| Objective: Alternative polyadenylation(APA)regulates early embryonic development through the formation of transcripts containing different lengths of 3’ untranslated region(3’UTR).CFIm complex(consisting of NUDT21,CPSF6 and CPSF7 subunits)is a vital component involved in APA and mediates the selection and recognition of alternative polyadenylation cleavage sites,thereby regulating 3’UTR length changes,determining transcriptome diversity and post-transcriptional regulatory outcomes.The aim of this study is to investigate the dynamic changes and functions of 3’UTR mediated by CFIm complex during early embryonic development,and to reveal the potential mechanism of APA in regulating mammalian early embryonic development.Methods: 1.Using the available single-cell transcriptome sequencing dataset,the variation of 3’UTR length in human and mouse pre-implantation embryos was analyzed by APAtrap,GO analysis and KEGG pathway enrichment analysis.2.To determine the expression and localization pattern of CFIm complex in human and mouse early embryos at different stages by immunofluorescence detection;3.To explore the effect of APA deficiency on mouse early embryonic development and the underlying regulatory network using si RNA microinjection into mouse zygotes to knock down CFIm complex and other key components of APA.Results: 1.Bioinformatic analysis of sc RNA-seq dataset showed that most of the polyadenylation sites in human and mouse pre-implantation embryos were located in the3’UTR,and the variation of 3’UTR length was related to the early embryonic development stage and was species specific.2.(1)NUDT21 was mainly localized in the nucleus of mouse early embryos,and its expression level increased at 8-cell stage and reached the highest level at blastocyst stage.The expression of CPSF6 was almost not observed in zygote,but it was highly expressed in the nucleus and cytoplasm from the2-cell stage,and the CPSF6 signal in the nucleus was maintained at a high level throughout the pre-implantation embryo stage.From the 4-cell stage,specific CPSF6 granules signal could be observed in the cytoplasm.CPSF7 mainly expressed in the cytoplasm of early embryos.(2)NUDT21 also mainly expressed in the nucleus in human early embryos,and the cortical localization of NUDT21 in D3 embryos was closely related to the quality of human early embryos.In addition to being localized in the nucleus of human D3 embryos,CPSF6 also showed a diffuse signal in the cytoplasm.CPSF6 highly expressed in the nucleus of blastocysts and displayed discontinuous punctate signals along cell-cell junctions.3.Microinjection of si RNA to knock down key complex components involved in APA(such as Nudt21,Cpsf6,Cstf1,Cstf2 t,Clp1,Cpsf4,etc.)in mouse zygotes resulted in early embryonic development arrest or fragmentation;The downstream targeted genes were selected by Smart-seq2 of mouse embryos,follow-up verification was carried out by RT-q PCR and immunofluorescence method.Conclusions: Alternative polyadenylation showed different patterns of 3’UTR dynamics during human and mouse early embryonic development,but RNA metabolism-related pathways were involved in both species.The expression of CFIm complex is specific in human and mouse early embryos,and the length of 3’UTR may be determined by the nuclear abundance of CFIm complex components,mainly NUDT21 and CPSF6,in early embryos.Importantly,dysfunction of key components of APA such as the CFIm complex can lead to arrested or fragmented mouse early embryos,indicating an important regulatory role of APA in early mammalian embryonic development. |
