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m6A-REFII: an antibody-independent tool for in situ visualizing cellular specific RNA m6A compatible with protein
Yujie Mi1, Chenyang Huang1, Donghong Liu1
1MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou, 310058, China.
Abstract:
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.
Insights
Scientists developed m6A-REFII, a novel tool for visualizing single N6-methyladenosine (m6A) modifications on specific RNAs within cells. This advancement allows for spatial characterization of m6A dynamics and interactions in real-time.
Area of Science:
- Molecular Biology
- Epigenetics
- Cell Biology
Background:
- N6-methyladenosine (m6A) is a prevalent RNA modification regulating cellular processes.
- Current methods lack spatial resolution for m6A visualization within cells.
- Understanding m6A spatial dynamics is crucial for deciphering its functions.
Purpose of the Study:
- To develop a non-antibody-based fluorescent imaging tool for in situ visualization of single m6A sites on specific RNAs.
- To enable spatial characterization of m6A modifications and their interactions within living cells.
- To provide a tool for studying m6A dynamics under various cellular conditions.
Main Methods:
- Development of m6A REading and Fluorescent Imaging In situ (m6A-REFII) tool.
- m6A-REFII integrates m6A reader, hybridization probe, and fluorescent signaling modules.
- Application of m6A-REFII for visualizing m6A sites, dynamic changes, and protein-RNA interactions.
Main Results:
- Successfully visualized m6A-modified RNAs at distinct subcellular locations using m6A-REFII.
- Demonstrated dynamic changes in m6A sites in response to CRISPR-mediated reduction and heat shock.
- Validated m6A-REFII compatibility with protein immunofluorescence, revealing scaffold attachment factor B binding to m6A-modified L1 RNA.
Conclusions:
- m6A-REFII is a novel tool for single-cell, single-m6A site visualization on specific RNAs.
- The tool facilitates in situ spatial characterization of m6A modifications and their biological relevance.
- m6A-REFII advances the study of m6A biology in complex cellular environments.
