Virus Internal Ribosome Entry Site Research Articles

Abstract 3482: Hypoxia induces translational regulation of lymphangiogenic growth factors

Abstract Hypoxia is a major condition for induction of angiogenesis during tumor development, nevertheless its role in lymphangiogenesis remain unclear. In this study, we investigate the role of hypoxia in the translational regulation of lymphangiogenic growth factors. The majority of cellular stresses such as hypoxia leads to an inhibition of mRNA translation in cell by the classical cap-dependent mechanism. However, several mRNAs are translated by an alternative mechanism mediated by internal ribosome entry sites (IRESs) located in their 5’ untranslated regions. We found IRES elements in mRNAs of lymphangiogenic growth factors such as VEGFA and FGF2, and demonstrated that hypoxia is able to activate IRES-mediated mRNA translation both in vitro and in vivo. Interestingly, our recent data demonstrated a strong induction of VEGFA and FGF2 IRES activities in vivo in pancreas adenocarcinoma CAPAN-1, that correlates with development of hypoxia area and lymphangiogenesis in primary tumors. Such an activation is not observed with the viral EMCV IRES used as a control, indicating a translational activation in response to tumoral hypoxia could be specific of angiogenic/lymphangiogenic factors IRESs. We are interested in the translational control of more specific of lymphangiogenic growth factors, VEGFC and VEGFD. We've shown, in CAPAN-1 model, that VEGFC and VEGFD translation correlates with hypoxia area development in vivo. To determine the presence of IRESs in VEGFC and -D mRNAs, we cloned the 5’ untranslated regions of murine and human VEGF-C and -D mRNAs in bicistronic lentivectors. First data suggest the presence of an IRES VEGFC and -D mRNAs. In further investigations, our purpose is to demonstrate that the process of IRES-dependent translation plays a pivotal role in the hypoxic response, and should lead to induction of lymphangiogenic factors, including VEGFC and VEGFD. Thus, cell proteins able to activate IRESs, called IRES trans-acting factors (ITAFs), would be crucial protagonists in the lymphangiogenesis process and potential taget for anti-angiogenic therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3482. doi:10.1158/1538-7445.AM2011-3482

The duck hepatitis virus 5'-UTR possesses HCV-like IRES activity that is independent of eIF4F complex and modulated by downstream coding sequences

Duck hepatitis virus (DHV-1) is a worldwide distributed picornavirus that causes acute and fatal disease in young ducklings. Recently, the complete genome of DHV-1 has been determined and comparative sequence analysis has shown that possesses the typical picornavirus organization but exhibits several unique features. For the first time, we provide evidence that the 626-nucleotide-long 5'-UTR of the DHV-1 genome contains an internal ribosome entry site (IRES) element that functions efficiently both in vitro and in mammalian cells. The prediction of the secondary structure of the DHV-1 IRES shows significant similarity to the hepatitis C virus (HCV) IRES. Moreover, similarly to HCV IRES, DHV-1 IRES can direct translation initiation in the absence of a functional eIF4F complex. We also demonstrate that the activity of the DHV-1 IRES is modulated by a viral coding sequence located downstream of the DHV-1 5'-UTR, which enhances DHV-1 IRES activity both in vitro and in vivo. Furthermore, mutational analysis of the predicted pseudo-knot structures at the 3'-end of the putative DHV-1 IRES supported the presence of conserved domains II and III and, as it has been previously described for other picornaviruses, these structures are essential for keeping the normal internal initiation of translation of DHV-1.

