Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045, USA.
Molecular Biology Graduate Program, University of Colorado-Denver Anschutz Medical Campus, Aurora, Colorado 80045, USA.
RNA. 2021 Apr;27(4):527-541. doi: 10.1261/rna.078543.120. Epub 2020 Dec 29.
Methylation at the N position of adenosine (mA) is one of the most abundant RNA modifications found in eukaryotes; however, accurate detection of specific mA nucleotides within transcripts has been historically challenging due to mA and unmodified adenosine having virtually indistinguishable chemical properties. While previous strategies such as methyl-RNA immunoprecipitation and sequencing (MeRIP-seq) have relied on mA-specific antibodies to isolate RNA fragments containing the modification, these methods do not allow for precise identification of individual mA residues. More recently, modified cross-linking and immunoprecipitation (CLIP)-based approaches that rely on inducing specific mutations during reverse transcription via UV cross-linking of the anti-mA antibody to methylated RNA have been used to overcome this limitation. However, the most utilized version of this approach, miCLIP, can be technically challenging to use for achieving high-complexity libraries. Here we present an improved methodology that yields high library complexity and allows for the straightforward identification of individual mA residues with reliable confidence metrics. Based on enhanced CLIP (eCLIP), our mA-eCLIP (meCLIP) approach couples the improvements of eCLIP with the inclusion of an input sample and an easy-to-use computational pipeline to allow for precise calling of mA sites at true single-nucleotide resolution. As the effort to accurately identify mAs in an efficient and straightforward way intensifies, this method is a valuable tool for investigators interested in unraveling the mA epitranscriptome.
腺苷 N 位的甲基化（mA）是真核生物中最丰富的 RNA 修饰之一；然而，由于 mA 和未修饰的腺苷在化学性质上几乎无法区分，因此在转录本中准确检测特定的 mA 核苷酸一直具有挑战性。虽然以前的策略，如甲基化 RNA 免疫沉淀和测序（MeRIP-seq），依赖于 mA 特异性抗体来分离含有修饰的 RNA 片段，但这些方法不能精确定位单个 mA 残基。最近，基于修饰的交联和免疫沉淀（CLIP）的方法已经被用于克服这一限制，这些方法依赖于通过抗 mA 抗体与甲基化 RNA 的 UV 交联在反转录过程中诱导特定突变。然而，这种方法中最常用的版本 miCLIP 在实现高复杂度文库时在技术上具有挑战性。在这里，我们提出了一种改进的方法，该方法可以产生高文库复杂度，并允许使用可靠的置信度指标来直接鉴定单个 mA 残基。基于增强型 CLIP（eCLIP），我们的 mA-eCLIP（meCLIP）方法将 eCLIP 的改进与包含输入样本和易于使用的计算管道相结合，以允许在真正的单核苷酸分辨率下精确调用 mA 位点。随着以高效和直接的方式准确识别 mAs 的努力的加强，这种方法是对解开 mA 转录后组感兴趣的研究人员的有价值的工具。
