title: 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6148, USA.
authors.forename: Ihab
authors.initials: I
authors.lastname: Younis
authors.forename: Michael
authors.initials: M
authors.lastname: Berg
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kaida
authors.forename: Kimberly
authors.initials: K
authors.lastname: Dittmar
authors.forename: Congli
authors.initials: C
authors.lastname: Wang
authors.forename: Gideon
authors.initials: G
authors.lastname: Dreyfuss
mesh_terms: D017398:Alternative Splicing / Q000187:drug effects; D000891:Anti-Infective Agents, Local / Q000737:chemistry / Q000494:pharmacology; D000927:Anticonvulsants / Q000737:chemistry / Q000494:pharmacology; D002710:Chlorhexidine / Q000737:chemistry / Q000494:pharmacology; D003022:Clotrimazole / Q000737:chemistry / Q000494:pharmacology; D005091:Exons; D005444:Flunarizine / Q000737:chemistry / Q000494:pharmacology; D017930:Genes, Reporter; D006367:HeLa Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D046228:Microarray Analysis; D015394:Molecular Structure; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D000891:Anti-Infective Agents, Local; D000927:Anticonvulsants; D012333:RNA, Messenger; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D003022:Clotrimazole; D002710:Chlorhexidine; D005444:Flunarizine