Rapid-Response Splicing Reporter Screens Identify Differential Regulators of Constitutive and Alternative Splicing

Abstract

Bioactive compounds have been invaluable for dissecting the mechanisms, regulation, and functions of cellular processes. However, very few such reagents have been described for pre-mRNA splicing. To facilitate their systematic discovery, we developed a high-throughput cell-based assay that measures pre-mRNA splicing by utilizing a quantitative reporter system with advantageous features. The reporter, consisting of a destabilized, intron-containing luciferase expressed from a short-lived mRNA, allows rapid screens (<4 h), thereby obviating the potential toxicity of splicing inhibitors. We describe three inhibitors (out of >23,000 screened), all pharmacologically active: clotrimazole, flunarizine, and chlorhexidine. Interestingly, none was a general splicing inhibitor. Rather, each caused distinct splicing changes of numerous genes. We further discovered the target of action of chlorhexidine and show that it is a selective inhibitor of specific Cdc2-like kinases (Clks) that phosphorylate serine-arginine-rich (SR) protein splicing factors. Our findings reveal unexpected activities of clinically used drugs in splicing and uncover differential regulation of constitutively spliced introns.