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N6-methyladenosine regulates maternal RNA maintenance in oocytes and timely RNA decay during mouse maternal-to-zygotic transition

Nature Cell Biology volume 24, pages 917–927 (2022)Cite this article

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Abstract

N6-methyladenosine (m6A) and its regulatory components play critical roles in various developmental processes in mammals. However, the landscape and function of m6A in early embryos remain unclear owing to limited materials. Here we developed a method of ultralow-input m6A RNA immunoprecipitation followed by sequencing to reveal the transcriptome-wide m6A landscape in mouse oocytes and early embryos and found unique enrichment and dynamics of m6A RNA modifications on maternal and zygotic RNAs, including the transcripts of transposable elements MTA and MERVL. Notably, we found that the maternal protein KIAA1429, a component of the m6A methyltransferase complex, was essential for m6A deposition on maternal mRNAs that undergo decay after zygotic genome activation and MTA transcripts to maintain their stability in oocytes. Interestingly, m6A methyltransferases, especially METTL3, deposited m6A on mRNAs transcribed during zygotic genome activation and ensured their decay after the two-cell stage, including Zscan4 and MERVL. Together, our findings uncover the essential functions of m6A in specific contexts during the maternal-to-zygotic transition, namely ensuring the stability of mRNAs in oocytes and the decay of two-cell-specific transcripts after fertilization.

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Fig. 1: ULI-MeRIP–seq for m6A in mouse oocytes and early embryos.
Fig. 2: m6A regulates the coding mRNAs during MZT.
Fig. 3: m6A participates in the regulation of TE transcripts during MZT.
Fig. 4: KIAA1429 is indispensable for the stability of maternal m6A+ mRNAs.
Fig. 5: Inhibition of m6A writing affects the decay of transient mRNAs.
Fig. 6: Inhibition of m6A writing affects the decay of MERVL.

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Data availability

All the MeRIP–seq, RNA-seq and ATAC–seq data generated in this study are summarized in Supplementary Table 1. The sequencing data reported in this paper have been deposited in the Genome Sequence Archive under project PRJCA004536, and the accession code is CRA003985. Previously published MeRIP-seq data of 500 ng starting RNA that were re-analysed here are available under accession code GSE116002. MeRIP–seq data of mESCs with DNaseI treatment re-analysed here are available under accession code GSE145315. mm9 reference genome (https://hgdownload.soe.ucsc.edu/goldenPath/mm9/bigZips/mm9.fa.gz) was used in all the data. Repeat elements were identified with RepeatMasker from the UCSC browser (http://hgdownload.cse.ucsc.edu/goldenPath/mm9/database/rmsk.txt.gz). Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

The code is provided at https://github.com/xiaocuixu/mouse_embryo_m6a.

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Acknowledgements

We thank Dr C. He for suggestions regarding the project. We thank Dr R. Le for helpful advice on the manuscript. We thank Dr C. Ning for helping with the bioinformatics analysis. This work was supported by the National Key R&D Program of China (2016YFA0100400 (S.G.), 2020YFA0113200 (Y.G.), 2018YFA0108900 (Y.G.) and 2021YFC2700600 (B.S.)), the National Natural Science Foundation of China (31922022 (Y.G.), 31721003 (S.G.), 32000418 (X.X.) and 31970796 (B.S.)), the Shanghai Municipal Medical and Health Discipline Construction Projects (2017ZZ02015), the Fundamental Research Funds for the Central Universities (1515219049 and 22120200410), Peak Disciplines (Type IV) of Institutions of Higher Learning in Shanghai, the Shanghai Rising-Star Program (22YF1436200 (Y.W.)) and the China Postdoctoral Science Foundation (2020M681382 (Y.W.) and 2021M692437 (X.X.)).

Author information

Author notes

  1. These authors contributed equally: You Wu, Xiaocui Xu, Meijie Qi.

Authors and Affiliations

  1. Institute for Regenerative Medicine, Shanghai East Hospital, Shanghai Key Laboratory of Signaling and Disease Research, School of Life Sciences and Technology, Tongji University, Shanghai, China

    You Wu, Chuan Chen, Rushuang Yan, Xuelian Liu, Yawei Gao & Shaorong Gao

  2. Frontier Science Center for Stem Cell Research, Tongji University, Shanghai, China

    You Wu, Xiaocui Xu, Yawei Gao & Shaorong Gao

  3. Shanghai Institute of Stem Cell Research and Clinical Translation, Shanghai, China

    You Wu, Yawei Gao & Shaorong Gao

  4. Clinical and Translational Research Center of Shanghai First Maternity and Infant Hospital, School of Life Sciences and Technology, Tongji University, Shanghai, China

