IRES Activity Research Articles

Translational control of the undifferentiated phenotype in ER‑positive breast tumor cells: Cytoplasmic localization of ERα and impact of IRES inhibition.

Using a series of potential biomarkers relevant to mechanisms of protein synthesis, we observed that estrogen receptor (ER)-positive breast tumor cells exist in two distinct yet interconvertible phenotypic states (of roughly equal proportion) which differ in the degree of differentiation and use of IRES-mediated translation. Nascently translated IGF1R in the cytoplasm positively correlated with IRES activity and the undifferentiated phenotype, while epitope accessibility of RACK1, an integral component of the 40S ribosomal subunit, aligned with the more differentiated IRES-off state. When deprived of soluble growth factors, the entire tumor cell population shifted to the undifferentiated phenotype in which IRES-mediated translation was active, facilitating survival under these adverse microenvironmental conditions. However, if IRES-mediated translation was inhibited, the cells instead were forced to transition uniformly to the more differentiated state. Notably, cytoplasmic localization of estrogen receptor α (ERα/ESR1) precisely mirrored the pattern observed with nascent IGF1R, correlating with the undifferentiated IRES-active phenotype. Inhibition of IRES-mediated translation resulted in both a shift in ERα to the nucleus (consistent with differentiation) and a marked decrease in ERα abundance (consistent with the inhibition of ERα synthesis via its IRES). Although breast tumor cells tolerated forced differentiation without extensive loss of their viability, their reproductive capacity was severely compromised. In addition, CDK1 was decreased, connexin 43 eliminated and Myc translation altered as a consequence of IRES inhibition. Isolated or low-density ER-positive breast tumor cells were particularly vulnerable to IRES inhibition, losing the ability to generate viable cohesive colonies, or undergoing massive cell death. Collectively, these results provide further evidence for the integral relationship between IRES-mediated translation and the undifferentiated phenotype and demonstrate how therapeutic manipulation of this specialized mode of protein synthesis may be used to limit the phenotypic plasticity and incapacitate or eliminate these otherwise highly resilient breast tumor cells.

Hsc70 regulates the IRES activity and serves as an antiviral target of enterovirus A71 infection

Enterovirus A71 (EV-A71) is a small positive-stranded RNA virus that causes human hand, foot and mouth disease (HFMD) and fatal neurological disorders in some cases without effective treatment. Here we show that heat shock cognate protein 70 (Hsc70), a molecular chaperone, displays pivotal role in viral infections. Knockdown of Hsc70 significantly suppresses viral replication evidenced by reducing not only the level of both viral replication intermediates (negative stranded RNA) and viral genomic RNA (positive stranded RNA), but also the level of viral protein expression; whereas ectopic expression of Hsc70 markedly promotes viral replication. Interestingly, depletion of Hsc70 decreases the IRES activity of EV-A71, and the ectopic expression of Hsc70 enhances the IRES activity accordingly. Further study shows that Hsc70 binds viral genomic RNA but does not directly interact with IRES. Moreover, we reveal that Hsc70 interacts with 2A protease and promotes eIF4G cleavage. More importantly, Hsc70 inhibitor Ver-155008 significantly protects cytopathic effects from EV-A71 infection and inhibits both IRES activity and viral reproduction in a dose-dependent manner. The cell viability assay shows that the IC50 and CC50 are 2.01 μM and 47.67 μM, respectively. These results demonstrate not only an important mechanism of Hsc70 in facilitating EV-A71 replication, but also a target for antiviral drug development.

