摘要/Abstract
![](http://sioc-journal.cn/Jwk_hxxb/fileup/0567-7351/PIC/20210402135758.png)
发展了亚硫酸钠介导的4-硫代尿苷(s4U)转化为胞苷(C)的方法, 用s4U标记新生RNA, 通过反应将s4U转化为C可成功检测新生RNA. 将新生RNA使用s4U标记后进行反应, 再使用m6A抗体对m6A片段进行富集, 从m6A中分析T到C突变位点便成功区分出含m6A的新生RNA. 该方法有希望应用于单个基因上m6A的动态变化检测, 有助于人们进一步了解m6A的修饰机制, 为m6A功能研究提供新的方法.
关键词: 亚硫酸钠, 新生RNA, 4-硫代尿苷, N6-甲基腺嘌呤
RNA sequencing can profile gene expression at steady-state level but is weak in studying temporal RNA dynamics. 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 (s4U-to-C) conversion. The RNA contained s4U is reacted in the buffer (10 mmol/L Na2SO3, 200 mmol/L NH4Cl and 200 mmol/L Na2HPO4/NaH2PO4, 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 m6A, 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 m6A on nascent RNA transcripts. 3×106 HEK293T cells were seeded per 10-cm cell dish and grown for 24 h. Then, s4U was added to the medium at a final concertation of 500 μmol/L chased for 0, 10, 15, 30, 60, 120 and 240 min. Total RNA was extracted using TRIzol reagent. 10 μg total RNA was fragmented using RNA fragmentation reagents at 70 ℃ for 10 min after gDNA was removed. Fragmented RNA was then subjected to s 4U reaction to realize the conversion of s4U to C. Subsequently, 5 μg treated RNA were taken to perform m 6A 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 m6A, the nascent m6A can be distinguished from the total m6A. This method can be a promising strategy to study the mechanism and function of m6A.
Key words: sodium sulfite, nascent RNA, 4-thiouridine, N6-methyladenosine
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