title: A high-throughput assay to identify small-molecule modulators of alternative pre-mRNA splicing.
abstract: Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB's molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this high-throughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.
authors.affiliation: Department of Biopharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL 60612, USA.
authors.forename: Ahmet Dirim
authors.initials: AD
authors.lastname: Arslan
authors.forename: Xiaolong
authors.initials: X
authors.lastname: He
authors.forename: Minxiu
authors.initials: M
authors.lastname: Wang
authors.forename: Emily
authors.initials: E
authors.lastname: Rumschlag-Booms
authors.forename: Lijun
authors.initials: L
authors.lastname: Rong
authors.forename: William T
authors.initials: WT
authors.lastname: Beck
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D045744:Cell Line, Tumor; D004354:Drug Screening Assays, Antitumor; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics; D038941:Polypyrimidine Tract-Binding Protein / Q000235:genetics / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics; D015203:Reproducibility of Results; D054852:Small Molecule Libraries
chemical_list: D009419:Nerve Tissue Proteins; C504334:PTBP2 protein, human; D012322:RNA Precursors; D054852:Small Molecule Libraries; D038941:Polypyrimidine Tract-Binding Protein