Novel fluorescence-based screen to identify small synthetic internal ribosome entry site elements

We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library o f bicistronic plasmids encoding the enhanced blue and green fluorescent pro teins (EBFP and EGFP) separated by randomized 50-nucleotide-long sequences was amplified in bacteria and delivered into mammalian cells via protoplast fusion, Cells that received functional IRES elements were isolated using t he EBFP and EGFP reporters and fluorescence-activated cell sorting, and sev eral small IRES elements were identified. Two of these elements were subseq uently shown to possess IRES activity comparable to that of a variant of th e encephalomyocarditis virus IRES element in a context-independent manner b oth in vitro and in vivo, and these elements functioned in multiple cell ty pes, Although no sequence or structural homology was apparent between the s ynthetic IRES elements and known viral and cellular IRES elements, the two synthetic IRES elements specifically blocked poliovirus (PV) IRES-mediated translation in vitro, Competitive protein-binding experiments suggested tha t these IRES elements compete with PV IRES-mediated translation by utilizin g some of the same factors as the PV IRES to direct translation. The utilit y of this fluorescent protein-based screen in identifying IRES elements wit h improved activity as well as in probing the mechanism of IRES-mediated tr anslation is discussed.