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PABPN1L mediates cytoplasmic mRNA decay as a placeholder during the maternal‐to‐zygotic transition

2020; Springer Nature; Volume: 21; Issue: 8 Linguagem: Inglês

10.15252/embr.201949956

1469-3178

Long‐Wen Zhao, Yezhang Zhu, Hao Chen, Yun‐Wen Wu, Shuai‐Bo Pi, Lu Chen, Li Shen, Heng‐Yu Fan,

Molecular Biology Techniques and Applications

Article17 June 2020free access Source DataTransparent process PABPN1L mediates cytoplasmic mRNA decay as a placeholder during the maternal-to-zygotic transition Long-Wen Zhao orcid.org/0000-0002-4964-9578 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Ye-Zhang Zhu orcid.org/0000-0001-6310-9108 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Hao Chen orcid.org/0000-0001-7436-5895 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yun-Wen Wu MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Shuai-Bo Pi MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Lu Chen MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Li Shen MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Heng-Yu Fan Corresponding Author [email protected] orcid.org/0000-0003-4544-4724 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Long-Wen Zhao orcid.org/0000-0002-4964-9578 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Ye-Zhang Zhu orcid.org/0000-0001-6310-9108 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Hao Chen orcid.org/0000-0001-7436-5895 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yun-Wen Wu MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Shuai-Bo Pi MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Lu Chen MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Li Shen MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Heng-Yu Fan Corresponding Author [email protected] orcid.org/0000-0003-4544-4724 MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Search for more papers by this author Author Information Long-Wen Zhao1,‡, Ye-Zhang Zhu1,‡, Hao Chen1, Yun-Wen Wu1, Shuai-Bo Pi1, Lu Chen1, Li Shen1 and Heng-Yu Fan *,1,2 1MOE Key Laboratory for Biosystems Homeostasis & Protection and Innovation Center for Cell Signaling Network, Life Sciences Institute, Zhejiang University, Hangzhou, China 2Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China ‡These authors contributed equally to this work *Corresponding author. Tel: +86 571 88981370; E-mail: [email protected] EMBO Rep (2020)21:e49956https://doi.org/10.15252/embr.201949956 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Maternal mRNA degradation is a critical event of the maternal-to-zygotic transition (MZT) that determines the developmental potential of early embryos. Nuclear Poly(A)-binding proteins (PABPNs) are extensively involved in mRNA post-transcriptional regulation, but their function in the MZT has not been investigated. In this study, we find that the maternally expressed PABPN1-like (PABPN1L), rather than its ubiquitously expressed homolog PABPN1, acts as an mRNA-binding adapter of the mammalian MZT licensing factor BTG4, which mediates maternal mRNA clearance. Female Pabpn1l null mice produce morphologically normal oocytes but are infertile owing to early developmental arrest of the resultant embryos at the 1- to 2-cell stage. Deletion of Pabpn1l impairs the deadenylation and degradation of a subset of BTG4-targeted maternal mRNAs during the MZT. In addition to recruiting BTG4 to the mRNA 3ʹ-poly(A) tails, PABPN1L is also required for BTG4 protein accumulation in maturing oocytes by protecting BTG4 from SCF-βTrCP1 E3 ubiquitin ligase-mediated polyubiquitination and degradation. This study highlights a noncanonical cytoplasmic function of nuclear poly(A)-binding protein in mRNA turnover, as well as its physiological importance during the MZT. Synopsis The maternal-effect factor PABPN1-like (PABPN1L) mediates maternal mRNA decay by acting as mRNA-binding adapter of the mammalian MZT licensing factor BTG4 in the cytoplasm. Female Pabpn1l null mice are infertile due to impaired deadenylation and degradation of a subset of maternal mRNAs during the MZT. PABPN1L recruits BTG4 and CCR4-NOT deadenylase to the 3ʹ-poly(A) tail of maternal transcripts. Arg-171 within the RNA recognition motif (RRM) is essential for the poly(A)-binding ability of PABPN1L. PABPN1L protects BTG4 from SCF-βTrCP1 E3 ubiquitin ligase-mediated polyubiquitination and degradation. Introduction An initial step of early embryonic development in all animals is the process called the “maternal-to-zygotic transition (MZT)”, by which developmental control passes from the maternal genome to the zygotic genome: The majority of maternal RNAs and proteins are eliminated, and the zygotic genome becomes transcriptionally active 1. The mechanisms that regulate the MZT have been extensively investigated in model organisms including Drosophila, zebrafish, and Xenopus, in which the embryos inherit large quantities of maternal materials due to the staggering volume of ooplasm 2-5. In these species, the MZT is accomplished when thousands of blastomeres have formed, and along these lines is otherwise called the “mid-blastula transition (MBT)” 6, 7. In mammals, however, oocytes are relatively small in size compared with those of other animal groups, and the MZT is “pre-blastula” and occurs as early as the 1–4 cell stage after fertilization 8-10. Since the maternal genome becomes transcriptionally silent when oocytes develop to their full size in antral ovarian follicles, the oocyte meiotic maturation, fertilization, and early embryo development, until zygotic gene activation (ZGA), are principally regulated by timely translational activation and degradation of specific maternally derived mRNAs stored in the ooplasm 5, 11, 12. Each of these events is tightly regulated and is interceded by a myriad of RNA-binding proteins that coat the mRNA from its birth in the nucleus until its eventual degradation in the cytoplasm 3, 13, 14. While the vast majority of eukaryotic mRNAs in somatic cells are polyadenylated immediately after their transcription in the nucleus 15, a large number of maternal mRNAs are stored in the growing oocyte with a short poly(A) tail of 20 to 40 nucleotides and are translationally repressed 16. Upon oocyte maturation or after fertilization, the poly(A) tail of these dormant mRNAs is elongated to 80–250 residues and the mRNAs are translationally activated 17. In both cases, the elongated poly(A) tails are covered by poly(A)-binding proteins (PABPs) 18, 19. Two structurally distinct groups of PABPs have been identified in vertebrates. A nuclear PABP (PABPN1), present in all cells of organisms, contains a single RNA recognition motif (RRM) in the central region and a nuclear localization signal (NLS) at the C-terminus 20. Conversely, a group of cytoplasmic PABPs (PABPCs) contains four RRMs at their N-terminus and a unique C-terminal PABP domain 21. According to textbook models, PABPN1 modulates polyadenylation, processing, and nuclear export of newly synthesized mRNAs, whereas PABPCs stabilize poly(A) RNA in the cytoplasm and also enhance translation 18. While these conventional roles are critically important, both PABP families expanded recently both in number and in function 22, 23. In Xenopus and mouse, an oocyte-specifically expressed cytoplasmic PABP, PABPC1L (also known as embryonic PAB, ePAB), stabilizes maternal mRNAs and promotes their translation 24-26. Knockout of the Pabpc1l gene in mice causes defects of oocyte meiotic maturation and ovulation 27, 28. Subsequently, a Pabpn1-like (Pabpn1l) gene was identified in Xenopus, mouse, and the human genome, with high mRNA expression in oocyte and early embryos; however, its physiological function remained unknown 29, 30. In addition, maternal mRNA deadenylation and degradation are the core event of the MZT and a prerequisite for ZGA. B-cell translocation gene-4 (BTG4), a meiotic cell cycle-coupled MZT licensing factor in mouse, recruits the CNOT7 catalytic subunit of CCR4-NOT deadenylase to maternal mRNAs and triggers their degradation 31-33. However, BTG4 per se does not contain an RNA-binding domain 34. How BTG4 interacts with RNA and mediates mRNA decay during the MZT is still unknown. In this study, we identified PABPN1L as a poly(A) adaptor of BTG4 and investigated the potential functional significance of these RNA-binding proteins during the MZT. Pabpn1l null female mice were sterile. Genetic deletion of Pabpn1l impairs the deadenylation and degradation of a subset of maternal mRNAs during the MZT. As a result, embryos derived from Pabpn1l−/− females arrested at the 1- to 2-cell stage after fertilization. The fact that Pabpn1l and Btg4 knockout mice phenotype each other provided in vivo evidence that PABPN1L mediated cytoplasmic mRNA decay during the MZT by recruiting BTG4 and