    Xiaocui Xu, Xiaochen Kou, Yanhong Zhao, Wenqiang Liu, Yanhe Li, Hong Wang & Shaorong Gao

  5. State Key Laboratory of Reproductive Medicine, Center for Global Health, Gusu School, Women’s Hospital of Nanjing Medical University, Nanjing Maternity and Child Health Care Hospital, Nanjing Medical University, Nanjing, China

    Meijie Qi, Mengying Li & Bin Shen

  6. Center for Reproductive Medicine, Division of Life Sciences and Medicine, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei, China

    Meijie Qi

  7. Women’s Hospital, Zhejiang University School of Medicine, Hangzhou, China

    Chuan Chen

  8. State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, China

    Meiling Zhang & Chengqi Yi

  9. Center for Reproductive Medicine, Cheeloo College of Medicine, Key Laboratory of Reproductive Endocrinology of Ministry of Education, Shandong University, Jinan, China

    Hongbin Liu

  10. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Junhong Xiang

  11. School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen, China

    Junhong Xiang

Authors
  1. You Wu
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  2. Xiaocui Xu
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Contributions

Y.G. and S.G. designed the project and directed all the experiments and bioinformatics analyses. B.S. provided cKO mice and technical direction. Y.W. carried out most experiments. X.X. carried out most bioinformatics analyses. M.Q. and M.L. carried out the generation, breeding and genotyping of Kiaa1429Zp3 cKO mice and Ythdf2Zp3-cKO mice. Y.W. and C.C. developed the ULI-MeRIP–seq method. M.Q. and B.S. performed mini-ATAC on oocytes. R.Y. and X.L. helped with embryo collection. X.K., Y.Z., W.L. and Y.L. performed microinjection and micromanipulation of embryos. M.Z. and C.Y. performed LC–MS/MS for quantification of m6A levels in embryos. H.L. provided Igf2bp2-KO mice. J.X. provided the STM2457 molecule. H.W. helped with cell culture work. Y.G., Y.W., X.X. and S.G. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Bin Shen, Yawei Gao or Shaorong Gao.

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Extended data

Extended Data Fig. 1 Validation of ULI-MeRIP-seq in mESCs.

a, Gel electrophoresis of 50 ng fragmented total RNA with different sonicated times tested by the High Sensitivity RNA ScreenTape system. b, S/N ratio of 0 min and 2 min sonication tested by qPCR, starting with 50 ng and 2 μg total RNA of mESCs. S/N, signal-to-noise ratio, represents IP over input for Klf4 versus Gapdh. Data are the presented as mean of two technical replicates from one experiment. The experiments for 2 μg were performed twice with similar trend (source data), and the experiments for 50 ng were performed once. Numbers above the bars were the mean value of S/N ratio. c, S/N ratio of m6A marked genes versus Gapdh with different antibody dosages tested by qPCR, starting with 50 ng total RNA of mESCs. Data are presented as mean value with 3 technical replicates. The experiments for antibody test were performed 3 times with similar trend (source data), and the data of one time are shown here. d, S/N ratio of pluripotency genes in 50 ng total RNA of mESCs tested by qPCR. S/N represents IP over input of target genes vs Gapdh. Data are the presented as mean of two technical replicates. The experiments were performed twice with similar trend (source data), and the data of one time are shown here. Numbers above the bars were the mean value of S/N ratio. e, Antisense and sense reads ratio at exons. ESC input and m6A-IP are the samples we produced using 50 ng RNAs. The control samples are from published data (GSE145315) of m6A-MeRIP-seq using DNase I treatment. f, The Pearson correlation coefficients (PCCs) showing a high correlation of the m6A signal from ULI-MeRIP-seq data between 50 ng and 2 μg starting amount of total RNA in mESCs. g, Sequence logo and p-values of the consensus motif of m6A peak centers located at genes. h, Density of m6A peak length in 2 μg and 50 ng starting RNA amount. i, UCSC browser tracks showing IP and input read distribution of representative pluripotency genes in 2 μg and 50 ng samples. Data in e-i represent results from three independent IP experiments of mESCs.

