Identification of a Selective Polymerase Enables Detection of N6-Methyladenosine in RNA

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• 作者：Emily M. Harcourt ; Thomas Ehrenschwender ; Pedro J. Batista ; Howard Y. Chang ; Eric T. Kool
• 刊名：Journal of the American Chemical Society
• 出版年：2013
• 出版时间：December 26, 2013
• 年：2013
• 卷：135
• 期：51
• 页码：19079-19082
• 全文大小：251K
• ISSN：1520-5126

文摘

N⁶-methyladenosine (m⁶A) is the most abundant mRNA modification and has important links to human health. While recent studies have successfully identified thousands of mammalian RNA transcripts containing the modification, it is extremely difficult to identify the exact location of any specific m⁶A. Here we have identified a polymerase with reverse transcriptase activity (from Thermus thermophilus) that is selective by up to 18-fold for incorporation of thymidine opposite unmodified A over m⁶A. We show that the enzyme can be used to locate and quantify m⁶A in synthetic RNAs by analysis of pausing bands, and have used the enzyme in tandem with a nonselective polymerase to locate the presence and position of m⁶A in high-abundance cellular RNAs. By this approach we demonstrate that the long-undetermined position of m⁶A in mammalian 28S rRNA is nucleotide 4190.