Maternal TDP43 Orchestrates Nuclear Speckle Assembly and Zygotic Splicing Activation During Oocyte-to-embryo Transition in Mouse [RIP-seq]

Zygotic splicing activation (ZSA) is an important means for mRNA post-transcriptional regulation during the maternal-to-zygotic transition (MZT) progress, ensuring normal embryonic development. However, the key factors and mechanisms of ZSA regulation are still unclear. Here, we found that nuclear speckle (NS), a key splicing region, is newly established at the 2-cell stage in mice, which is consistent with the period of ZSA. Moreover, NS and TDP43, an important component of paraspeckle, always have a partially adjacent and mutually exclusive localization relationship. TDP43 shuttles from the paraspeckle through liquid-liquid phase separation to perform its function: that is, it acts as a match-maker, binding to the transcribed RNAs on the one hand, and directly binding to the NS proteins on the other hand to ensure the correct assembly of NS. Maternal TDP43 deficiency leads to NS assembly failure in 2-cells, resulting in the inability of transcripts to skip; while injection of excessive TDP43 in zygotes leads to abnormal enlargement of NS in 2-cells, resulting in excessive skipping of transcripts. Both bidirectional ZSA disorders lead to 2-cell arrest in early embryogenesis. The ZSA defect caused by TDP43 deficiency also impairs the cell totipotency-pluripotency conversion. Collectively, our study discovered a NS upstream regulatory factor TDP43, which help maintain the balance of ZSA, providing a new perspective on post-transcriptional regulation of early embryos. Overall design: Ten embryos per sample were washed in 0.2 BSA in 1X PBS and collected for lysis in 4.2 µl lysis buffer (0.2% Triton X-100, RNase inhibitor, deoxyribonucleotide triphosphate (dNTPs), oligo-dT primers, and 1:1000 ERCC spike-in). Then the samples were reverse transcribed and amplificated using the Smart-seq2 method, as described previously. Sequencing libraries were constructed from 500 pg of amplified cDNA using TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503). Barcoded libraries were pooled and sequenced on the HiSeq X Ten platform with 150-bp paired-end reads.