Abstract
In angiosperms, microRNA156 (miR156) acts as an intrinsic, endogenous developmental timer for the age-dependent transition from the juvenile to the adult phase1,2,3. However, the mechanisms modulating the age-dependent expression pattern of miR156 are still poorly understood4. In this Article, we report that circular RNAs (ciMIR156Ds) derived from pri-miR156d negatively regulate miR156 levels in an aging-dependent manner in rice. The ciMIR156D levels increase as plants age, which is inversely correlated with the changes of pri-miR156d and miR156 abundance. Consistent with this observation, ciMIR156Ds deficiency caused by a spontaneous mutation increases pri-miR156d and miR156 levels, resulting in a delayed heading phenotype, whereas ciMIR156Ds overexpression has opposite effects, demonstrating that ciMIR156Ds are negative regulators of miR156. We further show that ciMIR156Ds form R-loops with MIR156D at the region where they derive in an aging-dependent manner, which reduces the occupancy of DNA-dependent RNA polymerase II at that location and hence impedes pri-miR156d elongation. These findings reveal a mechanism for regulating heading date by refining the aging-dependent expression of miR156.
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Data availability
Publicly available datasets used in this study include PlantcircBase v.7.0 (http://ibi.zju.edu.cn/plantcircbase/index.php) and Bedtools v.2.28.0 (https://bedtools.readthedocs.io/en/latest/index.html). DRIP–seq data used for R-loop identification in this study are available via ref. 31 (accession no. GSE111944). Pol II ChIP–seq data used in this study are available via ref. 34. RNA-seq datasets of NIL-sdh and Nip are available via the Sequence Read Archive of the National Center for Biotechnology Information (accession nos. PRJNA1171578 and PRJNA1171724). All other data supporting the findings of this study are available in the paper and its Supplementary Information and Supplementary Data 1–6. Source data are provided with this paper.
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Acknowledgements
We thank W. Zhang of Nanjing Agricultural University for providing R-loop results and Q. Song of Nanjing Agricultural University for analysing BSA results. This work was supported by BaGui Scholars Foundation of Guangxi (to Y.L.); National Natural Science Foundation of China (31970603 to Y.S.); National Institute of Health (GM127414 to B.Y.); and National Science Foundation (MCB-1818082 to B.Y.).
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Y.S., Y.Y., S.G., Z. Wang, X.L. and Z. Wei performed the experiments and analysed the data. Y.S., S.G. and H.W. modelled and wrote the paper. C.Z., S.G. and Q.X. carried out data analysis. Y.Q. helped create rice mutants. Y.L. and B.Y. jointly supervised the research and wrote the paper. All authors provided feedback on the paper.
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Extended data
Extended Data Fig. 1 The effect of the sdh allele on heading date.
a, c, The heading date of indicated genotypes under long-days (LDs) and short-days (SDs). The days towards heading of each plant were recorded as the number of days beginning from transplanting seedlings to soil to the appearance of the first panicle. b, d, The tiller numbers of indicated genotypes under LDs. Values in a–d are given as means ± SD. *, P < 0.05; **, P < 0.01 compared with the wild type by single-sided Student’s t-test. e, Phenotypes of 98-day-old Nipponbare (Nip) and NIL-sdh plants grown under LD conditions in paddy fields of Nanning (China) during maturation period.
Extended Data Fig. 2 Positioning cloning of SDH.
a, Identification of genomic regions possibly harboring causative mutations using MutMap. The X-axis represents the chromosome positions. The Y-axis represents SNP index for the mutant. The red arrow indicates initial mutation locus interval. b, The diagram of predicted genes between markers RM12584 and RM12590. The boxes represent exons, while the slim lines and bold lines represent introns and regions between genes, respectively. c, PCR analysis of sdh. Primer positions were indicated in Fig. 1d. WT: Gui649; sdh: SDH/sdh obtained from Gui649 x sdh crossing. d, Association analysis between phenotype segregation and DNA deletion in MIR156D in F2 progenies generated from Gui649 and sdh crossing. The analyzed samples were collected based on WT or delayed heading phenotypes and subject to PCR reactions using primer shown in (Fig. 1d).
