A mammalian cell-based assay for screening inhibitors of RNA cleavage.
RNA cleavage is a catalytic reaction which defines many types of RNA processing events, including those of metabolite-sensing riboswitch, self-splicing introns, mRNA splicing, tRNA processing, polyA-cleavage, and various small ribozymes such as hairpin and hammerhead ribozyme. In this chapter, we describe a general methodology for developing a mammalian cell-based high-throughput screening assay useful for identifying small molecules capable of inhibiting RNA cleavage in mammalian cells. In the specific assay described, a plasmid DNA vector in which the expression of a luciferase reporter gene is controlled by hammerhead ribozyme cleavage was stably introduced into the human 293 cell line. Such a cell line enabled the rapid screening of chemical compound libraries and the identification of cell membrane-permeable inhibitory molecules capable of blocking ribozyme cleavage. The general strategy described later could in principle be adapted to identify small molecule inhibitors of many types of RNA cleavage reactions.
Yen
Laising
L
Department of Pathology, Baylor College of Medicine, Houston, TX 77030, USA.
Stockwell
Brent R
BR
Mulligan
Richard C
RC
D001681:Biological Assay / Q000379:methods*; D002460:Cell Line; D002999:Clone Cells; D017930:Genes, Reporter; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D012313:RNA / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional / Q000187:drug effects*; D015203:Reproducibility of Results; D054852:Small Molecule Libraries / Q000032:analysis* / Q000494:pharmacology*
D016376:Oligonucleotides, Antisense; D054852:Small Molecule Libraries; D012313:RNA