A La Autoantigen Homologue Is Required for the Internal Ribosome Entry Site Mediated Translation of Giardiavirus

Translation of Giardiavirus (GLV) mRNA is initiated at an internal ribosome entry site (IRES) in the viral transcript. The IRES localizes to a downstream portion of 5′ untranslated region (UTR) and a part of the early downstream coding region of the transcript. Recent studies indicated that the IRES does not require a pre-initiation complex to initiate translation but may directly recruit the small ribosome subunit with the help of a number of trans-activating protein factors. A La autoantigen homologue in the viral host Giardia lamblia, GlLa, was proposed as one of the potential trans-activating factors based on its specific binding to GLV-IRES in vitro. In this study, we further elucidated the functional role of GlLa in GLV-IRES mediated translation in Giardia by knocking down GlLa with antisense morpholino oligo, which resulted in a reduction of GLV-IRES activity by 40%. An over-expression of GlLa in Giardia moderately stimulated GLV-IRES activity by 20%. A yeast inhibitory RNA (IRNA), known to bind mammalian and yeast La autoantigen and inhibit Poliovirus and Hepatitis C virus IRES activities in vitro and in vivo, was also found to bind to GlLa protein in vitro and inhibited GLV-IRES function in vivo. The C-terminal domain of La autoantigen interferes with the dimerization of La and inhibits its function. An over-expression of the C-terminal domain (200–348aa) of GlLa in Giardia showed a dominant-negative effect on GLV-IRES activity, suggesting a potential inhibition of GlLa dimerization. HA tagged GlLa protein was detected mainly in the cytoplasm of Giardia, thus supporting a primary role of GlLa in translation initiation in Giardiavirus.

Kaempferol inhibits enterovirus 71 replication and internal ribosome entry site (IRES) activity through FUBP and HNRP proteins

Flavonoids are associated with multiple biological and pharmacological activities, including anti-enterovirus activity. An internal ribosomal entry site (IRES) required for viral protein translation is a potential drug target for enterovirus 71 (EV71). Regulation translation initiation requires the interaction of IRES specific trans-acting host factors with viral IRES element. By evaluation of 12 flavonoids against EV71 infection, we found that (a) 7,8-dihydroxyflavone, kaempferol, quercetin, hesperetin and hesperidin exhibited more than 80% of cell survival and inhibition of EV71 infection; however, no anti-oxidative effects were noted from these flavonoids; (b) among them, only 7,8-dihydroxyflavone, kaempferol and hesperetin showed 40% of viral IRES activity; (c) kaempferol interfered with EV71 virus replication and pseudotyped virus production; and (d) FUBP1, FUBP3, HNRPD, HNRH1 and HNRPF proteins are associated with EV71 5′-UTR as shown using RNA affinity pull-down assay coupled with LC–MS/MS analysis. We firstly found that kaempferol may change the composition of these IRES associated trans-acting factors, and affect IRES function and EV71 virus replication. These studies help not only to understand the IRES function but also the mechanism by which drug induced cellular proteins are acting against EV71 infection.

The importance of being short: The role of rabies virus phosphoprotein isoforms assessed by differential IRES translation initiation

The rabies virus (RV) phosphoprotein P is a multifunctional protein involved in viral RNA synthesis and in counteracting host innate immune responses. We have previously shown that RV P gene expression levels can be regulated by using picornavirus internal ribosome entry site (IRES) elements. Here we exploited a particular feature of the foot-and-mouth disease virus (FMDV) IRES, namely, preferential initiation at a downstream initiation codon, to address the role of N-terminally truncated RV phosphoproteins usually generated in RV-infected cells through ribosomal leaky scanning. Recombinant RVs in which P synthesis was directed by the poliovirus or FMDV IRES produced full-length P (P1) or a truncated form (P2), as the dominant product, respectively. While the P2 overexpressing virus showed attenuated growth in interferon-incompetent cells, it was superior to the P1 overexpressing virus in preventing expression of host interferon-stimulated genes. This indicates that in RV infected cells the availability of the truncated P2 protein is critical for viral resistance to interferon.