Extended Data Fig. 3 Functional verification of SDH.
a, The relative expression levels of SPL genes in the flag leaves of Nip and NIL-sdh detected by RT-qPCR. b, The relative expression levels of pri-miR156d in the flag leaves of various genotypes detected by RT-qPCR. c, Heading dates of various genotypes under LDs. The days to heading of each plant were recorded as the number of days beginning from transplanting seedlings to soil to the appearance of the first panicle. d, The tiller numbers of various genotypic plants under LDs. Data in (c, d) are shown as means ± SD, *, P < 0.05 **, P < 0.01 by single-sided Student’s t-test. e, The diagram of structure of STTM, including the promoter of MIR156D, spacer linker between STTMs, and designed STTM156 in which a weak imperfect ‘stem-loop’ was included (marked by red). f, The relative expression levels of pri-miR156d in the flag leaves of various genotypes detected by RT-qPCR. Expression of Ubiquitin was used as an internal control, and the expression level of each gene in Nip was set as 1 in (a, b, f). Data are shown as means ± SD (n = 3), *, P < 0.05; **, P < 0.01 by single-sided Student’s t-test.
Extended Data Fig. 4 Identification and over-expression of ciMIR156Ds.
a, Diagram showing the positions of ciMIR156Ds at the MIR156D locus and their validation with divergent primers followed by Sanger sequencing. Dashed lines indicate in electropherogram represent reads matching the back-splicing junction. The typical locations of divergent primers used for circRNA-specific amplification from exon 3 by RT–PCR are shown as grey arrows. b, Schematic diagram of constructs used to over-express ciMIR156D (OE-miR156dcirc) harboring a region from the 1st to the 4th exon of MIR156D. The red lines indicate that miR156 and miR156* were mutated in the construct. Mutated miR156d and miR156* sequences were shown below. c, ciMIR156D transcript levels in 11 independent OE-miR156dcirc transgenic plants detected by RT-PCR. Nip was used as a positive control. Line #1, #4 and #6 were selected for subsequent experiments as OE-miR156dcirc rice.
Extended Data Fig. 5 The effects of circRNAs on expressions of miR156 and its target genes.
a, The relative expression levels of pri-miR156d in the flag leaves of various genotypes detected by RT-qPCR using primers located at 5th exon. b, miR156 and miR172 were detected in flag leaves of various genotypes using Northern blot. U6 was used as the loading control. c, The relative expression levels of SPL genes in the flag leaves of various genotypes detected by RT-qPCR. Ubiquitin was used as an internal control, and the expression levels of each gene in Nip were set as 1 in (a, c). Data are shown as means ± SD (n = 3), *, P < 0.05; **, P < 0.01; ns, not significant by single-sided Student’s t-test.
Extended Data Fig. 6 The effects of linear RNAs of OE-156dw/t3rd,intron on miR156.
a, Schematic diagram of constructs used to over-express truncated MIR156D harboring a region from the 1st to the 4th exon of MIR156D without 3rd intron (OE-156dw/t 3rd intron). The gene structure is represented by exons (boxes) and introns (lines), and the green box in the 1st exon indicates the pre-miR156d coding region. The red lines in 1st exon indicate that miR156 and miR156* were mutated in the construct, and mutated miR156d and miR156* sequences were shown in Supplementary Fig. 4b. The red dashed line indicates the deleted 3rd intron in the construct. b, The relative transgene transcript levels of OE-156dw/t 3rd intron and OE-156dcirn in the flag leaves of various genotypes detected by RT-qPCR using primers located at the 3rd exon. The transcript levels of pri-mR156d in Nip were set as 1. The line #10 was used for the subsequent data. c, The relative expression levels of ciMIR156Ds in the flag leaves of Nip and OE-156dw/t 3rd intron #10 detected by RT-qPCR. d, The relative expression levels of pri-miR156d in the flag leaves detected by RT-qPCR using primers located at 5th exon. e, The relative expression levels of miR156 in the flag leaves among NIP and OE-156dw/t 3rd intron #10 detected by stem loop RT-qPCR. f,g, The heading date of indicated genotypes under long days (LDs). Ubiquitin (b-d) or U6 (e) was used as an internal control. Data are shown as means ± SD (n = 3), *, P < 0.05; **, P < 0.01; ns, not significant by single-sided Student’s t-test.
