Dynamics of poly(A) tail length and non-A residues during the human oocyte-to-embryo transition

Poly(A) tail-mediated post-transcriptional regulation of maternal mRNA has been shown to be vital in the oocyte-to-embryo transition (OET) in flies, fish, frogs, and mice1-8. However, nothing is known about poly(A) tail dynamics for even a single gene during the human OET, because of the limited availability of human oocytes and embryos in combination with the low sensitivity of previous methods. Here, we systematically profiled the transcriptome-wide mRNA poly(A) tails in human oocytes at the germ-vesicle (GV), metaphase I (MI), and metaphase II (MII) stages, as well as pre-implantation embryos at the 1-cell (1C), 2-cell (2C), 4-cell (4C), 8-cell (8C), morula (MO), and blastocyst (BL) stages using single-oocyte/embryo PAIso-seq1 and PAIso-seq2 methods. We show that poly(A) tail length is highly dynamic during the OET, with BTG4 responsible for global deadenylation. Moreover, we reveal that non-A residues occur primarily in poly(A) tails of maternal RNA, which begin to increase at the MI stage, become highly abundant after fertilization (with U residues occurring in about two thirds, G residues in about one third, and C residues in about one fifth of mRNAs), and decline at the 8C stage. Importantly, we reveal that TUT4/7 can add U residues to deadenylated mRNA, which can then be re- polyadenylated to produce 5'-end and internal U residues. In addition, the re- polyadenylated mRNA can be stabilized through the addition of G residues by TENT4A/B. Finally, we demonstrate that U residues in poly(A) tails mark the maternal transcripts for quicker degradation in 8C human embryos compared to those without U residues. Together, our results not only reveal the dynamics of poly(A) tail length and non-A residues, but also provide mechanistic insights into the regulation of the length and the role of non-A residues during human OET. These findings further scientific understanding and open a new door for studying the human OET.