A toolkit for the identification of NEAT1_2/paraspeckle modulators

• Published in: Nucleic acids research Vol. 50; no. 20; p. e119
• Main Authors: An, Haiyan, Elvers, Karen T, Gillespie, Jason A, Jones, Kimberley, Atack, John R, Grubisha, Olivera, Shelkovnikova, Tatyana A
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 11.11.2022
• Subjects: Cell Nucleus - genetics; Drug Discovery; Humans; Methods Online; Paraspeckles - drug effects; Paraspeckles - genetics; Protein Kinase Inhibitors - pharmacology; RNA, Long Noncoding - chemistry; RNA, Long Noncoding - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkac771

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.