EndoVIA for quantifying A-to-I editing and mapping the subcellular localization of edited transcripts.

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Adenosine-to-inosine (A-to-I) editing is a key RNA modification impacting gene expression and cellular functions.
• Dysregulation of A-to-I editing is implicated in various diseases, including cancers.
• Current methods for studying A-to-I editing lack spatial and single-cell resolution.

Purpose of the Study:

• To introduce and detail the Endonuclease V Immunostaining Assay (EndoVIA) for in situ detection of A-to-I edited RNA.
• To enable visualization of A-to-I editing with subcellular localization and single-cell resolution.
• To overcome limitations of traditional RNA extraction and pooling methods.

Main Methods:

• Development and application of the Endonuclease V Immunostaining Assay (EndoVIA).
• Cell sample preparation for in situ staining.
• Staining of A-to-I edited transcripts using EndoVIA.
• Quantification of global inosine abundance.
• Visualization of subcellular localization of inosine-containing RNAs.

Main Results:

• EndoVIA allows selective detection and visualization of A-to-I edited RNA in situ.
• The method preserves subcellular localization and single-cell variation information.
• Global inosine abundance can be quantified.
• Subcellular localization of inosine-containing RNAs can be visualized at the single-molecule level.

Conclusions:

• EndoVIA is a powerful tool for studying A-to-I editing in situ.
• This method provides critical insights into the role of A-to-I editing in biological processes and disease.
• Understanding A-to-I editing at a single-cell and subcellular level is essential for disease mechanism research.