Extended Data Fig. 7 The pri-miR156d of Nip and NIL-sdh were not affected by NMD.
a, The representative diagram of Os07g0495900 gene structure. Exons and introns are indicated by boxes and lines respectively. The os07g0495900 mutant with a 4 bp deletion in the 5th exon was generated by CRISPR-Cas9 system. b, c, The relative levels of pri-miR156d (b) and miR156 (c) in Nip, os07g0495900, NIL-sdh (sdh) and os07g0495900 sdh were detected by RT-PCR and stem-loop RT-qPCR, respectively. Ubiquitin (UBQ) and U6 were used as the control in b and c, respectively. Data are shown as means ± SD (n = 3), *, P < 0.05; ns, not significant by single-sided Student’s t-test.
Extended Data Fig. 8 The cellular localization of circRNAs and epigenetic modifications of MIR156D region.
a, RT-qPCR analysis of circRNAs in the nuclear and cytosolic fractions in flag leaves of Nip. The nuclear abundance is set as 1. Data is shown as means ± SD (n = 3). b,c, The levels of H3K27ac (b) and H3K27me3 (c) at the MIR156D locus in wild-type and NIL-sdh. Four pairs of primers (used in Fig. 3a) around the deletion region were used to check the enrichment of H3K27ac or H3K27me3 modifications. The flag leaves were collected for ChIP-qPCR assays. The levels of H3K27ac and H3K27me3 of Actin gene in wild-type was set to 1.0. Three biological replicates were performed. Data is shown as means ± SD (n = 3). A single-sided Student’s t-test was used to generate the P values, *, P < 0.05; **, P < 0.01; ns, not significant.
Extended Data Fig. 9 Transcript levels of individual miR156 members in Nip and NIL-sdh.
The transcript levels of individual miR156 members in RNA-seq assay from 4th leaves of Nip and NIL-sdh were shown as TPM (Transcripts Per Kilobase of exon model per Million mapped reads) in heatmap, with three biological replicates for each sample.
Extended Data Fig. 10 The circRNA-R-loop may exist in other MIR156 loci of Arabidopsis and rice.
a,b, Diagram showing the circRNAs derived from MIR156F locus of Arabidopsis (a) and MIR156J locus of rice (b) overlap with the R-loop.
Supplementary information
Supplementary Information (download PDF )
Supplementary Table 1.
Supplementary Data 1 (download XLSX )
Summary of identified circRNAs in OsMIR156D locus.
Supplementary Data 2 (download XLSX )
The circRNAs derived from MIR156 locus of Arabidopsis and rice overlap with the R-loop.
Supplementary Data 3 (download XLSX )
The identified circRNAs located within a 2-kb range upstream and downstream of miRNAs in rice.
Supplementary Data 4 (download XLSX )
The identified circRNAs originated from miRNA loci of rice overlaps with R-loop.
Supplementary Data 5 (download XLSX )
The sequences of primers used in this study.
Supplementary Data 6 (download XLSX )
Source data for Supplementary Table 1.
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Su, Y., Yi, Y., Ge, S. et al. Circular RNAs derived from MIR156D promote rice heading by repressing transcription elongation of pri-miR156d through R-loop formation. Nat. Plants 11, 709–716 (2025). https://doi.org/10.1038/s41477-025-01961-7
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DOI: https://doi.org/10.1038/s41477-025-01961-7
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