Spatial Visualization of A-to-I Editing in Cells Using Endonuclease V Immunostaining Assay (EndoVIA).

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Adenosine-to-inosine (A-to-I) editing is a crucial post-transcriptional RNA modification catalyzed by ADARs.
• Dysregulation of A-to-I editing is linked to various diseases, including autoimmune disorders, neurological conditions, and cancer.
• The interplay between RNA localization and A-to-I editing is an emerging area with limited mechanistic understanding.

Purpose of the Study:

• To develop a novel method for visualizing and quantifying A-to-I edited RNA within intact cells.
• To overcome limitations of current in vitro methods that lose spatial and cellular heterogeneity information.
• To investigate the spatial landscape of A-to-I editing at the nanoscopic level.

Main Methods:

• Repurposing of endonuclease V (EndoV), a ribonuclease that specifically cleaves inosine bases.
• Development of an Endonuclease V Immunostaining Assay (EndoVIA) for cellular detection of edited RNA.
• Utilizing EndoVIA for spatial visualization and quantification of A-to-I edited transcripts.

Main Results:

• EndoVIA enables selective binding and detection of A-to-I edited RNA directly within cells.
• The assay provides spatial visualization of edited transcripts, revealing localization patterns.
• EndoVIA allows for rapid quantification of global inosine abundance and mapping of editing landscapes.

Conclusions:

• EndoVIA is a powerful new tool for studying A-to-I RNA editing in its native cellular context.
• This assay facilitates deeper understanding of the relationship between RNA editing, localization, and biological function.
• The findings open avenues for investigating the role of A-to-I editing in various physiological and pathological processes.