Novel Fluorescence-Based Screen To Identify Small Synthetic Internal Ribosome Entry Site Elements

Abstract

We report here a novel fluorescent protein-based screen to identify small, synthetic internal ribosome entry site (IRES) elements in vivo. A library of bicistronic plasmids encoding the enhanced blue and green fluorescent proteins (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 the EBFP and EGFP reporters and fluorescence-activated cell sorting, and several small IRES elements were identified. Two of these elements were subsequently shown to possess IRES activity comparable to that of a variant of the encephalomyocarditis virus IRES element in a context-independent manner both in vitro and in vivo, and these elements functioned in multiple cell types. Although no sequence or structural homology was apparent between the synthetic 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 that these IRES elements compete with PV IRES-mediated translation by utilizing some of the same factors as the PV IRES to direct translation. The utility of this fluorescent protein-based screen in identifying IRES elements with improved activity as well as in probing the mechanism of IRES-mediated translation is discussed.

ACKNOWLEDGMENTS

We thank Weimin Tsai for his superb technical help, Steve Landt for discussions regarding protoplast fusion, Stephen Smale for comments on the manuscript, and Thomas Belin for helpful discussions on Bayesian statistics. We also thank Iris Williams and Ingrid Schmid of the UCLA Core Flow Cytometry Laboratory.

This work was supported by NIH grant AI 45733 to A.D. A.V. was supported by NIH Medical Scientist Training Program grant NRSA/GM 08042-15 and the UCLA Microbial Pathogenesis Training Grant.