Quillin Alexandria L, Arnould Benoît, Knutson Steve D, Heemstra Jennifer M
Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63130, United States.
Merck Center for Catalysis, Princeton University, Princeton, New Jersey 08544, United States.
ACS Cent Sci. 2024 Jul 8;10(7):1396-1405. doi: 10.1021/acscentsci.4c00444. eCollection 2024 Jul 24.
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 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.
腺苷到次黄苷（A-to-I）编辑是最广泛的转录后RNA修饰之一，由作用于RNA的腺苷脱氨酶（ADARs）催化。A-to-I编辑在不同组织类型中存在差异，对众多生物学功能至关重要，其失调会导致自身免疫性疾病、神经疾病以及癌症。最近的证据还揭示了RNA定位与A-to-I编辑之间的联系，但对这种关系的潜在机制及其生物学影响的理解仍然有限。目前的方法主要依赖于对提取RNA的表征，而这最终会消除亚细胞定位和细胞间异质性。为应对这些挑战，我们重新利用了核酸内切酶V（EndoV），一种依赖镁的核糖核酸酶，它能切割编辑后RNA中的次黄苷碱基，以选择性地结合并检测细胞中的A-to-I编辑RNA。本文介绍了一种核酸内切酶V免疫染色测定法（EndoVIA），该工作流程可提供编辑转录本的空间可视化，能够快速定量总体次黄苷丰度，并在纳米水平绘制转录组内A-to-I编辑的图谱。
