亚硫酸钠介导的s^(4)U转化检测转录组新生RNA中m^(6)A

Sodium Sulfite-Mediated s^(4)U Conversion for m^(6)A Profiling of Nascent Transcriptome-Wide RNA

发展了亚硫酸钠介导的4-硫代尿苷(s^(4)U)转化为胞苷(C)的方法,用s^(4)U标记新生RNA,通过反应将s^(4)U转化为C可成功检测新生RNA.将新生RNA使用s^(4)U标记后进行反应,再使用m^(6)A抗体对m^(6)A片段进行富集,从m^(6)A中分析T到C突变位点便成功区分出含m^(6)A的新生RNA.该方法有希望应用于单个基因上m^(6)A的动态变化检测,有助于人们进一步了解m^(6)A的修饰机制,为m^(6)A功能研究提供新的方法.

RNA sequencing can profile gene expression at steady-state level but is weak in studying temporal RNA dynam-ics.A common method for nascent RNA analysis is metabolic labeling with nucleoside analog.We developed a method for nascent RNA sequencing based on sodium sulfite-mediated 4-thiouridine-to-cytidine(s^(4)U-to-C)conversion.The RNA con-tained s^(4)U is reacted in the buffer(10 mmol/L Na_(2)SO_(3),200 mmol/L NH_(4)Cl and 200 mmol/L Na2HPO_(4)/NaH_(2)PO_(4),pH 7.4)at 70℃ for 4 h,the yield can reach more than 90%.This method can efficiently distinguish nascent RNA information from total RNAs.SUC-seq is used to investigate the m^(6)A,which is the most prevalent modification in mammalian mRNA and has been shown to have an essential regulatory role in the control of gene expression.Here,we provide a time resolved high-resolution profiling of m^(6)A on nascent RNA transcripts.3×106 HEK293T cells were seeded per 10-cm cell dish and grown for 24 h.Then,s^(4)U was added to the medium at a final concertation of 500μmol/L chased for 0,10,15,30,60,120 and 240 min.Total RNA was extracted using TRIzol reagent.10μg total RNA was fragmented using RNA fragmentation reagents at 70℃ for 10 min after gDNA was removed.Fragmented RNA was then subjected to s^(4)U reaction to realize the conversion of s^(4)U to C.Subsequently,5μg treated RNA were taken to perform m^(6)A immunoprecipitation.After removing rRNA,library was constructed and perform next-generation sequencing.A large number of T to C mutations were observed in the mapping reads.The T to C mutation rate of the control RNA was kept at a low background level.The results show that our method can successfully distinguish nascent RNA from total RNA.Via analyzing the nascent RNA in m^(6)A,the nascent m^(6)A can be distinguished from the total m^(6)A.This method can be a promising strategy to study the mechanism and function of m^(6)A.