6A modifications. By aligning the YTH domain sequences in OsYTH10 and AtECT2, it was revealed that the conserved W431 and W444 in OsYTH10 are likely important sites affecting its function as an m6A reader protein (Fig. 1a). To confirm the significance of these sites in OsYTH10 for recognizing and binding m6A modifications, we expressed and purified GST protein, GST-tagged OsYTH10 protein, and three non-binding variants (GST-OsYTH10\u003csup\u003em1\u003c/sup\u003e, GST-OsYTH10\u003csup\u003em2\u003c/sup\u003e, and GST-OsYTH10\u003csup\u003em3\u003c/sup\u003e) with corresponding mutations (W431A, W444A, and W431A/W444A) (Fig. S1). Additionally, FAM 5\u0026rsquo;-end-labeled RNA probes with or without m6A modification were synthesized to assess the m6A-binding ability of YTH10 and its mutated forms using EMSA assays. The EMSA results confirmed the direct binding of OsYTH10 to m6A-modified RNA probes (Fig. 1b). Importantly, none of the three mutated OsYTH10 proteins were able to bind to the m6A-modified probe in vitro (Fig. 1c). Furthermore, 5\u0026rsquo;-end biotin-labeled RNA fragments, identical to the probe sequence used in EMSA assays, were designed and synthesized to further validate the recognition and binding of OsYTH10 to the m6A site through RNA-protein pull-down assays in vitro. GST-OsYTH10 specifically bound to the RNA fragments, while GST-OsYTH10ms and GST proteins did not (Fig. 1d), indicating that OsYTH10 binding to m6A-modified RNA is dependent on both of indispensable W431 and W444.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOsYTH10\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;negatively regulates heading date in rice\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the initial study, we used m6A-seq to identify differentially expressed genes in response to cadmium stress in rice, with YTH10 emerging as a key gene in the m6A-mediated regulatory network under this stress (Cheng et al. 2021). To investigate the function of \u003cem\u003eOsYTH10\u003c/em\u003e in rice, we generated two \u003cem\u003eOsYTH10\u003c/em\u003e mutants (\u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e2\u003c/em\u003e) using the CRISPR/Cas9 system, resulting in premature termination of OsYTH10 translation (Fig. 2a). Additionally, we developed \u003cem\u003eOsYTH10\u003c/em\u003e overexpressing plants (OE-\u003cem\u003eOsYTH10\u003c/em\u003e-1 and OE-\u003cem\u003eOsYTH10\u003c/em\u003e-2) by expressing 4 x Myc-tagged OsYTH10 under the 35S promoter. Subsequent qRT-PCR analysis confirmed significant upregulation of \u003cem\u003eOsYTH10\u003c/em\u003e expression in the overexpressing plants compared to wild-type (WT) seedlings (Fig. 2b). Western blot analysis with MYC antibody further validated the expression of OsYTH10-4 x Myc in the overexpressing plants (Fig. 2c). Surprisingly, phenotypic analysis did not reveal significant differences between \u003cem\u003eOsYTH10\u003c/em\u003e mutants and WT seedlings under cadmium stress (Fig. S2), indicating \u003cem\u003eOsYTH10\u003c/em\u003e mutants are insensitive to cadmium. However, both \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e2\u003c/em\u003e knockout mutants exhibited similar early heading phenotypes compared to WT plants under natural subtropical climatic conditions with short-day photoperiods in Nanchang, Jiangxi province (Fig. 2d). The \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e1\u003c/em\u003e and \u003cem\u003eosyth10\u003c/em\u003e-\u003cem\u003e2\u003c/em\u003e mutants initiated heading at 68 and 69 days after germination (DAG), respectively, representing 6-day and 5-day advancements compared to WT plants (Fig. 2e). Conversely, \u003cem\u003eOsYTH10\u003c/em\u003e overexpressing lines (OE-\u003cem\u003eOsYTH10\u003c/em\u003e-1 and OE-\u003cem\u003eOsYTH10\u003c/em\u003e-2) displayed delayed heading times of 83 and 82 DAG, corresponding to 9-day and 8-day delays relative to WT plants (Fig. 2d and e). These results demonstrate that \u003cem\u003eOsYTH10\u003c/em\u003e functions as a negative regulator of heading date in rice, with its overexpression significantly postponing the heading transition. Furthermore, m6A dot blot analysis revealed the lowest m6A levels in the mutant and the highest levels in YTH10 overexpressing plants, suggesting that YTH10 positively regulates m6A levels in rice (Fig. S3). m6A cumulative percent analysis also showed that m6A modified RNA was enriched in OE-\u003cem\u003eOsYTH10\u003c/em\u003e-1 using RIP with anti-IgG and anti-Myc (Fig. S4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression pattern and subcellular relocation of OsYTH10\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the new function of \u003cem\u003eOsYTH10\u003c/em\u003e, we initially examined its spatial and temporal transcription expression patterns in various tissues and at different growth stages in rice using RT-qPCR assays. The expression analysis of \u003cem\u003eOsYTH10\u003c/em\u003e revealed relatively high expression levels in the root, stem, leaf, sheath, and panicles (Fig. 3a). The subcellular localization of OsYTH10 in rice protoplasts was also investigated. The results showed GFP fluorescent signals in the cytoplasm and nucleus, overlapping with the mCherry fluorescent signals of the nuclear marker protein NLS-mCherry (Fig. 3b). Furthermore, nuclear-cytoplasmic fractionation assays confirmed the distribution of OsYTH10 protein in both the cytoplasm and nucleus, as shown by western blot analysis (Fig. S5). These results provide insights into the subcellular localization of OsYTH10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eOsYTH10\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;regulates the expression of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the regulatory mechanism of \u003cem\u003eOsYTH10\u003c/em\u003e on the rice heading stage, we initiated m6A-seq analysis using 30-day-old WT plants. The results revealed that m6A modified peaks were predominantly concentrated in the 3\u0026apos;UTR region (Fig. 4a), with the UGUAA motif showing the highest enrichment in this region (Fig 4b and c). Subsequent analysis of differentially expressed genes in the m6A-seq data identified 6 genes associated with late flowering, namely \u003cem\u003eOsDTH7\u003c/em\u003e, \u003cem\u003eOsCRCT\u003c/em\u003e, \u003cem\u003eOsGI\u003c/em\u003e, \u003cem\u003eOsRE1\u003c/em\u003e, \u003cem\u003eOsHBF1\u003c/em\u003e, and \u003cem\u003eOsHBF2\u003c/em\u003e, based on the phenotypic observations of \u003cem\u003eOsYTH10\u003c/em\u003e mutants and overexpressing plants. m6A-IP-qPCR results confirmed the presence of m6A modifications in these genes (Fig 4d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsYTH10 physically binds to the m6A modification of 3\u0026rsquo;UTR in \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e mRNA\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the regulatory target genes of \u003cem\u003eOsYTH10\u003c/em\u003e in \u003cem\u003evivo\u003c/em\u003e, we utilized OE-\u003cem\u003eOsYTH10\u003c/em\u003e-2 plants to perform RIP qPCR assays aimed at identifying candidate target genes that interact with OsYTH10 in \u003cem\u003evivo\u003c/em\u003e. The results indicated that only \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u0026nbsp;\u003c/em\u003ewere significantly enriched in the IP sample with the Myc antibody, suggesting a direct interaction between OsYTH10 and their 3\u0026apos;UTR mRNA (Fig. 5a). In \u003cem\u003evitro\u003c/em\u003e, FAM 5\u0026rsquo; end-labeled RNA probes with or without m6A modification based on the 3\u0026rsquo;UTR sequences of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e were designed for ESMA assays to validate the RIP-qPCR results. The EMSA results showed OsYTH10\u0026apos;s ability to bind to the m6A-modified 3\u0026apos;UTR in \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e (Fig. 5b-I).