Spatial Visualization of A-to-I Editing in Cells Using Endonuclease V Immunostaining Assay (EndoVIA).
Alexandria L QuillinBenoît ArnouldSteve D KnutsonJennifer M HeemstraPublished in: ACS central science (2024)
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.
Keyphrases
- crispr cas
- single cell
- induced apoptosis
- rna seq
- nucleic acid
- dna repair
- high throughput
- cell cycle arrest
- cell therapy
- squamous cell carcinoma
- cell death
- transcription factor
- endoplasmic reticulum stress
- room temperature
- bone marrow
- signaling pathway
- young adults
- microbial community
- blood brain barrier
- drug induced
- subarachnoid hemorrhage
- wastewater treatment
- dna damage
- childhood cancer