An, H., Elvers, K.T., Gillespie, J.A. et al. (4 more authors) (2022) A toolkit for the identification of NEAT1_2/paraspeckle modulators. Nucleic Acids Research, 50 (20). e119. ISSN 0305-1048
Abstract
Paraspeckles are ribonucleoprotein granules assembled by NEAT1_2 lncRNA, an isoform of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1). Dysregulation of NEAT1_2/paraspeckles has been linked to multiple human diseases making them an attractive drug target. However currently NEAT1_2/paraspeckle-focused translational research and drug discovery are hindered by a limited toolkit. To fill this gap, we developed and validated a set of tools for the identification of NEAT1_2 binders and modulators comprised of biochemical and cell-based assays. The NEAT1_2 triple helix stability element was utilized as the target in the biochemical assays, and the cellular assay (‘ParaQuant’) was based on high-content imaging of NEAT1_2 in fixed cells. As a proof of principle, these assays were used to screen a 1,200-compound FDA-approved drug library and a 170-compound kinase inhibitor library and to confirm the screening hits. The assays are simple to establish, use only commercially-available reagents and are scalable for higher throughput. In particular, ParaQuant is a cost-efficient assay suitable for any cells growing in adherent culture and amenable to multiplexing. Using ParaQuant, we identified dual PI3K/mTOR inhibitors as potent negative modulators of paraspeckles. The tools we describe herein should boost paraspeckle studies and help guide the search, validation and optimization of NEAT1_2/paraspeckle-targeted small molecules.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2022 The Authors. This is an Open Access article distributed under the terms of the Creative Commons Attribution Licence (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
Keywords: | Cell biology; Nucleic Acid Enzymology |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield) > Department of Neuroscience (Sheffield) |
Funding Information: | Funder Grant number BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL UNSPECIFIED |
Depositing User: | Symplectic Sheffield |
Date Deposited: | 24 Oct 2022 09:47 |
Last Modified: | 17 Jul 2024 10:21 |
Status: | Published |
Publisher: | Oxford University Press (OUP) |
Refereed: | Yes |
Identification Number: | 10.1093/nar/gkac771 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:192458 |