\u003c/p\u003e\n\u003cp\u003eFurthermore, to confirm whether OsYTH10 recognizes and binds to \u003cem\u003eOsDTH7\u003c/em\u003e mRNA in \u003cem\u003evivo\u003c/em\u003e, FA-CLIP assay was conducted using the leaves of 30-day-old OE-\u003cem\u003eOsYTH10\u003c/em\u003e-2 plants and anti-Myc magnetic beads. Data analysis revealed high enrichment of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e mRNA in the immunoprecipitation compared to the input, indicating binding of OsYTH10 to \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e mRNA. No enrichment was observed in the mRNA level of a non-related control gene. Additionally, FA-CLIP-seq analysis unveiled that OsYTH10\u0026apos;s binding sites on \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e transcripts contain m6A modifications, as analyzed using integrative genomics viewer (IGV) on the sequenced data (Fig. 6a), consistent with the previous m6A-seq result.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsYTH10 inhibits decay of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e mRNA\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe performed mRNA lifetime assays on \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e in vivo in both WT and \u003cem\u003eOsYTH10\u003c/em\u003e mutants treated with actinomycin D to examine the impact of mRNA m6A readers on the stability or degradation of Arabidopsis mRNA. The findings revealed that the mRNA degradation rates of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e were increased in \u003cem\u003eosyth10-1\u003c/em\u003e and \u003cem\u003eosyth10-2\u003c/em\u003e mutants compared to WT (Fig. 6b and c), suggesting that \u003cem\u003eOsYTH10\u003c/em\u003e mutation accelerates the decay of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e mRNA.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003em6A is the most abundant chemical modification in eukaryotic organisms and plays a crucial role in regulating plant and animal cells. Proteins known as m6A readers are essential for mediating the functions of m6A. While the m6A reader in Arabidopsis has been extensively studied, its role in rice remains largely unexplored. Our study reveals that OsYTH10 in rice regulates flowering by recognizing m6A in the 3\u0026apos;UTR region of mRNA, thereby maintaining the stability of mRNA for key flowering-related genes like \u003cem\u003eOsGI\u003c/em\u003e, \u003cem\u003eOsDTH7\u003c/em\u003e, and \u003cem\u003eOsCRCT\u003c/em\u003e (Fig. 2). Meanwhile, We utilized FA-CLIP to investigate OsYTH10. The FA-CLIP-seq analysis of the rice transcriptome revealed that OsYTH10 predominantly binds to the 3\u0026apos;UTR region of mRNA (Fig. 6). The primary binding motif of OsYTH10 was identified as UGUA, consistent with the AtECT2 target motif in Arabidopsis. Furthermore, our m6A-seq results indicated that m6A modifications in rice were predominantly located in the mRNA 3\u0026apos;UTR region with the major motif being UGUA (Fig. 6). These findings suggest that OsYTH10 may recognize m6A-modified UGUA motifs in the mRNA 3\u0026apos;UTR region to perform its reading function. To confirm this hypothesis, we designed two RNA probes, UGUAA and UGU(m6A)A, and conducted EMSA experiments with GST-OsYTH10, demonstrating that OsYTH10 could bind to these motifs (Fig. 1b and