CCR4-NOT deadenylase to the 3ʹ-poly(A) tail of maternal transcripts. Collectively, this study demonstrates new biochemical and physiological functions of poly(A)-binding proteins during the MZT in mammals. Results Maternal PABPN1L is crucial for the MZT Recent oocytic transcriptome analyses 35 have demonstrated that the Pabpn1l transcript was abundantly expressed in mouse oocytes, and its expression level was the highest among all PABPs detected (Fig EV1A). The oocyte-specific high-level expression of Pabpn1l was confirmed by quantitative RT–PCR (RT–qPCR) (Fig 1A). In spite of the fact that Pabpn1l mRNA levels were high in germinal vesicle (GV) oocytes, the expression of the PABPN1L protein was detected only after meiotic resumption; this expression reached a maximal level at the MII stage and quickly decreased after fertilization (Fig 1B). The expression window of PABPN1L spatiotemporally overlapped with the expression pattern of BTG4 and the MZT process 31 (Figs 1B and EV1B). To study the in vivo function of Pabpn1l, we generated a Pabpn1l knockout mouse strain utilizing the CRISPR-CAS9 system. This mutant line contained a 41-nucleotide deletion in exon 2 (E2) of the Pabpn1l gene. The deletion caused a reading-frame shift and created a premature stop codon (Fig EV1C). The absence of PABPN1L proteins in oocytes derived from the Pabpn1l−/− mice was confirmed by Western blot (Fig 1C). Click here to expand this figure. Figure EV1. Pabpn1l knockout in mouse and phenotype analyses A. mRNA expression profiles of 9 PABPs detected in mouse oocytes and early embryos by RNA-seq (GSE44183). The relative mRNA level of Pabpn1l in oocyte was set to 1.0, and fold changes of indicated mRNAs were normalized by Pabpn1l (in oocyte). n = 3 biological replicates. Error bars, SEM. B. Immunofluorescent staining of HA (green) and α-tubulin (red) in WT MII oocytes and zygotes microinjected with mRNAs encoding HA-PABPN1L. DNA was labeled by DAPI. Scale bar = 20 μm. C. A strategy for CRISPR/Cas9-based mouse Pabpn1l knockout. Stop, stop codon; sgRNA, synthetic single-guide RNA; E1, E2, E7, exon1, 2, 7. D. Representative images of ovaries from 4-month-old WT and Pabpn1l−/− mice. Scale bar = 500 μm. E. H&E staining results showing ovarian histology of 4-month-old WT and Pabpn1l−/− mice. Scale bar = 500 μm. Download figure Download PowerPoint Figure 1. Expression and function of PABPN1L during MZT A. Quantitative RT–PCR (RT–qPCR) results showing relative expression levels of mouse Pabpn1l in somatic tissues, oocytes, and preimplantation embryos. n = 3 biological replicates. Error bars indicate SEM. B. Western blot results of PABPN1L, BTG4, and phosphorylated ERK1/2 (pERK1/2) in oocytes and embryos. Total protein from 400 oocytes or embryos is loaded in each lane. DNA damage-binding protein 1 (DDB1) is blotted as a loading control. C. Western blot results of PABPN1L in MII oocytes of wild-type (WT) and Pabpn1l−/− females. DDB1 is blotted as a loading control. D. Cumulative pup numbers of WT and Pabpn1l−/− female mice. n = 5 for each genotype. Error bars, SEM. ***P < 0.001 by two-tailed Student's t-test. E. Quantification of preimplantation embryos derived from WT and Pabpn1l−/− females when WT embryos reached the corresponding stages. The number of analyzed embryos is indicated (n). Error bars, SEM. ***P < 0.001 by two-tailed Student's t-test. F. Representative images of the embryos collected from the oviducts of mated WT and Pabpn1l−/− females at the indicated time points after hCG injection. Scale bar = 100 μm. Data information: For all Western blot results, at least three independent experiments were done with consistent results. Source data are available online for this figure. Source Data for Figure 1 [embr201949956-sup-0009-SDataFig1.pdf] Download figure Download PowerPoint Pabpn1l−/− mice were viable and healthy. Males had normal fertility, but females were infertile (Fig 1D), although they had normal ovarian histology (Fig EV1D and E). After superovulation treatment, the Pabpn1l-deleted females ovulated regular numbers of MII oocytes (Fig EV2A–D). Furthermore, the fully grown GV oocytes isolated from Pabpn1l null females underwent normal in vitro meiotic maturation (Fig EV2E and F). Zygotes derived from WT and Pabpn1l−/− oocytes (Pabpn1l♀−/♂+) formed pronuclei with similar efficiency at 24 h after hCG injection (Fig EV2G and H). However, in contrast to the WT zygotes, Pabpn1l♀−/♂+ zygotes arrested at the 1- to 2-cell stage and failed to develop further (Fig 1E and F). These results indicated that Pabpn1l is a novel maternal-effect gene and is crucial for the MZT. Click here to expand this figure. Figure EV2. Phenotype analyses of Pabpn1l knockout oocytes A. Representative images of MII oocytes collected from oviducts of WT and Pabpn1l−/− mice at 16 h after hCG injection. Scale bar = 100 μm. B. Numbers of MII oocytes being ovulated by WT and Pabpn1l−/− mice (n = 4 for each genotype) after superovulation. Error bars, SEM. ns: non-significant, calculated by two-tailed Student's t-test. C. Confocal microscopy results showing spindle assembly and PB1 emission of oocytes collected from the oviducts of WT and Pabpn1l−/− mice. Scale bar = 20 μm. D. Rate of normal spindle assembly in mature WT and Pabpn1l−/−oocytes. E, F. GVBD (E) and PB emission (F) rates in cultured oocytes derived from WT and Pabpn1l−/−females. G. Immunofluorescent staining for α-tubulin (green) and DNA (red) in zygotes from WT and Pabpn1l−/− females 28 h after hCG injection. Scale bar = 20 μm. H. Percentage of zygotes with two pronuclei (PNs) from WT and Pabpn1l−/− females 28 h after hCG injection. Data information: In (D, E, F, and H), the number of analyzed oocytes is indicated (n). Error bars represent SEM. ns: non-significant, calculated by two-tailed Student's t-test. Download figure Download PowerPoint PABPN1L facilitates maternal mRNA clearance To understand the role of PABPN1L during the MZT, we subjected WT and Pabpn1l−/− oocytes, as well as the derived embryos at the 1-cell (zygote) and 2-cell stages, to global RNA-seq analyses. Gene expression levels were assessed as fragments per kilobase of transcript per million mapped reads (FPKM), and the relative mRNA copy number was evaluated using the External RNA Controls Consortium (ERCC) spike-in. All samples were analyzed in duplicate and showed a high correlation (Rmin = 0.88; Raverage = 0.93; Table EV1). Compared to WT, only three and six transcripts were increased or decreased more than 3-fold in Pabpn1l null oocytes at the GV stage, respectively (Fig 2A; Dataset EV1). In contrast, more transcripts were increased than decreased in Pabpn1l♀−/♂+ zygotes (1,424 versus 5) (Fig 2A; Dataset EV1). Gene set enrichment analysis of the increased transcripts at the zygote stage revealed that 1,414 of the 1,424 increased transcripts were those being degraded in WT zygotes (Fig 2B). Furthermore, there was a substantial overlap between the transcripts that accumulated in Btg4♀−/♂+ and Pabpn1l♀−/♂+ zygotes (Fig 2C), implying a collaboration between PABPN1L and BTG4 in maternal mRNA turnover. RT–qPCR results confirmed the RNA-seq data and indicated that previously reported Btg4-target transcripts in the MZT also accumulated after maternal Pabpn1l knockout (Fig 2D). To investigate the role of PABPN1L in mRNA turnover, a poly(A) tail (PAT) assay was performed, which recapitulates the poly(A) tail length changes in specific transcripts. The results showed the gradual shortening of poly(A) tails of the detected maternal mRNAs during the MZT in WT oocytes and embryos. However, the deadenylation of these transcripts was blocked or delayed after maternal Pabpn1l knockout (Fig 2E). These results indicated that PABPN1L is crucial for maternal mRNA clearance during the MZT. Figure 2. Transcriptome analyses in Pabpn1l-deleted oocyte and embryos during the MZT A. Scatter plot comparing the transcripts of GV oocytes and zygotes from WT and Pabpn1l−/− females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted GV oocytes or zygotes are highlighted in red or blue, respectively. B. Venn diagrams showing the overlap in transcripts that are significantly degraded during the MZT (FPKM[GV/zygote] > 3) of WT oocytes and transcripts that are accumulated in this process after Pabpn1l knockout (FPKM[Pabpn1l♀−/♂+/WT] in zygote) > 3). C. Venn diagrams showing the overlap in transcripts that are stabilized during the MZT in Pabpn1l♀−/♂+ and Btg4♀−/♂+ zygotes (FPKM[GV/zygote] > 3 in WT, but < 3 in KO). D. RT–qPCR results for relative expression levels of the indicated maternal transcripts in oocytes and zygotes from WT and Pabpn1l−/− females. n = 3 biological replicates. Error bars, SEM. *P < 0.05, **P < 0.01, and ***P < 0.001 by two-tailed Student's t-test. E. Poly(A) tail assay results showing poly(A)-tail length of indicated