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

Adenosine-to-inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions, and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed endonuclease V (EndoV), a magnesium-dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces an endonuclease V immunostaining assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

Spatial visualization of A-to-I Editing in cells using Endonuclease V Immunostaining Assay (EndoVIA).

Adenosine-to-Inosine (A-to-I) editing is one of the most widespread post-transcriptional RNA modifications and is catalyzed by adenosine deaminases acting on RNA (ADARs). Varying across tissue types, A-to-I editing is essential for numerous biological functions and dysregulation leads to autoimmune and neurological disorders, as well as cancer. Recent evidence has also revealed a link between RNA localization and A-to-I editing, yet understanding of the mechanisms underlying this relationship and its biological impact remains limited. Current methods rely primarily on in vitro characterization of extracted RNA that ultimately erases subcellular localization and cell-to-cell heterogeneity. To address these challenges, we have repurposed Endonuclease V (EndoV), a magnesium dependent ribonuclease that cleaves inosine bases in edited RNA, to selectively bind and detect A-to-I edited RNA in cells. The work herein introduces Endonuclease V Immunostaining Assay (EndoVIA), a workflow that provides spatial visualization of edited transcripts, enables rapid quantification of overall inosine abundance, and maps the landscape of A-to-I editing within the transcriptome at the nanoscopic level.

Current advances in photocatalytic proximity labeling.

Understanding the intricate network of biomolecular interactions that govern cellular processes is a fundamental pursuit in biology. Over the past decade, photocatalytic proximity labeling has emerged as one of the most powerful and versatile techniques for studying these interactions as well as uncovering subcellular trafficking patterns, drug mechanisms of action, and basic cellular physiology. In this article, we review the basic principles, methodologies, and applications of photocatalytic proximity labeling as well as examine its modern development into currently available platforms. We also discuss recent key studies that have successfully leveraged these technologies and importantly highlight current challenges faced by the field. Together, this review seeks to underscore the potential of photocatalysis in proximity labeling for enhancing our understanding of cell biology while also providing perspective on technological advances needed for future discovery.