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

Bicistronic vectors are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. Internal ribosome entry site (IRES) elements have been utilized to initiate expression of an additional protein from a bicistronic vector. The IRES element commonly used in current bicistronic vectors originates from the encephalomyocarditis virus (EMCV). As IRES-mediated translation is dependent on availability of IRES trans-acting factors, which vary between cell types and species, adequate gene expression from the EMCV IRES element is not always achieved. To identify a novel IRES element that mediates gene expression consistently with a higher efficiency than the EMCV IRES, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the vascular endothelial growth factor and type 1 collagen-inducible protein (VCIP) IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results suggest that VCIP-IRES containing vectors improve gene expression compared with those harboring an EMCV-IRES. This could increase the potential benefits of bicistronic vectors for experimental and therapeutic purposes.

All-atom knowledge-based potential for RNA structure prediction and assessment

Over the recent years, the vision that RNA simply serves as information transfer molecule has dramatically changed. The study of the sequence/structure/function relationships in RNA is becoming more important. As a direct consequence, the total number of experimentally solved RNA structures has dramatically increased and new computer tools for predicting RNA structure from sequence are rapidly emerging. Therefore, new and accurate methods for assessing the accuracy of RNA structure models are clearly needed. Here, we introduce an all-atom knowledge-based potential for the assessment of RNA three-dimensional (3D) structures. We have benchmarked our new potential, called Ribonucleic Acids Statistical Potential (RASP), with two different decoy datasets composed of near-native RNA structures. In one of the benchmark sets, RASP was able to rank the closest model to the X-ray structure as the best and within the top 10 models for ∼93 and ∼95% of decoys, respectively. The average correlation coefficient between model accuracy, calculated as the root mean square deviation and global distance test-total score (GDT-TS) measures of C3' atoms, and the RASP score was 0.85 and 0.89, respectively. Based on a recently released benchmark dataset that contains hundreds of 3D models for 32 RNA motifs with non-canonical base pairs, RASP scoring function compared favorably to ROSETTA FARFAR force field in the selection of accurate models. Finally, using the self-splicing group I intron and the stem-loop IIIc from hepatitis C virus internal ribosome entry site as test cases, we show that RASP is able to discriminate between known structure-destabilizing mutations and compensatory mutations. RASP can be readily applied to assess all-atom or coarse-grained RNA structures and thus should be of interest to both developers and end-users of RNA structure prediction methods. The computer software and knowledge-based potentials are freely available at http://melolab.org/supmat.html. fmelo@bio.puc.cl; mmarti@cipf.es Supplementary data are available at Bioinformatics online.

Crystal structures of complexes containing domains from two viral internal ribosome entry site (IRES) RNAs bound to the 70S ribosome

Internal ribosome entry site (IRES) RNAs are elements of viral or cellular mRNAs that bypass steps of canonical eukaryotic cap-dependent translation initiation. Understanding of the structural basis of IRES mechanisms is limited, partially due to a lack of high-resolution structures of IRES RNAs bound to their cellular targets. Prompted by the universal phylogenetic conservation of the ribosomal P site, we solved the crystal structures of proposed P site binding domains from two intergenic region IRES RNAs bound to bacterial 70S ribosomes. The structures show that these IRES domains nearly perfectly mimic a tRNA • mRNA interaction. However, there are clear differences in the global shape and position of this IRES domain in the intersubunit space compared to those of tRNA, supporting a mechanism for IRES action that invokes hybrid state mimicry to drive a noncanonical mode of translocation. These structures suggest how relatively small structured RNAs can manipulate complex biological machines.

RNA Virus Harnesses MicroRNAs to Seize Host Translation Control

Picornaviruses have evolved elaborate strategies to subvert host translation. In this issue of Cell Host and Microbe, Ho etal. (2011) report that enterovirus infection induces the synthesis of a transcription factor that enhances the synthesis of microRNA-141, which suppresses translation of the cap-binding protein, eIF4E, mRNA to inhibit cap-dependent translation.

