Single‐Cell Imaging of m6A Modified RNA Using m6A‐Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH‐PLA)

Abstract

AbstractN6‐methyladenosine (m6A) modification—the most prevalent mammalian RNA internal modification—plays key regulatory roles in mRNA metabolism. Current approaches for m6A modified RNA analysis limit at bulk‐population level, resulting in a loss of spatiotemporal and cell‐to‐cell variability information. Here we proposed a m6A‐specific in situ hybridization mediated proximity ligation assay (m6AISH‐PLA) for cellular imaging of m6A RNA, allowing to identify m6A modification at specific location in RNAs and image m6A RNA with single‐cell and single‐molecule resolution. Using m6AISH‐PLA, we investigated the m6A level and subcellular location of HSP70 RNA103‐m6A in response to heat shock stress, and found an increased m6A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6AISH‐PLA can serve in the study of m6A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis.