Maternal TDP43 Orchestrates Nuclear Speckle Assembly and Zygotic Splicing Activation During Oocyte-to-embryo Transition in Mouse [RNA-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. 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.

合子剪接激活（Zygotic Splicing Activation, ZSA）是母源-合子转换（Maternal-to-Zygotic Transition, MZT）过程中mRNA转录后调控的重要手段，对胚胎正常发育至关重要。然而，目前ZSA调控的关键因子与机制仍不明晰。
本研究发现，作为核心剪接调控区域的核斑（Nuclear Speckle, NS）会在小鼠2细胞期全新建立，这与ZSA的活跃时期高度一致。此外，核斑与副斑的重要组分TDP43（Transactive Response DNA-Binding Protein 43）始终呈现部分毗邻且相互排斥的定位模式。TDP43通过液液相分离从副斑穿梭至核斑发挥功能：作为分子桥接因子，一方面结合转录生成的RNA，另一方面直接与核斑蛋白结合，以保障核斑的正确组装。
母源TDP43缺失会导致2细胞期核斑组装失败，使转录本无法发生可变剪接外显子跳跃；而在合子中过量注射TDP43则会引发2细胞期核斑异常增大，造成转录本过度剪接跳跃。上述两种双向ZSA失衡均会导致胚胎早期出现2细胞期阻滞。TDP43缺失引发的ZSA缺陷还会损害细胞全能性向多能性的转换过程。
综上，本研究鉴定出核斑的上游调控因子TDP43，其可维持ZSA的动态平衡，为早期胚胎的转录后调控研究提供了全新视角。
每个样本取10枚胚胎，在含0.2%牛血清白蛋白（Bovine Serum Albumin, BSA）的1×磷酸盐缓冲液（Phosphate Buffered Saline, PBS）中洗涤后，收集于4.2 μl裂解缓冲液（含0.2% Triton X-100、RNase抑制剂、脱氧核糖核苷三磷酸（deoxyribonucleotide triphosphates, dNTPs）、寡聚dT引物以及1:1000比例的ERCC外源RNA对照（External RNA Controls Consortium, ERCC） spike-in）中进行裂解。随后参照既往报道的方法，采用Smart-seq2技术对样本进行逆转录与扩增。取500 pg扩增得到的cDNA，使用适配Illumina平台的TruePrep DNA Library Prep Kit V2（Vazyme, TD503）构建测序文库。将带有条码的文库混合后，在HiSeq X Ten测序平台上进行150 bp双端读长测序。