High-ThroughputLuciferase Reporter System for Detection of pre-mRNA Splicing
The Gideon Dreyfusslab has developed rapid-responseluciferase (firefly P. pyralis) reporterplasmids for use in high-throughput screening of pre-mRNA splicing. Thisreporter system can be used for identifying previously unknown factors andpathways involved in pre-mRNA splicing in a compound screen.
Their reporter system is comprised of two plasmids: Luc (intronless; CMV-LUC2CP/ARE and Luc-I (intron-containing; CMV-LUC2CP/intron/ARE). The Luc-I intron is a chimeric β-globin/immunoglobulin intron that hasbeen commonly used in constitutive splicing studies and has been optimized forhigh efficiency splicing. If intron splicing does not occur in Luc-I, theproduced luciferase is truncated and lacks enzymatic activity. The Luc plasmid(intronless) is useful for counter-screening, i.e. to filter out any compoundsthat are affecting other global cell processes, like translation.
Luciferasereporter construct maps
This luciferase reporter system was also designed for a short screeningtime (<4 hours) in order to avoid complicating effects from global toxicitydue to loss of splicing. To this end, destabilizing sequences were added toshorten the half-life of both the luciferase mRNA (3’UTR AUUUA [5 consecutive])and protein (C-terminal CL1 & PEST). These modifications ensure that anysignal from full-length luciferase produced prior to the start of the screen isquickly removed. This system should help researchers further elucidate the manyfactors that affect alternative splicing in mammalian cells.
- Younis, et al. Mol Cell Biol. 2010 Apr;30(7):1718-28.