Maternal mRNA Clearance Prevents Precocious Transcription and Genome Instability in Mouse Early Embryos [S4U-seq]

Oocytes accumulate abundant maternal mRNAs during the growth stage of meiotic maturation after transcriptional silencing, and early embryo development prior to zygotic genome activation (ZGA). During the oocyte-to-zygote transition, the selective and stepwise clearance of maternal transcripts is a prerequisite for the initiation of zygotic development. Maternal mutations in genes involved in mRNA clearance, including Pabpn1l, Cnot6l, and Btg4, lead to early embryonic arrest at the one-to-two cell stage in both mice and humans; however, the direct underlying mechanism is unclear. In the present study, optimized low-input sequencing methods of newly transcribed RNAs (neo-RNAs) were used, and the results provide evidence that the arrested zygotes abnormally accumulate transcription-associated DNA damages and R-loops in pronuclei. Ectopic R-loop formation and DNA strand breaks are accompanied by increased chromatin accessibility and leakage of maternal genes that should be turned off following fertilization. Abnormal R-loop accumulation causes the retardation of DNA replication and S-phase arrest during the first mitotic cell cycle. Overexpression of RNase H1 in maternal Pabpn1l knockout zygotes can remove genomic R-loops and partially reverse developmental defects. Collectively, the conjoint analysis of chromatin accessibility and neo-RNA transcription indicated that the failure of maternal mRNA clearance in mouse zygotes results in leakage transcription and extra R-loop formation, which is the primary reason for DNA replication stress, genome instability, and zygotic cell cycle arrest. Each sample contains 50 zygotes collected from mated female mice at 26 h after hCG injection were cultured in KSOM containing 200 μM 4-Thiouridine (S4U) (Sigma; T4509) for 2 h for labeling. After washing with DNase/RNase-free 0.2% BSA, samples were collected in DNase/RNase-free tubes. Total RNA was extracted using TRIzol reagent, and ribosomal RNA was removed using the rRNA depletion module (Abclonal; RK20348). The alkylation was performed using iodoacetamide (Sigma; I1149). Total RNA after iodoacetamide treatment was used as the input for library construction using the Fast RNA-seq Lib Prep Kit V2 (Abclonal; RK20306). DNA fragments were selected, and the final sequencing libraries were constructed from to 300-400 bp of the amplified cDNA. Sequencing data were extracted on the Illumina HiSeq X Ten platform in the 150 bp paired-end mode.