Regulation of enterovirus 2A protease-associated viral IRES activities by the cell's ERK signaling cascade: Implicating ERK as an efficiently antiviral target

In a previous study the ERK1/2 pathway was found to be crucially involved in positive regulation of the enterovirus A 71(EV-A71) IRES (vIRES), thereby contributing to the efficient replication of an important human enterovirus causing death in young children (<5yrs) worldwide. This study focuses on unraveling more about the detailed mechanism of ERK's involvement in this regulation of vIRES. Through the use of siRNAs and specifically pharmacological inhibitor U0126, the ERK cascade was shown to positively regulate EV-A71-mediated cleavage of eIF4GI that established the cellular conditions which favour vIRES-dependent translation. Site-directed mutagenesis of the viral 2A protease (2Apro) was undertaken to show that the positive regulation of virus replication by the ERK cascade was mediated through effects on both the cis-cleavage of the viral polyprotein by 2Apro and its trans-cleavage of cellular eIF4GI. This ERK-2Apro linked network coordinating vIRES efficiency was also found in other important human enteroviruses. This identification of the ERK cascade as having a key role in maintaining the 2Apro proteolytic activity required to maximize enterovirus IRES activity, expands current understanding of the diverse functions of the ERK signaling cascade in the regulation of viral translation, therefore providing a potentially comprehensive drug target for anti-enterovirus infection.

The Triticum Mosaic Virus 5' Leader Binds to Both eIF4G and eIFiso4G for Translation.

We recently identified a remarkably strong (739 nt-long) IRES-like element in the 5’ untranslated region (UTR) of Triticum mosaic virus (TriMV, Potyviridae). Here, we define the components of the cap-binding translation initiation complex that are required for TriMV translation. Using bio-layer interferometry and affinity capture of the native translation apparatus, we reveal that the viral translation element has a ten-fold greater affinity for the large subunit eIF4G/eIFiso4G than to the cap binding protein eIF4E/eIFiso4E. This data supports a translation mechanism that is largely dependent on eIF4G and its isoform. The binding of both scaffold isoforms requires an eight base-pair-long hairpin structure located 270 nucleotides upstream of the translation initiation site, which we have previously shown to be crucial for IRES activity. Despite a weak binding affinity to the mRNA, eIFiso4G alone or in combination with eIFiso4E supports TriMV translation in a cap-binding factor-depleted wheat germ extract. Notably, TriMV 5’ UTR-mediated translation is dependent upon eIF4A helicase activity, as the addition of the eIF4A inhibitor hippuristanol inhibits 5’ UTR-mediated translation. This inhibition is reversible with the addition of recombinant wheat eIF4A. These results and previous observations demonstrate a key role of eIF4G and eIF4A in this unique mechanism of cap-independent-translation. This work provides new insights into the lesser studied translation mechanisms of plant virus-mediated internal translation initiation.

The cis-acting replication element of the Hepatitis C virus genome recruits host factors that influence viral replication and translation.

The cis-acting replication element (CRE) of the hepatitis C virus (HCV) RNA genome is a region of conserved sequence and structure at the 3′ end of the open reading frame. It participates in a complex and dynamic RNA-RNA interaction network involving, among others, essential functional domains of the 3′ untranslated region and the internal ribosome entry site located at the 5′ terminus of the viral genome. A proper balance between all these contacts is critical for the control of viral replication and translation, and is likely dependent on host factors. Proteomic analyses identified a collection of proteins from a hepatoma cell line as CRE-interacting candidates. A large fraction of these were RNA-binding proteins sharing highly conserved RNA recognition motifs. The vast majority of these proteins were validated by bioinformatics tools that consider RNA-protein secondary structure. Further characterization of representative proteins indicated that hnRNPA1 and HMGB1 exerted negative effects on viral replication in a subgenomic HCV replication system. Furthermore DDX5 and PARP1 knockdown reduced the HCV IRES activity, suggesting an involvement of these proteins in HCV translation. The identification of all these host factors provides new clues regarding the function of the CRE during viral cycle progression.

The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach

The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5’-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2′-OMe modified antisense oligomers hybridizing to the 5'-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers’ binding were monitored by the Pb2+-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.

The Expression of CUGBP1 After Spinal Cord Injury in Rats.

