Abundant non-A residues in the poly(A) tail orchestrate the mouse oocyte-to-embryo transition

Non-A (U, G, and C) residues can be added to the 5-end, internal, and 3-end positions of poly(A) tails of RNA transcripts1-3, and some of these have been shown to regulate mRNA stability4, 5. The mammalian oocyte-to-embryo transition (OET) relies on post-transcriptional regulation of maternal RNA, because transcription is silent during this process until the point of zygotic genome activation (ZGA)6-9. Although the regulation of mRNA translation by poly(A) tail length plays an important role in the mammalian OET, the dynamics and functions of non-A residues in poly(A) tails are completely unknown. In this study, we profiled the genome-wide presence, abundance, and roles of non-A residues during the OET in mice using PAIso-seq1 and PAIso-seq22, 10, two complementary methods of poly(A) tail analysis. We found that non-A residues are highly dynamic in maternal mRNA, following a general pattern of beginning to increase at the MII stage, becoming highly abundant after fertilization with U residues in about half of poly(A) tails in 1-cell embryos, and declining in 2-cell embryos. We revealed that Btg4-mediated global maternal mRNA deadenylation created the substrates for U residue addition by Tut4/7 at their 3-ends and further re-polyadenylation. In addition, G residues can be added by Tent4a/b. Finally, we demonstrate that G residues stabilize the modified mRNA, while the U residues mark maternal RNA for faster degradation in 2-cell mouse embryos. Taken together, these findings demonstrate that non-A residues are abundant and re-sculpt the maternal transcriptome to initiate zygotic development, which reveals the functional importance of the post-transcriptional regulation mediated by non-A residues in mRNA poly(A) tails.