Source data

Extended Data Fig. 2 Validation of ULI-MeRIP-seq data quality in oocytes and embryos.

a, High enrichment of m6A in embryo IP samples tested by qPCR of GLuc versus CLuc. b, Scatterplots illustrating the distribution of PDP values in input (x-axis) and IP (y-axis) samples. The axes represent the ratio of the number of PCR duplicate reads to the total number of mapped reads. c, Scatterplots illustrating the distribution of NRF. The axes represent the ratios of the number of distinct genomic locations covered by reads to the total number of genomic locations covered by reads. d, Scatterplots illustrating the distribution of PBC1. The axes represent the ratios of the number of genomic locations covered by only one read to the number of distinct genomic locations covered by reads. e, Scatterplots illustrating the distribution of PBC2. The axes represent the ratios of the number of genomic locations covered by only one read to the number of genomic locations covered by only two reads. f, PCC between three independent IP replicates (and two replicates for 4 C) calculated by reads count. Data are presented as mean values. g, Heatmap depicting the Pearson correlation of different samples of the top 2000 transcripts ranked by CVs of fold enrichment (IP/input) levels of m6A at ± 200 bp around the stop codons. h, Saturation plot showing that high (FPKM > 1), medium (0.1<FPKM < 1) and low (0.01<FPKM < 1) levels of gene expression can be covered in input. i, m6A+ transcripts detected by MACS and MeTPeak. Unique represents m6A+ transcripts detected by either MACS or MeTPeak. Overlap represents m6A+ transcripts shared by MACS and MeTPeak. j, Density of m6A peak length in oocytes, early embryos and mESCs. k, Sequence logo and p-values of the consensus motif of m6A peak centers in each sample. l, [RRACH] density at exon peaks in each stage. Box plots are presented with horizontal line, median; box, interquartile range (IQR); whiskers, most extreme value within ±1.5 × IQR. m, Bar chart presenting the fraction of m6A peaks in different genomic regions. Data in a-m represent results from three independent IP experiments of GV to 2-cell and ESC, and two independent IP experiments of 4-cell.

Source data

Extended Data Fig. 3 m6A dynamics during MZT.

a, The number of transcripts harboring m6A (m6A + ) in each stage. b, Alluvial diagram showing the global dynamics of m6A + transcripts during MZT(n = 6940). Each line represents a transcript that is classified as a m6A + class in at least one stage. c, Bar chart showing m6A peak number in each sample. d, Alluvial diagram showing the global dynamics of m6A peaks during MZT (n = 43980). Each line represents a m6A peak in at least one stage. e, Heatmap showing normalized gene expression (left panel) and m6A modification status (right panel) of transcripts which gained m6A modification at L1C and L2C compared to GV and MII. f, The UCSC browser track showing IP and input reads of MD transcripts examples. g, The UCSC browser track showing IP and input reads of ZGA transcripts examples. h, Box plot showing [RRACH] motif density of ± 200 bp of stop codon in MD transcripts and ZGA transcripts with and without m6As. Box plots are presented with horizontal line, median; box, interquartile range (IQR); whiskers, most extreme value within ±1.5 × IQR. Two-tailed unpaired Wilcoxon test was used to calculate the p-values. i, Pie plot showing M-decay and Z-decay transcripts ratios of MD transcripts. Data in a-i represent results from three independent IP experiments of GV to 2-cell and ESC, and two independent IP experiments of 4-cell. Peaks of each stage are the intersect peaks of replicates or triplicates.

Source data

Extended Data Fig. 4 m6A profiles of TEs.

a, PCA showing m6A IP and input samples at all repetitive RNAs during MZT. Squares are input samples, and circles are m6A samples. b, Average RNA level of ERV, L1 and MaLR during MZT. c, Bar plots showing average RNA level (RPKM) for maternal expressed TEs in GV, MII (left) and zygotic expressed TEs in L2C and 4 C (right). d, Bar plots showing genomic copy number for maternal expressed TEs (left) and zygotic expressed TEs (right). e and f, Unmodified MTA(e) and MERVL(f) copies are not expressed or of low expression level in GV oocytes and 2-cell embryos. Full-length TE copies were classified into five groups according to expression levels. “1” represents copies with the lowest RNA level, while “5” represents copies with the highest level. Each group of copies was further classified according to m6A status. g and h, Sequence logo and p-values of the consensus motif of m6A peak centers at the MTA(g) and MERVL(h) locus. i and j, The UCSC browser track showing IP and input reads of MTA(i) and MERVL(j) during MZT. k, Average profile of m6A IP and input signal calculated by unique mapped reads for full-length MTA copies during MZT. l, The UCSC browser track showing example of MTA using only the unique mapped reads. m, Average profile of m6A IP and input signal calculated by unique mapped reads for full-length MERVL copies during MZT. n, The UCSC browser track showing example of MERVL using only the unique mapped reads during MZT. Data in a-n represent results from three independent IP experiments of GV to 2-cell and ESC, and two independent IP experiments of 4-cell.

