Hawley Ben R, Jaffrey Samie R
Department of Pharmacology, Weill Medical College, Cornell University, New York, New York.
Curr Protoc Mol Biol. 2019 Apr;126(1):e88. doi: 10.1002/cpmb.88. Epub 2019 Mar 15.
The most prevalent modified base in mRNA, N -methyladenosine (m A), is found in several thousand transcripts, typically near the stop codon, although it can occur anywhere in the mRNA. In addition, the highly similar nucleotide N ,2'-O-dimethyladenosine (m Am), which is difficult to distinguish from m A, occurs as the first transcribed nucleotide of certain transcripts. Both the m A and m Am modifications have been implicated in numerous biological processes, and their precise mapping is crucial to understanding their functions. To address this need, we developed miCLIP, a method that maps both m A and m Am at individual nucleotide resolution. Here we describe the miCLIP protocol, with slight improvements to the initially published protocol for both the experimental methodology and bioinformatics analysis. © 2019 by John Wiley & Sons, Inc.
信使核糖核酸（mRNA）中最常见的修饰碱基N -甲基腺苷（m⁶A）存在于数千个转录本中，通常靠近终止密码子，不过它也可能出现在mRNA的任何位置。此外，与m⁶A难以区分的高度相似的核苷酸N⁶,2'-O-二甲基腺苷（m⁶Am），作为某些转录本的首个转录核苷酸出现。m⁶A和m⁶Am修饰都与众多生物学过程有关，对它们进行精确的定位对于理解其功能至关重要。为满足这一需求，我们开发了miCLIP，这是一种能以单个核苷酸分辨率对m⁶A和m⁶Am进行定位的方法。在此，我们描述了miCLIP实验方案，在实验方法和生物信息学分析方面对最初发表的方案做了些许改进。© 2019约翰威立国际出版公司
