Thomas Justin M, Bryson Keri M, Meier Jordan L
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, United States.
Chemical Biology Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, United States.
Methods Enzymol. 2019;621:31-51. doi: 10.1016/bs.mie.2019.02.022. Epub 2019 Mar 12.
Posttranscriptional modifications of RNA represent an emerging class of regulatory elements in human biology. Improved methods for studying how these elements are controlled and where they occur has the potential to transform our understanding of gene expression in development and disease. Here we describe a chemical method for nucleotide resolution sequencing of N4-acetylcytidine (ac4C), a highly conserved modified nucleobase whose formation is catalyzed by the essential cytidine acetyltransferase enzyme NAT10. This approach enables the sensitive, PCR-amplifiable detection of individual ac4C sites from nanograms of unfractionated cellular RNA. The sensitive and quantitative nature of this assay provides a powerful tool to understand how cytidine acetylation is targeted, profile RNA acetyltransferase dynamics, and validate the sites and stoichiometry of ac4C in novel RNA species.
RNA的转录后修饰是人类生物学中一类新兴的调控元件。研究这些元件如何被调控以及它们在何处发生的改进方法，有可能改变我们对发育和疾病中基因表达的理解。在此，我们描述了一种用于N4-乙酰胞苷（ac4C）核苷酸分辨率测序的化学方法，ac4C是一种高度保守的修饰核苷碱基，其形成由必需的胞苷乙酰转移酶NAT10催化。这种方法能够从纳克级未分级的细胞RNA中灵敏地、可通过PCR扩增检测单个ac4C位点。该检测方法的灵敏性和定量特性提供了一个强大的工具，用于了解胞苷乙酰化的靶向方式、描绘RNA乙酰转移酶动力学，以及验证新RNA物种中ac4C的位点和化学计量。
