Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen

Abstract The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regula...

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Autores principales:	Jessica A. Hensel, Brent D. Heineman, Amy L. Kimble, Evan R. Jellison, Bo Reese, Patrick A. Murphy
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5d1ed72814604bcea9b2075dc80d131c
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spelling	oai:doaj.org-article:5d1ed72814604bcea9b2075dc80d131c2021-12-02T18:01:48ZIdentification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen10.1038/s41598-021-99079-12045-2322https://doaj.org/article/5d1ed72814604bcea9b2075dc80d131c2021-10-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-99079-1https://doaj.org/toc/2045-2322Abstract The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.Jessica A. HenselBrent D. HeinemanAmy L. KimbleEvan R. JellisonBo ReesePatrick A. MurphyNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Jessica A. Hensel Brent D. Heineman Amy L. Kimble Evan R. Jellison Bo Reese Patrick A. Murphy Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen
description	Abstract The extracellular matrix protein fibronectin (FN) is alternatively spliced in a variety of inflammatory conditions, resulting in increased inclusion of alternative exons EIIIA and EIIIB. Inclusion of these exons affects fibril formation, fibrosis, and inflammation. To define upstream regulators of alternative splicing in FN, we have developed an in vitro flow-cytometry based assay, using RNA-binding probes to determine alternative exon inclusion level in aortic endothelial cells. This approach allows us to detect exon inclusion in the primary transcripts themselves, rather than in surrogate splicing reporters. We validated this assay in cells with and without FN-EIIIA and -EIIIB expression. In a small-scale CRISPR KO screen of candidate regulatory splice factors, we successfully detected known regulators of EIIIA and EIIIB splicing, and detected several novel regulators. Finally, we show the potential in this approach to broadly interrogate upstream signaling pathways in aortic endothelial cells with a genome-wide CRISPR-KO screen, implicating the TNFalpha and RIG-I-like signaling pathways and genes involved in the regulation of fibrotic responses. Thus, we provide a novel means to screen the regulation of splicing of endogenous transcripts, and predict novel pathways in the regulation of FN-EIIIA inclusion.
format	article
author	Jessica A. Hensel Brent D. Heineman Amy L. Kimble Evan R. Jellison Bo Reese Patrick A. Murphy
author_facet	Jessica A. Hensel Brent D. Heineman Amy L. Kimble Evan R. Jellison Bo Reese Patrick A. Murphy
author_sort	Jessica A. Hensel
title	Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen
title_short	Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen
title_full	Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen
title_fullStr	Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen
title_full_unstemmed	Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen
title_sort	identification of splice regulators of fibronectin-eiiia and eiiib by direct measurement of exon usage in a flow-cytometry based crispr screen
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/5d1ed72814604bcea9b2075dc80d131c
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Identification of splice regulators of fibronectin-EIIIA and EIIIB by direct measurement of exon usage in a flow-cytometry based CRISPR screen

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