Single‐Cell Imaging of m<sup>6</sup>A Modified RNA Using m<sup>6</sup>A‐Specific In Situ Hybridization Mediated Proximity Ligation Assay (m<sup>6</sup>AISH‐PLA)
Xiaojun Ren, Ruijie Deng, Kaixiang Zhang, Yupeng Sun, Yue Li, Jinghong Li
Abstract
Abstract N 6 ‐methyladenosine (m 6 A) modification—the most prevalent mammalian RNA internal modification—plays key regulatory roles in mRNA metabolism. Current approaches for m 6 A modified RNA analysis limit at bulk‐population level, resulting in a loss of spatiotemporal and cell‐to‐cell variability information. Here we proposed a m 6 A‐specific in situ hybridization mediated proximity ligation assay (m 6 AISH‐PLA) for cellular imaging of m 6 A RNA, allowing to identify m 6 A modification at specific location in RNAs and image m 6 A RNA with single‐cell and single‐molecule resolution. Using m 6 AISH‐PLA, we investigated the m 6 A level and subcellular location of HSP70 RNA103‐m 6 A in response to heat shock stress, and found an increased m 6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m 6 AISH‐PLA can serve in the study of m 6 A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.