Extended Data Fig. 5 m6A maintains the high expression levels of marked genes and MTA mRNAs during oocyte development.

a, Immunostaining of KIAA1429 in Kiaa1429 Ctrl and Zp3-cKO GV oocytes. Scale bar = 20 μm. b, PCA of m6A IP and input for control and Kiaa1429 Zp3-cKO GV oocytes. c, Bar chart showing number of m6A + transcripts in control and Kiaa1429 Zp3-cKO GV oocytes. d, Relative m6A signal intensity in GV oocytes of control and Kiaa1429-cKO mice normalized by GLuc. e, Heatmap showing the normalized expression level of DEGs between Kiaa1429 Zp3-cKO and control GV oocytes. Gene number of DEGs was labeled in parentheses. f, The Pearson correlation showing a high correlation of the ATAC-seq signal between biological replicates. g, ATAC-seq replicates were pooled together for peak-calling. Bar plot showing peak coverage for pooled samples. h, ATAC signal of MTA in NSN and PSN types of GV oocytes for control and Kiaa1429-cKO mice for MTA groups of different expression level. Top1 represents MTA copies with highest expression level in WT GV oocytes. Data in f-h, n = 4 biological replicates of Ctrl NSN GV, 3 biological replicates of Ctrl PSN GV, 3 biological replicates of Kiaa-cKO NSN GV and 2 biological replicates of Kiaa-cKO PSN GV. Kiaa represents Kiaa1429.

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Extended Data Fig. 6 Potential roles of YTHDF2 and IGF2BP family proteins in oocytes.

a, Schematic diagram of RNA-seq for early embryos using Ythdf2 Zp3-cKO mice. b, Immunostaining of YTHDF2 in Ythdf2 Ctrl and Zp3-cKO GV oocytes. Scale bar = 20 μm. c, PCA of RNA-seq for MII oocytes and 2-cell embryos of Ythdf2 cKO and control mice. d, Heatmap of normalized expression levels of maternal decay genes for MII oocytes and 2-cell embryos in control and Ythdf2 cKO mice. The classification of M-decay and Z-decay transcripts for Ythdf2 cKO samples are according to single-cell RNA-seq data. Gene numbers were labeled in parentheses. e, Gene expression level (FPKM) from RNA-seq data showing Igf2bp1/2/3 expression level during oocyte and early embryo development. f, RIP-qPCR showing fold enrichment of IGF2BP2 and IGF2BP3 at MTA and SINEb1 RNAs over IgG. Data shown represent 2 biological replicates for IGF2BP3-RIP and 1 biological replicate for IGF2BP2-RIP; value for each biological replicate represents the mean of two technical replicates. g, MTA expression level relative to Actin in GV oocytes of Igf2bp2 WT and KO mice. Data shown represent 2 biological replicates; value for each biological replicate represents the mean of two technical replicates. Data in c and d, n = 5 biological replicates of MII oocytes and Ctrl 2-cell embryos. n = 4 biological replicates of Ythdf2 cKO 2-cell embryos.

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Extended Data Fig. 7 Knockdown of m6A writers in early embryos affects the decay of 2 C mRNAs.

