m6A-REFII: an antibody-independent tool for in situ visualizing cellular specific RNA m6A compatible with protein immunofluorescence staining

As one of the most prevalent messenger RNA modifications in eukaryotes, N 6 -methyladenosine (m 6 A) regulates various physiological and pathological processes. The RNA m 6 A sequencing techniques have significantly advanced the understanding of m 6 A biology, however, there is a lack of tools to in situ spatially characterize RNA m 6 A and its interacting components inside cells. Spatial information could significantly promote our understanding of the m 6 A dynamics and functions in complex biological contexts. While traditional m 6 A antibody immunostaining method images global cellular m 6 A sites, we here develop a non-antibody-based fluorescent imaging tool to in situ visualize single m 6 A site of specific cellular RNA, named m 6 A REading and Fluorescent Imaging In situ (m 6 A-REFII), which contains m 6 A reader module, hybridization probe module for targeting specific m 6 A-locating RNA sequence, and fluorescent signaling module. We successfully leveraged m 6 A-REFII to visualize m 6 A-modified RNAs at distinct subcellular localizations as well as to reveal the dynamic changes of targeted m 6 A sites in response to CRISPR-mediated m 6 A reduction and heat shock stimuli. Furthermore, we demonstrated the compatibility of m 6 A-REFII with protein immunofluorescence staining and validated the finding that the scaffold attachment factor B protein binds m 6 A-modified long interspersed element-1 RNA. Collectively, m 6 A-REFII is a new tool empowering visualization of single m 6 A modification on specific RNAs at the single-cell level.