RNA splicing and alternative polyadenylation profile during sheep zygotic genome activation†

Zygotic genome activation (ZGA) is a critical biological step in mammalian early embryo development. However, ZGA initiation in sheep and the related sophisticated RNA metabolism remains largely unknown. Here, we observed extensive alterations in gene expression and DNA methylation patterns, along with elevated levels of RNA polymerase II (RNAPII) and its phosphorylation at serine 2 (RNAPII-Ser2P) at the 16-cell stage. Moreover, the embryos were blocked at the 16-cell stage embryo when treated with α-Amanitin, indicating that ZGA is initiated at the 16-cell stage in sheep in vitro fertilized embryos. To uncover the sophisticated RNA metabolism during ZGA, we conducted weighted gene co-expression network analysis and identified 1957 critical maternal genes, including TET3, UHRF1, and KIF2C. Using dapars analysis, we discovered 1058 and 933 lengthened alternative polyadenylation (APA) events during ZGA in sheep and mice. Specifically, genes exhibiting shorten APA were highly expressed at sheep 16-cell stage embryos and mouse 2-cell stage embryos. During ZGA in sheep and mice, 2675 and 1963 genes showed exon skipping, an alternative splicing (AS) events, which is related to RNA binding, translation, gamete generation, and reproduction. Of note, inhibition of AS led to 2-cell block in mice and 8/16-cell block in sheep. Moreover, 5-EU, RNAPII, and RNAPII-Ser2P signal were decreased in AS inhibited 2-cell embryos in mice, suggesting AS might regulate the ZGA process by crosstalk with RNAPII. In conclusion, our data confirmed ZGA initiation at the 16-cell stage embryos, and provides insights into the complex RNA metabolism during ZGA in mammals.