摘要/Abstract

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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