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

Abstract

Adenosine-to-inosine (A-to-I) editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a prevalent post-transcriptional modification that is vital for numerous biological functions. Given that this modification impacts global gene expression, RNA localization, and innate cellular immunity, dysregulation of A-to-I editing has unsurprisingly been linked to a variety of cancers and other diseases. However, our current understanding of the underpinning mechanisms that connect dysregulated A-to-I editing and disease processes remains limited. Widely used methods require RNA extraction and pooling that ultimately erases subcellular localization and cell-to-cell variation, which may be critical to understanding misregulation. To overcome these challenges, we recently developed Endonuclease V Immunostaining Assay (EndoVIA) to selectively detect and visualize A-to-I edited RNA in situ. In this chapter, we describe in detail how to prepare cell samples, stain A-to-I edited transcripts with EndoVIA, quantify global inosine abundance, and visualize the subcellular localization of inosine-containing RNAs at the single molecule level.