c). Additionally, Biotin-RNA Pulldown experiments showed that UGU(m6A)A successfully pulled down OsYTH10, while UGUAA did not (Fig. 1d), indicating that OsYTH10 binds to UGUA motifs by recognizing m6A modifications. Previous studies have shown that editing \u003cem\u003eOsYTH10\u003c/em\u003e in Dongjin led to changes in rice plant height. In this study, we observed that knockdown of \u003cem\u003eOsYTH10\u003c/em\u003e in NIP caused rice to flower earlier under long-day conditions, while overexpression of \u003cem\u003eOsYTH10\u003c/em\u003e resulted in late flowering, indicating a crucial role of \u003cem\u003eOsYTH10\u003c/em\u003e in regulating rice flowering. The function of m6A modification primarily relies on its reader proteins. Research on human YTH family proteins has demonstrated their involvement in pre-mRNA splicing, nuclear export, mRNA degradation, and mRNA translation efficiency (Hsu et al. 2017; Roundtree and He 2016; Shi et al. 2017; Wang et al. 2014). In Arabidopsis, AtECT2 is known to maintain mRNA stability (Wei et al. 2018), while in rice, YTH07 binds to mRNAs modified by m6A and reduces their translation efficiency (Cui et al. 2024). Flowering time in rice is controlled by numerous genes, so we focused on the genes associated with rice flowering time among the target genes of OsYTH10. We analyzed the mRNA abundance of these genes in \u003cem\u003eosyth10-1\u003c/em\u003e versus WT and found that the abundance of these genes in \u003cem\u003eosyth10-1\u003c/em\u003e was lower than in WT. Given the high homology between OsYTH10 and AtECT2, we inferred that OsYTH10 has the capability of maintaining mRNA stability.\u003c/p\u003e\n\u003cp\u003eTreating rice seedlings with actinomycin D showed that\u003cem\u003e\u0026nbsp;OsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e degraded more rapidly in the \u003cem\u003eosyth10-1\u003c/em\u003e strain compared to WT (Fig. 6b and c). RIP-qPCR confirmed that OsYTH10 can bind to the mRNAs of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e. These findings suggest that OsYTH10 maintains mRNA stability of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e by interacting with their transcripts, and that deletion of \u003cem\u003eOsYTH10\u003c/em\u003e accelerates the degradation of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e, resulting in an early flowering phenotype.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study investigates the functional role of OsYTH10, an m6A-binding protein, in regulating rice flowering under prolonged light exposure. Genetic analyses demonstrate that OsYTH10 serves as a key modulator of flowering time by stabilizing transcripts of \u003cem\u003eOsDTH7\u003c/em\u003e and \u003cem\u003eOsGI\u003c/em\u003e. Given that precise control of flowering timing directly impacts yield potential, OsYTH10 emerges as a promising molecular target for breeding rice varieties adapted to regions with extended daylight cycles, offering a strategic avenue to improve grain production.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Biological Breeding-National Science and Technology Maior Project (20302022ZD040010202), the National Natural Science Foundation of China (32160485), and the Jiangxi Province Double Thousand Plan by Science and Technology Innovation High-Talent Project (JXSQ2023201057).