transcripts in WT and Pabpn1l-deleted oocytes and embryos. The poly(A) tails of Actin are used as an internal control. Each sample was prepared from 100 oocytes or embryos. Plots show the averaged relative signal intensity (y-axis) and length of the PCR products based on mobility (x-axis). Download figure Download PowerPoint Maternal PABPN1L is essential for ZGA Because maternal Pabpn1l deletion causes zygotic developmental arrest, we further examined the potential effect of maternal PABPN1L on ZGA. Compared to WT, 269 and 718 transcripts were at abnormally high or low levels in Pabpn1l♀−/♂+ 2-cell embryos, respectively (Fig 3A; Dataset EV1). Gene set enrichment analysis revealed that there are 718 transcripts showed decreased levels in Pabpn1l♀−/♂+ 2-cell embryos when compared with WT (WT/Pabpn1l♀−/♂+ ≥ 3). Among these transcripts, 419 belonged to genes that are activated during ZGA in the WT 2-cell embryos (Fig 3B). Figure 3. Maternal PABPN1L is essential for ZGA A. Scatter plot comparing the transcripts of 2-cell embryos from WT and Pabpn1l−/− females. Transcripts that increased or decreased by more than 3-fold in Pabpn1l-deleted oocytes or embryos are highlighted in red or blue, respectively. B. Venn diagram and schematic showing the overlap in transcripts that are activated during zygote-to-2-cell transition in WT (FPKM[2-cell/zygote] > 3) and the transcripts that are decreased in Pabpn1l♀−/♂+ compared to WT at the 2-cell stage (FPKM[WT/Pabpn1l♀−/♂+] > 3 in 2-cell embryos). C. 5-Ethynyl uridine (EU) fluorescence (green) showing RNA transcription in WT and Pabpn1l♀−/♂+ 2-cell embryos. Some WT embryos were treated with α-amanitin as early as the zygote stage and cultured to the 2-cell stage. The phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2) (red) is co-stained to label the RNA polymerase II activity. Nuclei are labeled by DAPI (blue). Scale bar = 20 μm. D. Quantification of EU and pS2 signals in (C). The numbers of analyzed embryos are indicated (n). Error bars, SEM. ***P < 0.001 by two-tailed Student's t-test. E. Relative intensity of tdTomato signals in (F). The numbers of analyzed embryos are indicated (n). Error bars, SEM. ***P < 0.001 by two-tailed Student's t-test. F. Representative images of MuERV-L::tdTomato relative to GFP signal in the same embryo when WT embryos reached the corresponding stages. Zygotes were injected with the MuERV-L::tdTomato reporter plasmid and polyadenylated Gfp mRNA (as a positive control of microinjection), then allowed to develop in vitro. Cultured embryos were imaged at 24 and 40 h after hCG injection. DIC, differential interference contrast. Scale bar = 100 μm. Download figure Download PowerPoint To evaluate global transcription activity during ZGA, we performed 5′-ethynyl uridine (EU) staining assay in which the EU labeled newly synthesized RNAs in the 2-cell embryos. Phosphorylated RNA polymerase II CTD repeat YSPTSPS (pS2, also a marker of transcription activation) was co-stained with the EU. As a negative control, some 2-cell embryos were pre-treated with α-amanitin, an RNA polymerase II inhibitor, before ZGA (Fig 3C and D). EU and pS2 signals were detected in the nuclei of Pabpn1l♀−/♂+ embryos but were weaker than those in WT embryos. These results indicated that maternal Pabpn1l deletion impairs the transcription activation in 2-cell embryos. Results of RT–qPCR indicated that the expression pattern of representative zygotic genes was similar to those revealed by the RNA-seq analysis (Appendix Fig S1A). Previous studies showed that a large number of retrotransposons are expressed as a feature of 2-cell embryos undergoing ZGA 36, 37. At the 2-cell stage, the murine endogenous retrovirus with a leucine tRNA primer (MuERV-L) element was transiently transcribed at the 2-cell stage in WT embryos but was transcribed at a remarkably lower level after maternal Pabpn1l deletion (Appendix Fig S1B). Similarly, transcription of MuERV-L target genes, including Guca1a, Tead4, Tdpoz1/4, and Zfp352, was also blocked after maternal Pabpn1l deletion (Appendix Fig S1B). We also microinjected zygotes (WT and Pabpn1l♀−/♂+) with the MuERV-L::tdTomato reporter plasmid (MuERV-L 5′-long terminal repeat (LTR) promoters upstream of the red fluorescent protein tdTomato) as previously described 36, 38 and monitored the expression of tdTomato during culture. TdTomato expression was observed in WT 2-cell embryos but not