Ybx1 binding sites and Ybx1-binding m5C sites in 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. Overall design: Examination of Ybx1 binding sites, and Ybx1-binding m5C sites in zebrafish embryo. RIP-seq: Briefly, 500 zebrafish embryos at shield stage (4 hpf) were resuspended with 2 ml lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 0.4 U/μl RNasin) by rotating at 4°C for 30 min and centrifuged at 15,000 g for 15 min. The supernatant was collected and pre-cleared with protein A Dynabeads by rotation at 4°C for 1 h. 50 μl pre-cleared embryo lysate was saved as Input and the left were incubated with affinity purified rabbit polyclonal anti-Ybx1 antibody (prepared by AbMax, China) and 30 μl Protein A Dynabeads for 4 h at 4°C or overnight. After washing with1 ml ice-cold NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 0.4U/µl RNasin) for eight times and once with 1ml ice-cold 1× PK buffer (100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA , 0.2% SDS), the beads was treated in 200 ul PK buffer containing 20 ul proteinase K (Roche, 0311582001) for 1 h at 55°C. The solution was collected and subjected to RNA extraction with Acid Phenol: ChCl3 (pH4.3~4.7) and ethanol precipitation. The Input RNA was extracted by using TRIzol reagent. Both the Input and IP RNA were treated by TURBOâ„¢ DNase (Invitrogen, AM2238). iCLIP-seq: 1000 zebrafish embryos at 4 hpf were irradiated twice with 0.8 J/cm2 (Stratalinker 2400, Stratagene), lysed and subjected to mild fragmentation. Crosslinked RNA-protein complexes were immunoprecipated using polyclonal Ybx1 antibody (Abmax) and protein A dynabeads. RNA was extracted and subjected to library construction using Smarter smRNA-Seq kit (Takara). All the libraries were sequenced using HiSeq2500 (Illumina) in paired-read mode, creating reads with a length of 150 bp. RNA-RIP-BisSeq: Briefly, 500 zebrafish embryos at shield stage (4 hpf) were resuspended with 2 ml lysis buffer (150 mM KCl, 10 mM HEPES pH 7.6, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 protease inhibitor cocktail, 0.4 U/μl RNasin) by rotating at 4°C for 30 min and centrifuged at 15,000 g for 15 min. The supernatant was collected and pre-cleared with protein A Dynabeads by rotation at 4°C for 1 h. 50 μl pre-cleared embryo lysate was saved as Input and the left were incubated with affinity purified rabbit polyclonal anti-Ybx1 antibody (prepared by AbMax, China) and 30 μl Protein A Dynabeads for 4 h at 4°C or overnight. After washing with1 ml ice-cold NT2 buffer (200 mM NaCl, 50 mM HEPES pH 7.6, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, 0.4U/µl RNasin) for eight times and once with 1ml ice-cold 1× PK buffer (100 mM Tris-HCl pH 7.4, 50 mM NaCl, 10 mM EDTA , 0.2% SDS), the beads was treated in 200 ul PK buffer containing 20 ul proteinase K (Roche, 0311582001) for 1 h at 55°C. The solution was collected and subjected to RNA extraction with Acid Phenol: ChCl3 (pH4.3~4.7) and ethanol precipitation. The purified mRNA was used for RNA-BisSeq library construction. Around 200 ng mRNA premixed with the in vitro transcribed Dhfr mRNA at a ratio of 300:1 (Dhfr mRNA serves as methylation conversion control) was fragmented to ~100-nt fragments for 1 min at 90°C in 10× RNA Fragmentation Reagent (Ambion), then stopped by 10× RNA stop solution (Ambion), and precipitated with 100% ethanol. The RNA pellet was resuspended in 100 µl bisulfite solution (pH 5.1), which is a 100:1 mixture of 40% sodium bisulfite (Sigma) and 600 µM hydroquinone (Sigma) and subjected to heat incubation at 75°C for 4.5 h. The reaction mixture was desalted by passing through Nanosep with 3K Omega 500/pk columns (PALL Corporation) with centrifugation. After washed with nuclease-free water and centrifuged for five times, the RNA was finally disolved in 75 ?l nuclease-free water and then desulfonated by incubation with an equal volume of 1 M Tris-HCl (pH 9.0) at 75 °C for 1 h. After ethanol precipitation, the RNA was resuspended in 11 µl of RNase-free water and subjected to library construction. Reverse transcription was carried out with superscript II Reverse Transcriptase (Invitrogen) and ACT random hexamers. The following procedures were performed with the KAPA Stranded mRNA-Seq Kit (KAPA) according to the manufacturer?s instructions.Libraries were sequenced using HiSeq2500 (Illumina) in paired-read mode, creating reads with a length of 125 bp.