Structural analysis provides insights into the modular organization of picornavirus IRES

Picornavirus RNA translation is driven by the internal ribosome entry site (IRES) element. The impact of RNA structure on the foot-and-mouth disease virus (FMDV) IRES activity has been analyzed using Selective 2'Hydroxyl Acylation analyzed by Primer Extension (SHAPE) and high throughput analysis of RNA conformation by antisense oligonucleotides printed on microarrays. SHAPE reactivity revealed the self-folding capacity of domain 3 and evidenced a change of RNA structure in a defective GNRA mutant. A modified RNA conformation of this mutant was also evidenced by RNA accessibility to oligonucleotides. Interestingly, comparison of nucleotide reactivity with RNA accessibility revealed that SHAPE reactive nucleotides corresponding to the GNRA motif were not accessible to their respective target oligonucleotides. The differential response was observed both in domain 3 and the entire IRES. Our results demonstrate distant effects of the GNRA motif in the domain 3 RNA conformation, and highlight the modular organization of a picornavirus IRES.

Structurally conserved five nucleotide bulge determines the overall topology of the core domain of human telomerase RNA

Telomerase is a unique ribonucleoprotein complex that catalyzes the addition of telomeric DNA repeats onto the 3′ ends of linear chromosomes. All vertebrate telomerase RNAs contain a catalytically essential core domain that includes the template and a pseudoknot with extended helical subdomains. Within these helical regions is an asymmetric 5-nt internal bulge loop (J2a/b) flanked by helices (P2a and P2b) that is highly conserved in its location but not sequence. NMR structure determination reveals that J2a/b forms a defined S-shape and creates an ∼90 ° bend with a surprisingly low twist (∼10 °) between the flanking helices. A search of RNA structures revealed only one other example of a 5-nt bulge, from hepatitis C virus internal ribosome entry site, with a different sequence but the same structure. J2a/b is intrinsically flexible but the interhelical motions across the loop are remarkably restricted. Nucleotide substitutions in J2a/b that affect the bend angle, direction, and interhelical dynamics are correlated with telomerase activity. Based on the structures of P2ab (J2a/b and flanking helices), the conserved region of the pseudoknot (P2b/P3, previously determined) and the remaining helical segment (P2a.1–J2a.1 refined using residual dipolar couplings and the modeling program MC-Sym) we have calculated an NMR-based model of the full-length pseudoknot. The model and dynamics analysis show that J2a/b serves as a dominant structural and dynamical element in defining the overall topology of the core domain, and suggest that interhelical motions in P2ab facilitate nucleotide addition along the template and template translocation.

Alternative Ways to Think about Cellular Internal Ribosome Entry

Internal ribosome entry sites (IRESs) are specialized mRNA elements that allow recruitment of eukaryotic ribosomes to naturally uncapped mRNAs or to capped mRNAs under conditions in which cap-dependent translation is inhibited. Putative cellular IRESs have been proposed to play crucial roles in stress responses, development, apoptosis, cell cycle control, and neuronal function. However, most of the evidence for cellular IRES activity rests on bicistronic reporter assays, the reliability of which has been questioned. Here, the mechanisms underlying cap-independent translation of cellular mRNAs and the contributions of such translation to cellular protein synthesis are discussed. I suggest that the division of cellular mRNAs into mutually exclusive categories of "cap-dependent" and "IRES-dependent" should be reconsidered and that the implications of cellular IRES activity need to be incorporated into our models of cap-dependent initiation.