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJianmin Bian and Haohua He conceived the project and designed the experiments. Jun Yang, Zhihao Chen and Peng Wang executed the experiments. Jun Yang, Mvuyeni Nyasulu, Qin Cheng, Xiaopeng He, Jie Xu, Junru Fu, Dahu Zhou, Linjuan Ouyang and Jianmin Bian analyzed the data. Jun Yang, Mvuyeni Nyasulu, Peng Wang and Jianmin Bian wrote the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the conclusions of this article are provided within the article (and its additional files), and the raw sequence data for this article are deposited in the NCBI (BioProject accession no. PRJNA1247134). Data generated in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eArribas-Hernandez L, Rennie S, Schon M, Porcelli C, Enugutti B, Andersson R, Nodine MD, Brodersen P (2021) The YTHDF proteins ECT2 and ECT3 bind largely overlapping target sets and influence target mRNA abundance, not alternative polyadenylation. Elife 10.\u003c/li\u003e\n \u003cli\u003eArribas-Hernandez L, Simonini S, Hansen MH, Paredes EB, Bressendorff S, Dong Y, Ostergaard L, Brodersen P (2020) Recurrent requirement for the m(6)A-ECT2/ECT3/ECT4 axis in the control of cell proliferation during plant organogenesis. Development 147.\u003c/li\u003e\n \u003cli\u003eBhat SS, Bielewicz D, Jarmolowski A, Szweykowska-Kulinska Z (2018) N(6)-methyladenosine (m(6)A): Revisiting the Old with Focus on New, an Arabidopsis thaliana Centered Review. Genes (Basel) 9.\u003c/li\u003e\n \u003cli\u003eCai L, Cui S, Jin T, Huang X, Hou H, Hao B, Xu Z, Cai L, Hu Y, Yang X, Zhou L, Yu T, Tian Y, Liu X, Chen L, Liu S, Jiang L, Zhou S, Wan J (2023) The N(6)-methyladenosine binding proteins YTH03/05/10 coordinately regulate rice plant height. Plant Sci 329:111546.\u003c/li\u003e\n \u003cli\u003eChen Y, Wan R, Zou Z, Lao L, Shao G, Zheng Y, Tang L, Yuan Y, Ge Y, He C, Lin S (2023) O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins. Nat Cell Biol 25:1676-1690.\u003c/li\u003e\n \u003cli\u003eCheng Q, Wang P, Wu G, Wang Y, Tan J, Li C, Zhang X, Liu S, Huang S, Huang T, Yang M, He H, Bian J (2021) Coordination of m(6)A mRNA methylation and gene transcriptome in rice response to cadmium stress. Rice (N Y) 14:62.\u003c/li\u003e\n \u003cli\u003eCui S, Song P, Wang C, Chen S, Hao B, Xu Z, Cai L, Chen X, Zhu S, Gan X, Dong H, Hu Y, Zhou L, Hou H, Tian Y, Liu X, Chen L, Liu S, Jiang L, Wang H, Jia G, Zhou S, Wan J (2024) The RNA binding protein EHD6 recruits the m(6)A reader YTH07 and sequesters OsCOL4 mRNA into phase-separated ribonucleoprotein condensates to promote rice flowering. Mol Plant 17(6):935-954.\u003c/li\u003e\n \u003cli\u003eDuan HC, Wei LH, Zhang C, Wang Y, Chen L, Lu Z, Chen PR, He C, Jia G (2017) ALKBH10B Is an RNA N(6)-Methyladenosine Demethylase Affecting Arabidopsis Floral Transition. Plant Cell 29:2995-3011.\u003c/li\u003e\n \u003cli\u003eHsu PJ, Zhu Y, Ma H, Guo Y, Shi X, Liu Y, Qi M, Lu Z, Shi H, Wang J, Cheng Y, Luo G, Dai Q, Liu M, Guo X, Sha J, Shen B, He C (2017) Ythdc2 is an N(6)-methyladenosine binding protein that regulates mammalian spermatogenesis. Cell Res 27:1115-1127.\u003c/li\u003e\n \u003cli\u003eLee KP, Liu K, Kim EY, Medina-Puche L, Dong H, Di M, Singh RM, Li M, Qi S, Meng Z, Cho J, Zhang H, Lozano-Duran R, Kim C (2024) The m6A reader ECT1 drives mRNA sequestration to dampen salicylic acid-dependent stress responses in Arabidopsis. Plant Cell 36:746-763.\u003c/li\u003e\n \u003cli\u003eLi F, Zhao D, Wu J, Shi Y (2014) Structure of the YTH domain of human YTHDF2 in complex with an m(6)A mononucleotide reveals an aromatic cage for m(6)A recognition. Cell Res 24:1490-1492.\u003c/li\u003e\n \u003cli\u003eLiang D, Lin WJ, Ren M, Qiu J, Yang C, Wang X, Li N, Zeng T, Sun K, You L, Yan L, Wang W (2022) m(6)A reader YTHDC1 modulates autophagy by targeting SQSTM1 in diabetic skin. Autophagy 18:1318-1337.\u003c/li\u003e\n \u003cli\u003eLiang Z, Riaz A, Chachar S, Ding Y, Du H, Gu X (2020) Epigenetic Modifications of mRNA and DNA in Plants. Mol Plant 13:14-30.\u003c/li\u003e\n \u003cli\u003eLim WA, Pawson T (2010) Phosphotyrosine signaling: evolving a new cellular communication system. Cell 142:661-667.