Biology subjects

Arnould, B.

Publications and source records attributed to Arnould, B..

3 recordsLinked to original sources

bioRxiv2025

Illuminating the Role of A-to-I Editing in Gastric Cancer using EndoVIA 2.0

Background & AimsAdenosine-to-inosine (A-to-I) RNA editing is an essential post-transcriptional modification catalyzed by ADAR enzymes, and emerging evidence suggests its dysregulation can contribute to cancer. However, technical barriers have hindered spatial analysis of editing activity in formalin-fixed paraffin-embedded (FFPE) tissues--an abundant but challenging sample type. Here, we introduce EndoVIA 2.0, an optimized immunostaining assay that enables spatial detection of edited RNAs in FFPE tissues. MethodsUsing human Endonuclease V (hEndoV) as a molecular affinity reagent, we developed a calcium-dependent staining workflow compatible with crosslinked tissues. EndoVIA 2.0 was validated in cell lines with altered ADAR1 expression, fresh frozen tissues, gastric organoids, and FFPE tissue sections. Quantitative imaging was combined with machine-learning segmentation and spatial analysis. ResultsEndoVIA 2.0 successfully detected A-to-I editing changes in ADAR1 knockout and overexpression models and revealed differential editing patterns across tissues. In gastric organoids and FFPE tissues, the assay spatially resolved editing heterogeneity and distinguished ADAR1-deficient from ADAR1-sufficient cells. Application to long-archived lung and breast cancer FFPE tissues suggest EndoVIA 2.0s broad utility and potential of capturing disease-associated hyper-editing in malignant samples. ConclusionsEndoVIA 2.0 enables robust, spatial detection of A-to-I editing in FFPE tissues--circumventing the limitations of RNA extraction and unlocking access to archived clinical specimens. This platform lays the foundation for mapping RNA editing dynamics in cancer progression and may support future biomarker discovery.

molecular biology↗

bioRxiv2025

Affinity-guided labeling reveals P2X7 nanoscale membrane redistribution during microglial activation

ATP-gated purinergic P2X7 receptors are crucial ion channels involved in inflammation. They sense abnormal ATP release during stress or injury and are considered promising clinical targets for therapeutic intervention. However, despite their predominant expression in immune cells such as microglia, there is limited information on P2X7 membrane expression and regulation during inflammation at the single-molecule level, necessitating new labeling approaches to visualize P2X7 in native cells. Here, we present X7-uP, an unbiased, affinity-guided P2X7 chemical labeling reagent that selectively and covalently biotinylates endogenous P2X7 in BV2 cells, a murine microglial cell line, allowing subsequent labeling with streptavidin-Alexa 647 tailored for super-resolution imaging. We uncovered a nanoscale microglial P2X7 redistribution mechanism where evenly spaced individual receptors in quiescent cells undergo upregulation and clustering in response to the pro-inflammatory agent lipopolysaccharide and ATP, leading to synergistic interleukin-1{beta} release. Our method thus offers a new approach to revealing endogenous P2X7 expression at the single-molecule level. Impact statementAn affinity-guided chemical strategy enabling highly specific biotinylation of P2X7 receptors reveals, by super-resolution microscopy, how the nanoscale organization of endogenous P2X7 in BV2 microglial cells dynamically changes upon activation.

neuroscience↗

bioRxiv2024

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.

molecular biology↗