Abstract
Functional RNAs often exhibit distinct subcellular localization, but studying circular RNA (circRNA) localization has been challenging due to their extensive sequence overlap with linear mRNAs. We developed amplicon-based circular RNA fluorescence in situ hybridization (abcFISH), a method that employs an optimized rolling circle amplification (RCA) strategy targeting back-splicing junction (BSJ) sites for robust, high-specificity, single-molecule circRNA imaging. abcFISH enables quantitative, multiplexed imaging in cells and tissues, allowing us to reveal alternative circularization patterns from the single locus; uncover cell-type-specific circPOLR2A(9,10) expression related to a combinatorial effect of RNA-binding proteins; map the distinct spatial distribution patterns of multiple circRNAs in neurons and brain tissues; and show the functional interplay of circRNA Cdr1as and lncRNA Cyrano co-localization. Furthermore, we applied abcFISH to validate the efficacy of therapeutic double-stranded circRNA aptamers (ds-cRNAs), simultaneously visualizing AAV-delivered ds-cRNAs and a consequent reduction in astrocyte infiltration around transduced cells. Collectively, abcFISH establishes a robust and user-friendly toolkit for deciphering circRNA localization and function in vivo.
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Abstract
Functional RNAs often exhibit distinct subcellular localization, but studying circular RNA (circRNA) localization has been challenging due to their extensive sequence overlap with linear mRNAs. We developed amplicon-based circular RNA fluorescence in situ hybridization (abcFISH), a method that employs an optimized rolling circle amplification (RCA) strategy targeting back-splicing junction (BSJ) sites for robust, high-specificity, single-molecule circRNA imaging. abcFISH enables quantitative, multiplexed imaging in cells and tissues, allowing us to reveal alternative circularization patterns from the single locus; uncover cell-type-specific circPOLR2A(9,10) expression related to a combinatorial effect of RNA-binding proteins; map the distinct spatial distribution patterns of multiple circRNAs in neurons and brain tissues; and show the functional interplay of Cdr1as and Cyrano co-localization. Furthermore, we applied abcFISH to validate the efficacy of therapeutic double-stranded circRNA aptamers (ds-cRNAs), simultaneously visualizing AAV-delivered ds-cRNAs and a consequent reduction in astrocyte infiltration around transduced cells. Collectively, abcFISH establishes a robust and user-friendly toolkit for deciphering circRNA localization and function in vivo.
Competing Interest Statement
L.-L. C., Y.-X. L. and B.-W. J. are named as inventors on patents related to abcFISH enables multiplexed, single-molecule visualization of circular RNA spatial heterogeneity held by CAS Center for Excellence in Molecular Cell Science. The other authors declare no competing interests.
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