\u003c/li\u003e\n \u003cli\u003eMa L, Chen T, Zhang X, Miao Y, Tian X, Yu K, Xu X, Niu Y, Guo S, Zhang C, Qiu S, Qiao Y, Fang W, Du L, Yu Y, Wang J (2021) The m(6)A reader YTHDC2 inhibits lung adenocarcinoma tumorigenesis by suppressing SLC7A11-dependent antioxidant function. Redox Biol 38:101801.\u003c/li\u003e\n \u003cli\u003eRoundtree IA, He C (2016) RNA epigenetics--chemical messages for posttranscriptional gene regulation. Curr Opin Chem Biol 30:46-51.\u003c/li\u003e\n \u003cli\u003eRoundtree IA, Luo GZ, Zhang Z, Wang X, Zhou T, Cui Y, Sha J, Huang X, Guerrero L, Xie P, He E, Shen B, He C (2017) YTHDC1 mediates nuclear export of N(6)-methyladenosine methylated mRNAs. Elife 6:e31311.\u003c/li\u003e\n \u003cli\u003eShen L, Liang Z, Wong CE, Yu H (2019) Messenger RNA Modifications in Plants. Trends Plant Sci 24:328-341.\u003c/li\u003e\n \u003cli\u003eShi H, Wang X, Lu Z, Zhao BS, Ma H, Hsu PJ, Liu C, He C (2017) YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA. Cell Res 27:315-328.\u003c/li\u003e\n \u003cli\u003eSong P, Wei L, Chen Z, Cai Z, Lu Q, Wang C, Tian E, Jia G (2023) m(6)A readers ECT2/ECT3/ECT4 enhance mRNA stability through direct recruitment of the poly(A) binding proteins in Arabidopsis. Genome biology 24:103.\u003c/li\u003e\n \u003cli\u003eTang J, Chen S, Jia G (2023) Detection, regulation, and functions of RNA N(6)-methyladenosine modification in plants. Plant Commun 4:100546.\u003c/li\u003e\n \u003cli\u003eTang J, Yang J, Lu Q, Tang Q, Chen S, Jia G (2022) The RNA N(6) -methyladenosine demethylase ALKBH9B modulates ABA responses in Arabidopsis. J Integr Plant Biol 64:2361-2373.\u003c/li\u003e\n \u003cli\u003eWang H, Niu R, Zhou Y, Tang Z, Xu G, Zhou G (2023) ECT9 condensates with ECT1 and regulates plant immunity. Front Plant Sci 14:1140840.\u003c/li\u003e\n \u003cli\u003eWang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, Fu Y, Parisien M, Dai Q, Jia G, Ren B, Pan T, He C (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505:117-120.\u003c/li\u003e\n \u003cli\u003eWang Y, Zhao JC (2016) Update: Mechanisms Underlying N(6)-Methyladenosine Modification of Eukaryotic mRNA. Trends Genet 32:763-773.\u003c/li\u003e\n \u003cli\u003eWei LH, Song P, Wang Y, Lu Z, Tang Q, Yu Q, Xiao Y, Zhang X, Duan HC, Jia G (2018) The m(6)A Reader ECT2 Controls Trichome Morphology by Affecting mRNA Stability in Arabidopsis. Plant Cell 30:968-985.\u003c/li\u003e\n \u003cli\u003eWu J, Peled-Zehavi H, Galili G (2020) The m(6) A reader ECT2 post-transcriptionally regulates proteasome activity in Arabidopsis. New Phytol 228:151-162.\u003c/li\u003e\n \u003cli\u003eWu M, Nie F, Liu H, Zhang T, Li M, Song X, Chen W (2022) The evolution of N(6)-methyladenosine regulators in plants. Methods 203:268-275.\u003c/li\u003e\n \u003cli\u003eXu C, Liu K, Ahmed H, Loppnau P, Schapira M, Min J (2015) Structural Basis for the Discriminative Recognition of N6-Methyladenosine RNA by the Human YT521-B Homology Domain Family of Proteins. J Biol Chem 290:24902-24913.\u003c/li\u003e\n \u003cli\u003eYang H, Chiang C, Luo Q, Chen C, Huang J, Zhu L, Zheng D (2022) YT521-B homology domain family proteins as N6-methyladenosine readers in tumors. Front Genet 13:934223.\u003c/li\u003e\n \u003cli\u003eYang J, He R, Qu Z, Gu J, Jiang L, Zhan X, Gao Y, Adelson DL, Li S, Wang ZY, Zhu Y, Wang D (2023) Long noncoding RNA ARTA controls ABA response through MYB7 nuclear trafficking in Arabidopsis. Dev Cell 58:1206-1217 e1204.\u003c/li\u003e\n \u003cli\u003eYuan S, Xi S, Weng H, Guo MM, Zhang JH, Yu ZP, Zhang H, Yu Z, Xing Z, Liu MY, Ming DJ, Sah RK, Zhou Y, Li G, Zeng T, Hong X, Li Y, Zeng XT, Hu H (2023) YTHDC1 as a tumor progression suppressor through modulating FSP1-dependent ferroptosis suppression in lung cancer. Cell Death Differ 30:2477-2490.