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N 6 -methyladenosine (m 6 A) modification-the most prevalent mammalian RNA internal modification-plays key regulatory roles in mRNA metabolism. Current approaches for m 6 A modified RNA analysis limit at bulk-population level, resulting in a loss of spatiotemporal and cell-to-cell variability information. Here we proposed a m 6 A-specific in situ hybridization mediated proximity ligation assay (m 6 AISH-PLA) for cellular imaging of m 6 A RNA, allowing to identify m 6 A modification at specific location in RNAs and image m 6 A RNA with single-cell and single-molecule resolution. Using m 6 AISH-PLA, we investigated the m 6 A level and subcellular location of HSP70 RNA103-m 6 A in response to heat shock stress, and found an increased m 6 A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m 6 AISH-PLA can serve in the study of m 6 A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis., (© 2021 Wiley-VCH GmbH.)

Aims: Pulmonary arterial hypertension (PAH) is a fatal cardiovascular disease with a cancer-like phenotype. Competing endogenous RNA (ceRNA) networks extensively involve in its pathological processes. But rare ceRNA networks and profound molecular mechanisms have been revealed in PAH. The aim of this study was to illuminate the ceRNA networks in PAH., Materials and Methods: In this work, we have chosen the idiopathic PAH as an example. GSE15197 (mRNA) and GSE56914 (miRNA) from the Gene Expression Omnibus (GEO) were selected to explore key genes and novel ceRNA networks in PAH by a series of integrated bioinformatic analysis. To be more scientific, a part of pairs in identified ceRNA network were detected in hypoxia-induced HPASMCs. And the dual-luciferase assay was performed to certify the relationship between miRNAs and mRNAs., Key Findings: Totally, 311 differentially expressed genes (DEGs) were identified and functional enrichment analysis illuminated that the majority of DEGs were enriched in proliferation, anti-apoptosis, inflammation and cancer-related pathways. And 10 hub genes were determined via Cytohubba after PPI network construction. Sequentially, with stepwise reverse prediction and pan-cancer co-expression analysis from mRNA to LncRNA in TargetScan, miRNet, ENCORI (Starbase V3.0) databases, a crucially ceRNA network was identified including 14 LncRNAs, 2 miRNAs, and 3 mRNAs. Further, in hypoxia-induced HPASMCs, the alterations of mRNAs, miRNAs and LncRNAs and their relationship were in accordance with the results we identified., Significance: Consequently, the unique hub genes and ceRNA network we proposed may advance our understanding of the molecular mechanisms in PAH., (Copyright © 2021 Elsevier Inc. All rights reserved.)

N6‐methyladenosine (m6A) modification—the most prevalent mammalian RNA internal modification—plays key regulatory roles in mRNA metabolism. Current approaches for m6A modified RNA analysis limit at bulk‐population level, resulting in a loss of spatiotemporal and cell‐to‐cell variability information. Here we proposed a m6A‐specific in situ hybridization mediated proximity ligation assay (m6AISH‐PLA) for cellular imaging of m6A RNA, allowing to identify m6A modification at specific location in RNAs and image m6A RNA with single‐cell and single‐molecule resolution. Using m6AISH‐PLA, we investigated the m6A level and subcellular location of HSP70 RNA103‐m6A in response to heat shock stress, and found an increased m6A modified ratio and an increased distribution ratio in cytoplasm under heat shock. m6AISH‐PLA can serve in the study of m6A RNA in single cells for deciphering epitranscriptomic mechanisms and assisting clinical diagnosis. [ABSTRACT FROM AUTHOR]

Titelbild: Single-Cell Imaging of m6A Modified RNA Using m6A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH-PLA) (Angew. In ihrer Zuschrift auf S. 22828 postulieren Jinghong Li et al. eine leistungsstarke In-situ-Imaging-Methode, die als "m SP 6 sp A-specific in situ hybridization mediated proximity ligation assay" (m SP 6 sp AISH-PLA) bezeichnet wird und die Identifizierung von m SP 6 sp A-Modifikationen an spezifischen Stellen in RNA und die Bildgebung von m SP 6 sp A-RNA mit Einzelzell- und Einzelmolekül-Auflösung ermöglicht. In ihrer Zuschrift auf S. 22828 postulieren Jinghong Li et al. eine leistungsstarke In-situ-Imaging-Methode, die als "m SP 6 sp A-specific in situ hybridization mediated proximity ligation assay" (m SP 6 sp AISH-PLA) bezeichnet wird und die Identifizierung von m SP 6 sp A-Modifikationen an spezifischen Stellen in RNA und die Bildgebung von m SP 6 sp A-RNA mit Einzelzell- und Einzelmolekül-Auflösung ermöglicht. [Extracted from the article]

In their Communication on page 22646, Jinghong Li et al. propose a powerful in situ imaging method, termed m SP 6 sp A-specific in situ hybridization mediated proximity ligation assay (m SP 6 sp AISH-PLA), that enables the identification of m SP 6 sp A modifications at a specific location in RNAs and the imaging of m SP 6 sp A RNA with single-cell and single-molecule resolution. Keywords: in situ imaging; m6A; proximity ligation; single-molecule; spatiotemporal information EN in situ imaging m6A proximity ligation single-molecule spatiotemporal information 22589 22589 1 10/06/21 20211011 NES 211011 B N b SP B 6 b sp B -methyladenosine b (m SP 6 sp A)-modified RNA analysis is limited at the bulk-population level, thus not giving access to spatiotemporal and cell-to-cell variability information. Cover Picture: Single-Cell Imaging of m6A Modified RNA Using m6A-Specific In Situ Hybridization Mediated Proximity Ligation Assay (m6AISH-PLA) (Angew. [Extracted from the article]