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 IRESmediated 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.Internal ribosome entry site (IRES)-mediated translation was first recognized in picornaviruses as a mechanism of translation that is distinct from cap-dependent translation of cellular RNAs (19,27). Picornaviruses such as poliovirus (PV), encephalomyocarditis virus (EMCV), and rhinovirus were shown to possess a cis-acting region, the IRES, within the viral 5Ј untranslated region (5Ј UTR) that could mediate translation in the absence of a cap structure at the 5Ј end of the RNA. Subsequently, it was shown that a variety of other viruses, including hepatitis C virus (HCV), as well as certain cellular mRNAs, such as those encoding BiP, c-myc, and the G subunit of eukaryotic initiation factor 4 (eIF4G), also directed translation via IRES elements (21,38,42). All of these IRES elements span hundreds of nucleotides in length and possess complex secondary and tertiary structures.An understanding of the mechanisms of IRES-mediated translation has come about in recent years due mainly to in vitro studies of viral IRES elements. In particular, an in vitro assay for the formation of 48S preinitiation complexes has demonstrated that viral IRES elements do not require eIF4E, the cap-binding protein, for complex formation. In addition, various viruses require different combinations of other canonical translation factors; the EMCV IRES, for example, requires eIF4A and a portion of eIF4G for 48S complex formation, while the HCV IRES does not require either of these factors (28,29). Although these studies have shed light on the minimum required factors for viral IRES-mediated translation, it is apparent that t...

Searching for IRES

“…Relatively small IRES elements have also been synthetically derived using a bicistronic vector with 50-nt-long randomly generated sequences inserted between two reporter genes (Venkatesan and Dasgupta 2001). Although two of the sequences that exhibited IRES activity showed no sequence homology with any transcripts in the public databases, the findings did show that a relatively small sequence is needed for IRES activity.…”

Improved co-expression of multiple genes in vectors containing internal ribosome entry sites (IRESes) from human genes

“…25 As it has been reported that the nature of the upstream gene influences the translation efficiency of the viral IRES, 7 it would also be important to determine if our observations are also applicable when different therapeutic genes are introduced upstream and downstream of these cellular IRESes. The applicability of other cellular as well as artificial IRESes 26 for the co-expression of multiple genes for gene therapy can also be explored.…”

Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae

“…In addition, Dasgupta et al (34) identified two other (50-nt) sequences by using a method that differed primarily in how the constructs were introduced into cells. For both screens, cells were transfected with dicistronic constructs encoding two different fluorescent proteins.…”