\u003c/li\u003e\n \u003cli\u003eZaccara S, Jaffrey SR (2020) A Unified Model for the Function of YTHDF Proteins in Regulating m(6)A-Modified mRNA. Cell 181:1582-1595 e1518.\u003c/li\u003e\n \u003cli\u003eZhou B, Liu C, Xu L, Yuan Y, Zhao J, Zhao W, Chen Y, Qiu J, Meng M, Zheng Y, Wang D, Gao X, Li X, Zhao Q, Wei X, Wu D, Zhang H, Hu C, Zhuo X, Zheng M, Wang H, Lu Y, Ma X (2021) N(6) -Methyladenosine Reader Protein YT521-B Homology Domain-Containing 2 Suppresses Liver Steatosis by Regulation of mRNA Stability of Lipogenic Genes. Hepatology 73:91-103.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"RNA m6A, OsYTH10, Flowering Time, Rice","lastPublishedDoi":"10.21203/rs.3.rs-6593659/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6593659/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRNA m6A modification plays a crucial role in plant growth and crop yield. Proteins that can recognize m6A modifications, known as m6A reader proteins, are essential for the regulatory functions of m6A in gene expression. Among mRNA modification, methylation of internal adenosine N6 positions (m6As) is the most prevalent. The functional impact of m6A modifications largely relies on reader proteins. In this study, we identified OsYTH10, a member of the rice YTH-domain family protein, as a key player in m6A binding. The m6A-binding activity of OsYTH10 is mediated by its YTH structural domain. Deletion of OsYTH10 function results in early flowering in rice. Through FA-CLIP and m6A-seq analysis, we discovered that OsYTH10 binds to m6A in the 3'UTR region of mRNA. Our findings reveal that OsYTH10 stabilizes the mRNAs of target genes \u003cem\u003eOsDTH7\u003c/em\u003eand \u003cem\u003eOsGI\u003c/em\u003e, thereby regulating the normal flowering process in rice under prolonged sunlight conditions. This study sheds light on the critical role of OsYTH10 in m6A-mediated gene regulation and its impact on flowering time in rice.\u003c/p\u003e","manuscriptTitle":"The Critical Role of OsYTH10 in Promoting Early Flowering of Rice Under Long Sunlight","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 06:47:59","doi":"10.21203/rs.3.rs-6593659/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revisions","date":"2025-06-04T05:44:18+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-05-08T23:22:55+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-08T09:38:17+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-05T15:53:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Theoretical and Applied Genetics","date":"2025-05-05T06:40:07+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"theoretical-and-applied-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"taag","sideBox":"Learn more about [Theoretical and Applied Genetics](https://www.springer.com/journal/122)","snPcode":"122","submissionUrl":"https://submission.nature.com/new-submission/122/3","title":"Theoretical and Applied Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"83597809-0bcf-4d88-a717-7774cc3b26e6","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T16:03:29+00:00","versionOfRecord":{"articleIdentity":"rs-6593659","link":"https://doi.org/10.1007/s00122-025-05041-4","journal":{"identity":"theoretical-and-applied-genetics","isVorOnly":false,"title":"Theoretical and Applied Genetics"},"publishedOn":"2025-09-24 15:57:11","publishedOnDateReadable":"September 24th, 2025"},"versionCreatedAt":"2025-05-13 06:47:59","video":"","vorDoi":"10.1007/s00122-025-05041-4","vorDoiUrl":"https://doi.org/10.1007/s00122-025-05041-4","workflowStages":[]},"version":"v1","identity":"rs-6593659","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6593659","identity":"rs-6593659","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}