The importance of m 6 A topology in chicken embryo mRNA; a precise mapping of m 6 A at the conserved chicken ß-actin zipcode
The importance of m 6 A topology in chicken embryo mRNA; a precise mapping of m 6 A at the conserved chicken ß-actin zipcode
复制标题
m 6 A拓扑结构在鸡胚mRNA中的重要性；
DOI：
10.1101/2022.03.04.483006
复制
发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Baron F
中科院分区：
文献类型：
--
作者：
Baron F
mRNA中的n6 -甲基腺苷（m6A）几乎调节着mRNA生命周期的每个阶段，使用m6A特异性甲基化RNA免疫沉淀与下一代测序（MeRIPSeq）或m6A个体核苷酸分辨率交联和免疫沉淀（miCLIP）的高通量检测mRNA中甲基化位点的方法的发展彻底改变了m6A的研究领域。这两种方法都是基于碎片化mRNA的免疫沉淀。然而，有充分的证据表明，抗体通常具有非特异性活性，因此使用抗体独立的方法验证已鉴定的m6A位点将是非常可取的。我们根据鸡胚MeRIPSeq结果和我们的rna - epimodificationdetection and base - recognition （RedBaron）抗体独立试验的数据，对鸡β-actin邮政编码中的m6A位点进行了定位和定量。我们还证明，β-肌动蛋白邮政编码中该位点的甲基化增强了ZBP1在体外的结合，而附近腺苷的甲基化则消除了这种结合。这表明m6A可能在调节β-肌动蛋白mRNA的局部翻译中发挥作用，m6A增强或抑制解读蛋白RNA结合的能力突出了在核苷酸分辨率上检测m6A的重要性。
N6-methyladenosine (m6A) in mRNA regulates almost every stage in the mRNA life cycle, and the development of methodologies for the high-throughput detection of methylated sites in mRNA using m6A-specific methylated RNA immunoprecipitation with next-generation sequencing (MeRIPSeq) or m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) have revolutionized the m6A research field. Both of these methods are based on immunoprecipitation of fragmented mRNA. However, it is well documented that antibodies often have nonspecific activities, thus verification of identified m6A sites using an antibody-independent method would be highly desirable. We mapped and quantified the m6A site in the chicken β-actin zipcode based on the data from chicken embryo MeRIPSeq results and ourRNA-EpimodificationDetection andBase-Recognition(RedBaron) antibody-independent assay. We also demonstrated that methylation of this site in the β-actin zipcode enhances ZBP1 binding in vitro, while methylation of a nearby adenosine abolishes binding. This suggests that m6A may play a role in regulating localized translation of β-actin mRNA, and the ability of m6A to enhance or inhibit a reader protein's RNA binding highlights the importance of m6A detection at nucleotide resolution.