in Pabpn1l♀−/♂+ embryos, which arrested at the 1- to 2-cell stage (Fig 3E and F). In contrast, Gfp mRNAs co-injected with the MuERV-L 5ʹ-LTR reporter were equally expressed in all embryos (Fig 3F). In summary, these results indicated that maternal PABPN1L-mediated biochemical processes, most likely maternal mRNA turnover, are a prerequisite for ZGA in mouse early embryos. PABPN1L binds both RNA and BTG4 and is involved in deadenylating maternal mRNAs Based on these results, we hypothesized that PABPN1L may function as an RNA-binding adapter of BTG4 during the MZT. Thus, BTG4-RNA immunoprecipitation (RIP) assays were performed in the presence and absence of PABPN1L. Since it was technically challenging to perform extensive biochemical experiments in oocytes due to their low number, we co-expressed PABPN1L and BTG4 in HeLa cells, which do not endogenously express these proteins, and in line with oocytes/zygotes, the PABPN1L possesses cytoplasm distributed characteristic in HeLa cells (Figs EV3A and EV1B). The results showed that representative transcripts, which were commonly targeted by BTG4 and PABPN1L according to RNA-seq results, effectively interacted with BTG4 only in the presence of PABPN1L (Fig 4A). As a negative control, mRNAs were not enriched by the RRM-deleted or Arg-171 (an essential RNA-binding residue in the RRM, discussed below)-mutated PABPN1L in the RIP assay (Figs 4A and EV4B). Click here to expand this figure. Figure EV3. PABPN1L interacts with BTG4 A. Immunofluorescent staining of HA (green) and DNA (red) in HeLa cells transfected with a HA-PABPN1L expressing plasmid. The white dotted box showing the PABPN1L signal was zoomed out on the right panel. Scale bar = 10 μm. B. A diagram showing of mouse BTG4 with regions of predicted disorder. C. Diagrams and co-IP results showing that BTG4 binds with PABPN1L and its N-terminal-deleted form (NterΔ). Lysates from HeLa cells expressing HA-BTG4 and FLAG-PABPN1L were immunoprecipitated with an anti-FLAG antibody. At least three independent experiments were done with consistent results. Source data are available online for this figure. Download figure Download PowerPoint Figure 4. PABPN1L is an RNA-binding adapter of BTG4 involved in deadenylating maternal mRNAs A. RNA immunoprecipitation (RIP) results showing the interaction of BTG4 with indicated transcripts, in the presence or absence of PABPN1L (full length, RRM-deleted, or R171A mutant). HeLa cells were co-transfected with plasmids expressing FLAG-tagged PABPN1L and HA-tagged BTG4 for 48 h before immunoprecipitating with an anti-HA antibody. RNAs recovered from the immunoprecipitants were subjected to RT–qPCR of the indicated transcripts. Fold change values of both input and IP samples were normalized by RIP results of HA-BTG4 and FLAG-PABPN1L co-expression groups. n = 3 biological replicates. Error bars, SEM. The P-value represents the two-tailed Student's t-test comparing the RIP results of BTG4 with the indicated transcripts in the presence of PABPN1L, *P < 0.05, **P < 0.01, and ***P < 0.001. B. Diagrams of mouse BTG4 constructs. C, D. Co-IP and Western blot results showing interactions between PABPN1L and BTG4. Lysates from HeLa cells expressing HA-BTG4 (WT or mutants shown in (B)) and FLAG-PABPN1L were immunoprecipitated with an anti-HA antibody. The immunoprecipitated proteins are detected by Western blot with the indicated antibodies. E. Diagrams of mouse BTG2 and BTG4 constructs, and co-IP results showing that PABPN1L binds to BTG4 but not BTG2. F. Diagrams and co-IP results showing BTG4 binding to PABPN1L. Lysates from HeLa cells expressing HA-BTG4 and FLAG-PABPN1L (full length and C-terminal deleted) were immunoprecipitated with an anti-FLAG antibody. The immunoprecipitated proteins are detected by Western blot with the indicated antibodies. G. Diagrams of PABPN1 and PABPN1L constructs, and co-IP results showing that PABPN1L, but not PABPN1, binds to BTG4. Data information: For all Western blot results, at least three independent experiments were done with consistent results. Source data are available online for this figure. Source Data for Figure 4 [embr201949956-sup-0010-SDataFig4.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Role of Arg-171 of mouse PABPN1L in RNA binding A. A table showing the Tm in Fig 6A–C. B. Sequence alignment of mouse PABPN1L (MusPABPN1L) and Citrus sinensis PABPN1(CsPABPN1). The broken circl