Transcriptomics analysis of gene expression in wide type, ybx1 MO, pabpc1a MO zebrafish embryo sample.

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the embryo undergoes dramatic reprogramming to convert maternal environment to embryonic-driven programing. However, how the maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) in zebrafish early embryos, we show that m5C methylated maternal mRNAs display higher stability during MZT. We identify that the Y box-binding protein 1 (Ybx1) prefers to recognizing m5C-modified mRNAs through p-p interaction with a key residue Trp45 in its cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Cooperated with an mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates a novel mechanism of RNA m5C methylation-regulated maternal mRNA stability during zebrafish MZT, highlighting the critical role of m5C mRNA methylation in early development. Examination of [1] gene expressive levels in wide type and ybx1 MO zebrafish embryo [2] gene expressive levels in pabpc1a MO zebrafish embryo [3] mRNA lifetime in wide type zebrafish embryo. Total RNA was isolated from zebrafish embryos harvested at different stages using TRIzol Reagent (Ambion). mRNA was extracted with using Dynabeads mRNA Purification Kit (Ambion) and subjected to TURBO DNase (Invitrogen) treatment at 37C for 30 min and ethanol precipitation. Thus purified mRNA was used for RNA-Seq library construction. Libraries were directly constructed using the KAPA Stranded mRNA-Seq Kit (KAPA) and were sequenced using HiSeq2500 (Illumina) in paired-read mode, creating reads with a length of 125 bp. Synchronously developing embryos were treated with 0.2 ng of Pol II inhibition alpha-amanitin (Sigma) at one-cell stage and were collected at indicated time points. 100 embryos were collected for each time point in duplicates. The total RNA was extracted by Trizol (Invitrogen) and used for RNA-Seq. An equal amount of external RNA control consortium (ERCC) RNA spike-in control (Thermo Fisher) was added to the total RNA samples as internal controls. The RNA was subjected to ribosomal RNA depletion with Ribo-Zero rRNA Removal Kit (Illumina) followed by library construction using NEB Next® Ultra? RNA library Prep Kit (NEB). RNA stability profiling was generated from two biological replicates.