Saplaoura Eleftheria, Perrera Valentina, Colot Vincent, Kragler Friedrich
Max Planck Institute of Molecular Plant Physiology;
Department of Molecular Medicine, Medical School, University of Padua.
J Vis Exp. 2020 May 14(159). doi: 10.3791/61231.
Secondary base modifications on RNA, such as mC, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.
RNA上的二级碱基修饰，如5-甲基胞嘧啶（mC），会影响被修饰的RNA分子的结构和功能。甲基化RNA免疫沉淀测序（MeRIP-seq）是一种旨在富集甲基化RNA并最终鉴定被修饰转录本的方法。简要来说，将超声破碎后的RNA与针对5-甲基胞嘧啶的抗体孵育，并在蛋白G磁珠的协助下进行沉淀。然后对富集的片段进行测序，并根据 reads 的分布和峰检测来定位潜在的甲基化位点。MeRIP可应用于任何生物体，因为它不需要任何先前的序列或修饰酶知识。此外，除了片段化之外，RNA无需进行任何其他化学或温度处理。然而，与其他方法不同，MeRIP-seq不能提供甲基化位点的单核苷酸预测，尽管甲基化区域可以缩小到几个核苷酸。使用不同的修饰特异性抗体可使MeRIP针对RNA上存在的不同碱基修饰进行调整，从而扩展了该方法的可能应用范围。
