In situ visualization of m(6)A sites in cellular mRNAs

N (6)-methyladenosine (m(6)A) is an abundant RNA modification which plays critical roles in RNA function and cellular physiology. However, our understanding of how m(6)A is spatially regulated remains limited due to a lack of methods for visualizing methylated transcripts of interest in cells. Here, we develop DART-FISH, a method for in situ visualization of specific m(6)A sites in target RNAs which enables simultaneous detection of both m(6)A-modified and unmodified transcript copies. We demonstrate the ability of DART-FISH to visualize m(6)A in a variety of mRNAs across diverse cell types and to provide information on the location and stoichiometry of m(6)A sites at single-cell resolution. Finally, we use DART-FISH to reveal that m(6)A is not sufficient for mRNA localization to stress granules during oxidative stress. This technique provides a powerful tool for examining m(6)A-modified transcript dynamics and investigating methylated RNA localization in individual cells.