Base-resolution sequencing of m6A in RNA

 描述(申请人提供)：DNA和组蛋白上的可逆化学修饰在调控真核生物的基因表达方面起着关键作用。在我们的工作之前，还没有显示出对RNA进行可逆的化学修饰会影响基因表达的例子。2011年，我们发现了第一个RNA去甲基酶FTO，它属于AlkB家族铁和2-酮戊二酸(2-KG)依赖的双加氧酶，与人类脂肪质量肥胖有关。FTO催化哺乳动物信使RNA(MRNA)和其他核RNA N6-甲基腺苷(M6A)最常见的内部修饰的氧化去甲基化。这一结果与随后m6A的转录组范围的作图结合在一起，重新唤起了人们对mRNA修饰研究的兴趣。我们还鉴定了甲基转移酶核心复合体以及能够选择性识别m6A修饰的mRNA的蛋白质；一系列阅读器蛋白与含有m6A的mRNA的结合影响目标mRNA的翻译状态和寿命。虽然世界各地的实验室正在研究m6A在各种生物过程中的功能作用，但我们还没有克服RNA中m6A研究的一个重大技术障碍：目前还没有高通量测序方法可以检测m6A的确切位置并揭示每个位点上m6A的修饰百分比。在目前的应用中，我们提出了两种新的方法来进行m6A的转录组范围的碱基分辨测序：i)ADAR介导的腺苷脱氨反应，区分未修饰的A和m6A；ii)甲基转移酶辅助的腺苷化学标记，以区分未修饰的A和m6A。在逆转录(RT)和随后的扩增过程中，这两种方法都会将RNA中未修饰的A转换为不同的碱基。M6A上甲基的存在阻碍了转化，从而使我们能够在测序中区分A和m6A。这些方法将在选定的生物系统中得到验证，并将以斑马鱼为模型，研究m6A在母体向合子转变过程中可能区分母体和合子mRNA的潜在作用。我们相信这些方法的可获得性将为未来RNA中m6A的研究提供使能工具。

 DESCRIPTION (provided by applicant): Reversible chemical modifications on DNA and histones play critical roles in regulating gene expression in eukaryotes. Prior to our work, no example of reversible chemical modifications on RNA that could affect gene expression had been shown. In 2011, we discovered the first RNA demethylase, FTO, a protein belonging to the AlkB family iron- and 2-ketoglutarate (2-KG)-dependent dioxygenases and which is associated with human fat mass obesity. FTO catalyzes oxidative demethylation of the most prevalent internal modifications of mammalian messenger RNA (mRNA) and other nuclear RNA, N6-methyladenosine (m6A). This result, taken together with subsequent transcriptome-wide mapping of m6A, has revived research interest in the investigation of mRNA modifications. We have also characterized the methyltransferase core complex as well as proteins that can selectively recognize m6A-modified mRNA; the binding of m6A-containing mRNA by a family of the reader proteins affects the translation status and lifetime of the target mRNA. While research is ongoing to investigate the functional roles of m6A in various biological processes in laboratories around the world, we have yet to overcome a significant technology hurdle for the study of m6A in RNA: at present no high-throughput sequencing method exists that can detect the exact locations of m6A and reveal the modification percentage of m6A at each site. In the current application, we propose two new methods for the transcriptome-wide, base-resolution sequencing of m6A: i) ADAR-mediated adenosine deamination that differentiates unmodified A from m6A; ii) methyltransferase-assisted chemical labeling of adenosine to identify unmodified A from m6A. Both methods convert unmodified A in RNA into a different base during reverse transcription (RT) and subsequent amplification. The presence of the methyl group on m6A hinders the conversion, thereby allowing us to differentiate A from m6A in sequencing. These methods will be validated in selected biological systems and will be used to investigate potential roles of m6A that may differentiate maternal from zygotic mRNA during maternal to zygotic transition using zebrafish as a model. We believe the availability of these methods will provide enabling tools to future research of m6A in RNA.