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Review
. 2013 Feb 1;5(2):a011569.
doi: 10.1101/cshperspect.a011569.

The current status of vertebrate cellular mRNA IRESs

Affiliations
Review

The current status of vertebrate cellular mRNA IRESs

Richard J Jackson. Cold Spring Harb Perspect Biol. .

Abstract

Internal ribosome entry sites/segments (IRESs) were first discovered over 20 years ago in picornaviruses, followed by the discovery of two other types of IRES in hepatitis C virus (HCV), and the dicistroviruses, which infect invertebrates. In the meantime, reports of IRESs in eukaryotic cellular mRNAs started to appear, and the list of such putative IRESs continues to grow to the point in which it now stands at ~100, 80% of them in vertebrate mRNAs. Despite initial skepticism from some quarters, there now seems universal agreement that there is genuine internal ribosome entry on the viral IRESs. However, the same cannot be said for cellular mRNA IRESs, which continue to be shrouded in controversy. The aim of this article is to explain why vertebrate mRNA IRESs remain controversial, and to discuss ways in which these controversies might be resolved.

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Figures

Figure 1.
Figure 1.
Secondary structure models of the three main classes of picornavirus IRESs, with the eIF4G and PTB (polypyrimidine tract binding protein) binding sites also shown. (A) Secondary structures of the designated core IRESs, with individual structural domains labeled according to standard nomenclature. The dotted lines represent 5′-UTR sequences outside the core IRES boundaries, and the dashed line at 3′-end of the Aichivirus structure shows 5′-proximal viral coding sequences. The dark gray rectangle represents the ∼25 nt pyrimidine-rich tract at the 3′-end of the IRESs, the small red rectangle shows the authentic initiation site AUG, and the blue rectangle represents the putative ribosome recruitment AUG of Type I IRESs (at nt 586 in poliovirus type 1). The binding site of the central domain of eIF4G (p50 fragment—see Fig. 2A) on each IRES, as determined by footprinting and tethered hydroxyl radical probing, is shown in light gray, with the amino and carboxyl termini indicated (in red) to show the orientation of binding (Kolupaeva et al. 2003; de Breyne et al. 2009; Yu et al. 2011). (B) Sites and orientation of PTB binding, as determined by tethered hydroxyl radical probing. The interaction sites of each RBD (RNA-binding domain) of PTB-1 are shown on the three IRES secondary structure maps, using the same color coding as in Figure 2B, namely: RBD-1 in green, RBD-2 in pink, RBD-3 in blue and RBD-4 in yellow (Kafasla et al. 2009, 2010). The Aichivirus results showed that RBD-1 interacts strongly with the apical regions of both domains I and J, suggesting that these regions are closer to each other than can be shown on a two-dimensional diagram (Yu et al. 2011), which explains the elongated depiction of RBD-1. No contacts between RBD-4 and the Aichivirus IRES were detected.
Figure 2.
Figure 2.
Domain structures of eIF4GI and polypyrimidine tract binding protein (PTB). (A) Domain structure of the longest isoform (1599 amino acids) of eIF4GI (blue) with associated eIF4E (magenta), showing the interaction sites of poly(A) binding protein (PABP) in green, and eIF3 (gold). The two sites of potential interaction with eIF4A are shown, although there is usually only a single bound eIF4A. The sites at which eIF4GI is cleaved by poliovirus 2A protease and FMDV L-protease are shown; the 2A cleavage site defines the amino termini of the carboxy-terminal two-thirds fragment (p100) of eIF4GI, and the central one-third domain (p50). The sequence-independent RNA-binding motif at the amino terminus of p100 (and p50) is highlighted in orange; this motif is necessary for scanning, but is not required for internal initiation on the EMCV IRES (Ali and Jackson 2001; Prévôt et al. 2003). (B) Domain structure of PTB-1. The amino-terminal ∼55 amino acid residues have nuclear import and export signals, but play no part in RNA-binding. The positions of the 4 RBDs (RNA-binding domains) are shown. RBDs-2 and -3 are longer than the other two RBDs because their RNA-binding surface has an additional β-strand. The linkers between the RBDs are flexible except for that between RBDs-3 and -4, which interact with each other in a back-to-back configuration, and act as a coordinated pair (Oberstrass et al. 2005). PTB-2 and PTB-4 differ from the canonical PTB-1 in having inserts (arising from alternative splicing) of 19 or 26 amino acids, respectively, at residue 298 of PTB-1.
Figure 3.
Figure 3.
Mechanisms by which monocistronic mRNAs can arise from the dual luciferase plasmid construct commonly used to test for IRESs. In all three subpanels the Renilla luciferase (RLuc) ORF is shown in blue, the Photinus luciferase (FLuc) ORF in yellow, and the cellular mRNA 5′-UTR under test for IRES activity is in red. Panel (B) is on a reduced scale. (A) Configuration of the dual luciferase construct and its essential elements (promoter, chimeric intron, polyA addition signal, and enhancer). The downstream SV40 enhancer strongly activates transcription from the SV40 promoter, but can also activate transcription from any cryptic promoter element present in the putative IRES (as shown by the dashed line), thereby generating a monocistronic FLuc mRNA. (B) Transcription from a promoter in the vector backbone, near the pMB1 origin, also gives rise to monocistronic FLuc mRNA if the putative IRES has 3′-splice sites. The diagram shows two of the most abundant spliced RNA products found (Lemp et al. 2012), with the retained (exon) sequences shown in green, and the spliced out introns in red. About half of such monocistronic FLuc mRNAs are devoid of upstream AUG triplets, and so could give rise to high FLuc expression via cap-dependent scanning. These unanticipated mRNAs are equally abundant irrespective of whether the SV40 promoter is absent (as depicted) or present (Lemp et al. 2012). (C) Generation of a monocistronic FLuc mRNA by splicing from the 5′-splice donor site of the chimeric intron to 3′-splice site(s) fortuitously present in the putative IRES.
Figure 4.
Figure 4.
Highly sensitive murine leukaemia virus test for the presence of 3′-splice sites in putative IRESs (Baranick et al. 2008). The putative IRES and GFP reporter are inserted into the proviral DNA (driven by a CMV promoter) downstream from the env gene. The normal MLV splicing pattern (in the absence of the IRES-GFP insert) is shown in black, above the gene map. The presence of a 3′-splice site in the putative IRES promotes the alternative splicing pattern shown in red below the gene map. Consequently, there is very high GFP expression (i.e., apparent IRES activity) from the resulting capped monocistronic mRNA, but viral replication is severely impaired because of the decrease in full-length unspliced RNA for packaging, coupled with the reduction in gag, pol, and env protein synthesis. In contrast, with the EMCV IRES, GFP production is significantly lower (though nevertheless 10- to 20-fold greater than background), but there is no inhibitory effect on viral replication. The sensitivity of this assay is because of the double readout of (1) GFP expression (i.e., apparent IRES activity), and (2) viral replication.

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