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 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 k

SUBMITTER: Hensel JA 

PROVIDER: S-EPMC8494765 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature