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, N⁶-methyladenosine (m⁶A) regulates various physiological and pathological processes. The RNA m⁶A Sequencing techniques have significantly advanced the understanding of m⁶A biology, however, there is a lack of tools to in situ spatially characterize RNA m⁶A and its interacting components inside cells. Spatial information could significantly promote our understanding of the m⁶A dynamics and functions in complex biological contexts. While traditional m⁶A antibody immunostaining method images global cellular m⁶A sites, we here develop a non-antibody-based fluorescent imaging tool to in situ visualize single m⁶A site of specific cellular RNA, named m⁶A REading and Fluorescent Imaging In situ (m⁶A-REFII), which contains m⁶A reader module, hybridization probe module for targeting specific m⁶A-locating RNA sequence, and fluorescent signaling module. We successfully leveraged m⁶A-REFII to visualize m⁶A-modified RNAs at distinct subcellular localizations as well as to reveal the dynamic changes of targeted m⁶A sites in response to CRISPR-mediated m⁶A reduction and heat shock stimuli. Furthermore, we demonstrated the compatibility of m⁶A-REFII with protein immunofluorescence staining and validated the finding that the scaffold attachment factor B protein binds m⁶A-modified long interspersed element-1 RNA. Collectively, m⁶A-REFII is a new tool empowering visualization of single m⁶A modification on specific RNAs at the single-cell level.

fluorescent in situ m6A imaging; m6A-RNA and protein co-imaging; m6A-RNA subcellular localization; single m6A site visualization; spatial m6A information.