Effects of magnesium ions on two EMCV IRES key activity fragments

Using NMR magnetization transfer from water and ammonia-catalyzed exchange of the imino protons, changes have been monitored in base-pair kinetics induced by Mg(2 + ) in two key activity fragments r(CACCUGGCGACAGGUG) and r(GGCCAAAAGCC) of the encephalomyocarditis virus internal ribosome entry site. For r(CACCUGGCGACAGGUG), the addition of Mg(2 + ) reveals two types of base-pairs: r(U(545)·A) and r(G(546)·C), in the first category, have lifetimes only slightly higher in the presence of Mg(2 + ), whereas their dissociation constants are substantially reduced. This behavior has been termed proximal. The base-pairs r(G(553)·C) and r(G(554)·C), in the second category, have lifetimes substantially higher in the presence of Mg(2 + ), whereas their dissociation constants remain almost constant. This behavior has been termed distal. Mg(2 + ) has a specific effect on r(CACCUGGCGACAGGUG), the magnitude of which is progressively modulated from the proximal region of the 16-mer towards its distal region. For r(GGCCAAAAGCC), an intermediate behavior is found for base-pairs r(G(565)·C) and r(G(572)·C). Their lifetimes are slightly higher in the presence of Mg(2 + ) and their dissociation constants are significantly lower, a behavior resembling that of the 16-mer proximal region. These results indicate that Mg(2 + ) diffusively moves around r(GGCCAAAAGCC).

GTP-independent tRNA Delivery to the Ribosomal P-site by a Novel Eukaryotic Translation Factor

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA(i)(Met) occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.

Translation Driven by Picornavirus IRES Is Hampered from Sindbis Virus Replicons: Rescue by Poliovirus 2A Protease

Alphavirus replicons are very useful for analyzing different aspects of viral molecular biology. They are also useful tools in the development of new vaccines and highly efficient expression of heterologous genes. We have investigated the translatability of Sindbis virus (SV) subgenomic mRNA bearing different 5′-untranslated regions, including several viral internal ribosome entry sites (IRESs) from picornaviruses, hepatitis C virus, and cricket paralysis virus. Our findings indicate that all these IRES-containing mRNAs are initially translated in culture cells transfected with the corresponding SV replicon but their translation is inhibited in the late phase of SV replication. Notably, co-expression of different poliovirus (PV) non-structural genes reveals that the protease 2A (2A pro) is able to increase translation of subgenomic mRNAs containing the PV or encephalomyocarditis virus IRESs but not of those of hepatitis C virus or cricket paralysis virus. A PV 2A pro variant deficient in eukaryotic initiation factor (eIF) 4GI cleavage or PV protease 3C, neither of which cleaves eIF4GI, does not increase picornavirus IRES-driven translation, whereas L protease from foot-and-mouth disease virus also rescues translation. These findings suggest that the replicative foci of SV-infected cells where translation takes place are deficient in components necessary to translate IRES-containing mRNAs. In the case of picornavirus IRESs, cleavage of eIF4GI accomplished by PV 2A pro or foot-and-mouth disease virus protease L rescues this inhibition. eIF4GI co-localizes with ribosomes both in cells electroporated with SV replicons bearing the picornavirus IRES and in cells co-electroporated with replicons that express PV 2A pro. These findings support the idea that eIF4GI cleavage is necessary to rescue the translation driven by picornavirus IRESs in baby hamster kidney cells that express SV replicons.

Efficacy and safety of a modified vaccinia Ankara (MVA) vectored plague vaccine in mice

The efficacy and safety of plague vaccines based on the modified vaccinia Ankara (MVA) viral vector was evaluated. MVA recombinants were constructed expressing Yersinia pestis antigens under the translational control of the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES) and/or fused to the tissue plasminogen activator (tPA) secretory signal. A MVA/ Y. pestis recombinant that expressed a truncated version of the low-calcium response V antigen (MVA/IRES/tPA/V 307), conferred significant protection (87.5–100%) against intranasal or intraperitoneal challenge with CO92 (encapsulated) or Java 9 (non-encapsulated) strains of Y. pestis, respectively. In contrast, a MVA/ Y. pestis recombinant that expressed the full-length V antigen provided only 37.5% protection against challenge with CO92 or Java 9 strains, respectively. Interestingly, a MVA/ Y. pestis recombinant that expressed the capsular protein (F1) did not elicit significant antibody titers but still conferred 50% and 25% protection against CO92 or Java 9 challenge, respectively. The MVA/ Y. pestis recombinant viruses did not demonstrate any mortality or morbidity in SCID mice. Based on their safety and efficacy in mice, these MVA/ Y. pestis recombinants are candidates for further development as biodefense and public health vaccines.

A Modular Approach to Synthetic RNA Binders of the Hepatitis C Virus Internal Ribosome Entry Site

Natural products that target the RNA components of bacterial ribosomes, and thereby act as antibiotics that shut down microbial protein synthesis, have provided a rich source of inspiration for the design and synthesis of small molecule ligands directed at RNA targets.[1, 2] A prominent example of a privileged scaffold for RNA recognition occurring in natural aminoglycoside antibiotics is 2-deoxystreptamine (2-DOS),[3] which contains a rigid framework of hydrogen bond donors among which the rigid cis-1,3 arrangement of amino groups is responsible for selective interaction with structural motifs in RNA targets (Figure 1).[4] In an approach to reduce the complexity of chemical library synthesis involving the highly functionalized 2-DOS scaffold, we have recently developed the 3,5-diaminopiperidine heterocycle (DAP) as a structural mimetic of the RNA-recognizing pharmacophore of the 2-DOS scaffold (Figure 1). Structure-guided design had been applied to discover a series of antibacterial DAP-triazine derivatives that act on the same ribosomal RNA target as the natural aminoglycoside antibiotics which initially served as the inspiration for the conception of the DAP compounds.[5, 6]

Structure of the three-way helical junction of the hepatitis C virus IRES element

The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important conformational principles in the folding of three-way helical junctions. It is formally a 3HS(4) junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS(1)HS(3) and 2HS(2)HS(2) junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg(2+) ions in low micromolar concentrations, and suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e., helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique and stable fold.

Robust and generic RNA modeling using inferred constraints: a structure for the hepatitis C virus IRES pseudoknot domain.

RNA function is dependent on its structure, yet three-dimensional folds for most biologically important RNAs are unknown. We develop a generic discrete molecular dynamics-based modeling system that uses long-range constraints inferred from diverse biochemical or bioinformatic analyses to create statistically significant (p < 0.01) nativelike folds for RNAs of known structure ranging from 45 to 158 nucleotides. We then predict the unknown structure of the hepatitis C virus internal ribosome entry site (IRES) pseudoknot domain. The resulting RNA model rationalizes independent solvent accessibility and cryo-electron microscopy structure information. The pseudoknot domain positions the AUG start codon near the mRNA channel and is tRNA-like, suggesting the IRES employs molecular mimicry as a functional strategy.

Manipulation of ribosomes by viral RNAs

Viruses employ a variety of strategies to manipulate the basic molecular machinery of their hosts and to coordinate important events during infection. In many cases, virally encoded RNAs are at the heart of these strategies. One example of this are in viral internal ribosome entry sites (IRES) RNAs, which recruit, position, and activate the host cell's translation machinery to the viral RNA. The correct functioning of IRESs is critical for the successful infection of these viruses, which include hepatitis C virus, polio, HIV‐1, and others. In addition, IRESs are used by some cellular mRNAs and are thus are a potentially important mechanism of post‐transcriptional gene regulation. The intergenic region (IGR) IRESs of the Dicistroviridae viruses use the most streamlined IRES mechanism identified to date, binding directly to the ribosome and manipulating it into initiating translation of the viral message. As such, the IGR IRESs have been dubbed “molecular hijackers.” We are using the IGR IRESs as a model to understand some of the basic molecular tenets of IRES RNA structure, IRES‐ribosome interactions and IRES function, as well as to understand the basic workings of the ribosome that are potentially important for post‐transcription regulation, in general. We have developed a detailed model for the mechanism of the IGR IRESs using results from cryo‐EM, X‐ray crystallography, and other structural, functional, and biophysical methods. I will present new data from both structural and functional studies that test our mechanistic model and also suggest new ideas for how these RNA‐based molecular hijackers work.