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 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.
Molecular Biology Institute, University of California, Los Angeles 90095, USA.
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Venkatesan
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Dasgupta
D001483:Base Sequence; D002459:Cell Fusion; D002460:Cell Line; D005434:Flow Cytometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis; D011523:Protoplasts; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism*
D008164:Luminescent Proteins; D012335:RNA, Ribosomal; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins