Zygotic Splicing Activation of the Transcriptome is a Crucial Aspect of Maternal‐to‐Zygotic Transition and Required for the Conversion from Totipotency to Pluripotency

Abstract During maternal‐to‐zygotic transition (MZT) in the embryo, mRNA undergoes complex post‐transcriptional regulatory processes. However, it is unclear whether and how alternative splicing plays a functional role in MZT. By analyzing transcriptome changes in mouse and human early embryos, dynamic changes in alternative splicing during MZT are observed and a previously unnoticed process of zygotic splicing activation (ZSA) following embryonic transcriptional activation is described. As the underlying mechanism of RNA splicing, splicing factors undergo dramatic maternal‐to‐zygotic conversion. This conversion relies on the key maternal factors BTG4 and PABPN1L and is zygotic‐transcription‐dependent. CDK11‐dependent phosphorylation of the key splicing factor, SF3B1, and its aggregation with SRSF2 in the subnuclear domains of 2‐cell embryos are prerequisites for ZSA. Isoforms generated by erroneous splicing, such as full‐length Dppa4, hinder normal embryonic development. Moreover, alternative splicing regulates the conversion of early embryonic blastomeres from totipotency to pluripotency, thereby affecting embryonic lineage differentiation. ZSA is an essential post‐transcriptional process of MZT and has physiological significance in generating new life. In addition to transcriptional activation, appropriate expression of transcript isoforms is also necessary for preimplantation embryonic development.


Figure S2 .
Figure S2.Dynamic pattern of alternative splicing during the development of oocytes and preimplantation embryos in mouse and human.A: The alternative splicing events (ASEs) of different stages from mouse germinal vesicle (GV) oocytes and 4-cell embryos are shown with a volcano map.B: The ASEs of different stages from human zygotes and morula embryos are shown with a volcano map.C: The verification of different isoforms in 2-cell mouse embryos by reverse transcription-polymerase chain reaction (RT-PCR), and the visualization results from the Integrative Genomics Viewer.D: The ratio of different ASEs at different stages of mouse and human embryos.

Figure S3 .
Figure S3.Different clusters of exon IncLevel from Mfuzz in mouse.Profiles of scaled IncLevels (ILs) for each exon within each Mfuzz cluster in mouse.The color of each profile line corresponds to the "Mfuzz membership score" of the exon, i.e. how similar it is to the profile of the median values (from orange-yellow [low similarity] to purple-red [high similarity]).The number of events (n) is indicated for each cluster.

Figure S4 .
Figure S4.Different clusters of exon IncLevel from Mfuzz in human.Profiles of scaled IncLevels (ILs) for each exon within each Mfuzz cluster in human.The color of each profile line corresponds to the "Mfuzz membership score" of the exon, i.e. how similar it is to the profile of the median values (from orange-yellow [low similarity] to purple-red [high similarity]).The number of events (n) is indicated for each cluster.

Figure S6 .
Figure S6.Effects of PlaB and OTS964 on alternative splicing and embryonic development.A: The working model of the effects of pladienolide B (PlaB) and OTS964 on SF3B1.B and C: Schematic diagram of oocytes and embryo cultures in vitro.All oocytes and embryos were collected in vivo.D: Comparison of germinal vesicle breakdown (GVBD) rates in cultured oocytes treated with dimethyl sulfoxide (DMSO) or PlaB.E: The rates of polar body emission (PBE) in cultured oocytes treated with DMSO or PlaB.When oocytes had undergone GVBD within 6 h, they were selected for further culture.F: The development rates of 2-cell to 4-cell embryos after treatment with DMSO or PlaB.The numbers of analyzed embryos are indicated (n).n = 3 biological replicates.Error bars, SEM; n.s.: non-significant.***P < 0.001 by two-tailed Student's t-test.

Figure S7 .
Figure S7.Expression patterns of SRSF2, SF3B1, CDK11, and Cyclin L1 in oocytes and early embryos.A and B: The expression pattern of Srsf2 and Sf3b1 during the maternal-to-zygotic transition (MZT) is derived from Smart-seq2 and Ribo-seq data.Translation efficiency analysis was calculated by the ratio of Ribo-seq and Smart-seq2 (FPKM + 1/FPKM + 1).C: Western blotting results of SF3B1 in Zygote and 2-cell embryos.D: Immunofluorescent staining showing the speckles of SRSF2 and DAPI in Zygote, 2-cell, 4-cell, and 8-cell stages.Scale bar, 20 μm.E: Quantification of the number of SRSF2 speckles per 2-cell embryo in Fig 3D.F and G: The expression pattern of Cdk11 and Cyclin L1 during the maternal-to-zygotic transition (MZT) is derived from Smart-seq2 and Ribo-seq data.Translation efficiency analysis was calculated by the ratio of Ribo-seq and Smart-seq2 (FPKM + 1/FPKM + 1).H: Quantification of the number of SRSF2 speckles per 2-cell embryo in Fig 4E.

Figure S8 .
Figure S8.Analyses of the correlations between the indicated biological replicates of zygotes, 2-cell embryos, and 4-cell embryos treated with DMSO or PlaB (100 nM).The numbers correspond to R 2 (Pearson's correlation coefficient) values between the indicated replicates and the correlation strength is indicated by the color code.

Table S5 . Maternal and zygotic splicing factors defined by RNA-seq datasets from Btg4- and Pabpn1l-knockout mice
(in a separate Excel file)