a, Schematic diagram of knockdown assay. b, Expression of Mettl3/14/16 and Kiaa1429 during early development. c, Knockdown efficiency measured by RT-qPCR. NC, non-target. KD, writer knockdown. Data shown represent 3 independent experiments for Mettl3 and Mettl14 KD and 2 independent experiments for Mettl16 KD; value for each biological replicate represents the mean of two or three technical replicates (shown in source data). d, Development rate of NC and KD embryos. Data are presented as mean values from two biological replicates (n = 30 each). e, Morphology of NC and KD blastocysts. Scare bar = 100 μm. f, Expression change ratio of ZGA m6A + (n = 756) and m6A- (n = 359) transcripts upon KD at middle-2-cell and morula stages. g and h, DEGs between NC and writer KD groups in middle 2-cell (g) and morula (h) stages. DEG number are indicated. Representative 2 C genes are labeled. i, Heatmap of expression (left), and m6A status (right) of DEGs in WT oocytes and embryos. j and k, Expression of Zscan4 (j) and MERVL (k) in NC and KD embryos tested by qPCR. Data are presented as mean values from two biological replicates, with 2 technical replicates for each. l, MERVL level in NC and KD embryos at middle-2-cell and morula stages. m, The decay of Zscan4 and MERVL after Actinomycin D treatment. Data are presented as mean values from two technical replicates of a single experiment. No biological replicates due to material limitation. n, The abundance of H3K9me3 at MERVL and Zscan4 genomic locus in NC and KD morula. Data are presented as mean values from two biological replicates with 2 technical replicates for each. o, The change of METTL3 protein in different KD embryos measured by immunofluorescence. Data are presented as mean values ± s.e.m. n = 10, 14, 10, 22, 16 and 10 biologically independent embryos. P-values are calculated by one-tailed unpair Student’s t test, showed above the bars. Data in f-l represent results from two biological replicates. Box plots in f,l are presented with horizontal line, median; box, IQR; whiskers, most extreme value within ±1.5 × IQR. Two-tailed unpaired Wilcoxon test was used to calculate the p-value.

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Extended Data Fig. 8 METTL3 inhibitor STM2457 affects embryo development and decay of transient genes.

a, Ratio of embryo types at IVF 19 h with and without STM2457 treatment. n = embryo number tested. b, Ratio of embryo types at E4.5 in vitro with DMSO and STM2457 treatment. n = embryo number tested. c, Embryo photos of DMSO and STM2457 treatment at E3.5 and E4.5 in vitro culturing. Scale bar = 100μm. d, m6A marked transcript number in L2C with DMSO and STM2457 treatment. e, Normalized m6A signal intensity in L2C with DMSO and STM2457 treatment. f, RNA signal level of ± 200 bp of stop codon of m6A IP and input for MD m6A + (n = 1089) and ZGA m6A + (n = 756) transcripts defined in Fig. 2a, with DMSO and STM2457 treatment after fertilization. Box plots are presented with horizontal line, median; box, interquartile range (IQR); whiskers, most extreme value within ±1.5 × IQR. P-values are calculated by two-tailed unpaired Wilcoxon test, showed above the boxes. g, PCA of RNA-seq for embryos with DMSO and STM2457 treatment. h, Proportion of m6A- and m6A + transcripts in sustained and transient expressed ZGA transcripts. Two-tailed Fisher’s exact test was used to calculate the p-value. i, Normalized m6A IP and input signal of sustained and transient m6A + transcripts, with DMSO and STM2457 treatment after fertilization. The IP and input signal were scaled by GLuc and CLuc, respectively. j, S/N ratio (targets / Gapdh) in L2C embryos with DMSO and STM2457 treatment after fertilization. Data shown represent 2 biological replicates; value for each biological replicate represents the mean of three technical replicates. k, RT-qPCR results of Zscan4 expression level relative to Actin. l, MERVL RNA level at 4 C and morula relative to 2 C. Data are the mean of MERVL RPKM. m, RT-qPCR results of MERVL RNA level relative to Actin. Data in d-f,i,j represent results from ULI-MeRIP-seq for L2C embryos with two individual replicates. Data in k, m are presented as mean values ± SEM. n = 3 biologically independent samples. P-values are calculated by two-tailed unpair Student’s t test, showed above the bars.

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Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1. Mapping information for RNA and ATAC–seq samples. Supplementary Table 2. Peak number and spike-in reads number. Supplementary Table 3. Gene list for MD and ZGA genes. Supplementary Table 4. DEGs in Kiaa1429 Zp3-cKO GV oocytes. Supplementary Table 5. DEGs in writer-KD 2C embryos and morulae. Supplementary Table 6. DEGs related to STM2457 treatment. Supplementary Table 7. Embryo background and collection information. Supplementary Table 8. Oligo and primer sequences.

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Wu, Y., Xu, X., Qi, M. et al. N6-methyladenosine regulates maternal RNA maintenance in oocytes and timely RNA decay during mouse maternal-to-zygotic transition. Nat Cell Biol 24, 917–927 (2022). https://doi.org/10.1038/s41556-022-00915-x

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