Study on the regulation of mRNA tailing during the maternal-to-zygotic transition in vertebrates

Abstract

During the maternal-to-zygotic transition (MZT), maternal transcriptome is degraded and replaced by de novo-synthesized zygotic transcripts in a highly coordinated manner. As zygotic genome is kept inactivated in fertilized eggs, maternal transcripts direct the early events of development and are regulated by post-transcriptional mechanisms such as cytoplasmic polyadenylation and differential RNA degradation. Timely decay of these maternal mRNAs is important because maternal transcripts must remain stable before the onset of zygotic genome activation (ZGA), but should be removed after ZGA so as not to interfere with the subsequent developmental events. However, it remains unknown how mRNA stability is temporally coordinated during early development in vertebrates.

To understand how mRNA tailing influences the transcriptome dynamics during the MZT, I applied a new high-throughput sequencing method called TAIL-seq to early zebrafish embryos. This method allows me to investigate poly(A) tail length and additional 3′ end modification at the genomic scale. I found that poly(A) tails of maternal mRNAs elongate globally after fertilization, whereas several groups of maternal transcripts remain unchanged or are rapidly deadenylated shortly after fertilization, establishing the temporal regulation of maternal gene expression. After the onset of ZGA, maternal mRNAs are massively deadenylated and degraded. Interestingly, mRNA uridylation is dramatically induced during the MZT. Such induction of uridylation is consistently observed in mouse and Xenopus embryos but not in Drosophila, suggesting the conserved role of uridylation in vertebrate mRNA decay pathway.

Morpholino-mediated knockdown experiments and high-throughput mRNA sequencing revealed that mRNA uridylation plays a critical role in the progression of the MZT. When TUT4 (ZCCH11) and TUT7 (ZCCHC6), previously identified as mRNA uridylation enzymes in human cells, are knocked down in zebrafish, maternal mRNA clearance is significantly delayed, leading to impaired zygotic transcription and developmental defects during gastrulation. Maternal mRNAs with short poly(A) are preferentially targeted by TUT4 and TUT7, whereas maternal transcripts with long poly(A) are less affected by uridylation activity. I further found the essential role of uridylation in early embryonic development in Xenopus laevis by knockdown of TUT7. Wild-type TUT7 but not catalytically inactive TUT7 mRNA rescues the gastrulation defects and the uridylation reduction in the TUT7 morphants, indicating the specificity of the developmental phenotype caused by TUT7 morpholinos.

Based on my results, I propose that uridylation has a conserved role in maternal mRNA clearance during early embryonic development in vertebrates. During the MZT, mRNA uridylation is essential for precise clearance of maternal transcripts with short poly(A) tails, thereby directing the progression of early development.