CUG-binding protein 1, a member of the CELF (CUGBP and embryonic lethal abnormal vision-like factor) family of RNA-binding proteins, is shown to be multifunctional, regulating many posttranscriptional processes including alternative splicing, deadenylation, mRNA decay, and translation. Recently, CUGBP1 is found to represses p27 IRES activity and inhibits expression of endogenous p27 in cultured breast cancer cells. However, the roles of CUGBP1 in central nervous system injury remain unknown. In our study, we performed acute spinal cord injury (SCI) model in adult rats in order to research the expression changes of CUGBP1 in spinal cord. Western blot analysis showed a marked upregulation of CUGBP1 after SCI. Immunohistochemistry analysis revealed a wide distribution of CUGBP1 in the spinal cord. Double immunofluorescence staining indicated that CUGBP1 immunoreactivity was increased predominantly in neurons and astrocytes after SCI. Moreover, colocalization of CUGBP1/proliferating cell nuclear antigen (PCNA) was detected in GFAP positive cells. We also examined the expression profiles of p27, which was up-regulated after SCI. To further understand whether CUGBP1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that CUGBP1 expression was positively correlated with PCNA expression, and the p27 expression was negatively correlated with CUGBP1 expression following LPS stimulation. Our results suggest that CUGBP1 might be implicated in the pathophysiology of spinal cord after SCI.

The 5′-UTR of DDB2 harbors an IRES element and upregulates translation during stress conditions

DDB2 is a tumor-inhibiting factor not only involved a major DNA repair mechanism in the Nucleotide Excision Repair (NER), but also correlated with cell apoptosis in the DNA damage response pathway. During serum-starvation, we noted that the translation levels of DDB2 were increased. To evaluate whether the 5′-UTR of DDB2 harbors an IRES element, we used a bicistronic luciferase plasmid with the 5′-UTR of DDB2 inserted between two cistron coding regions. We found that DDB2 5′-UTR could initiate the downstream reporter, demonstrating that the 5′-UTR of DDB2 contained an IRES. The 5′-UTR of DDB2 was predicted into a relatively stable secondary structure by the Mfold program. We deleted the stem-loops in turn to analyze the core part of IRES and found that full length of the 5′-UTR was significant for the IRES activity. Furthermore, our data demonstrated that the DDB2 IRES activity was promoted during stress conditions. These results reveal a novel mechanism contributing to DDB2 expression.

0170 : VEGF-D translational regulations promotes tumor lymphatic vessels plasticity

The lymphatic vasculature has a major role in physiology. It drains the interstitial fluids and performs immune surveillance by transporting immunity cells. The lymphatic vessels are also involved in pathological conditions such as chronic inflammatory diseases, lymphedema and cancer. Lymphangiogenesis, the growth of new lymphatic vessels, is induced by the lymphangiogenic growth factors VEGF-C and VEGF-D. Recent studies suggest that VEGF-D is also involved in lymphatic dilatation through prostaglandin signaling pathways. In this study, we demonstrated the molecular mechanism involved in lymphatic dilatation. We identified a stress-induced translational regulation of VEGF-D expression through an IRES activation under heat shock conditions. We demonstrated that VEGF-D IRES activity was dependent of prostaglandin pathway as Cox-2 inhibitors are able to abolish the IRES-promoted VEGF-D protein synthesis, and lymphatic vessels dilatation. Using plasmon surface resonance on biotinylated VEGF-D mRNA coupled to mass spectrometry, we identified the IRES transacting factor (ITAF) that allows the recruitment of the ribosome to promote VEGF-D translational initiation: the Nucleolin. Our results bring new insights on the VEGF-D regulation and on the lymphatic vessels plasticity. As lymphatic vessels play a crucial role in inflammation by performing immune cells tissue trafficking, understanding the regulations of lymphatic vasodilatation is a crucial step toward an innovative inflammatory disease therapy.

Magnesium lithospermate B and rosmarinic acid, two compounds present in Salvia miltiorrhiza, have potent antiviral activity against enterovirus 71 infections

The aim of this study was to identify the active ingredients responsible for the anti-EV71 activity produced by Salvia miltiorrhiza extracts. A pGS-EV71 IRES-based bicistronic reporter assay platform was used for rapid analysis of compounds that could specifically inhibit EV71 viral IRES-mediated translation. The analysis identified 2 caffeic acid derivatives, magnesium lithospermate B (MLB) and rosmarinic acid (RA), which suppressed EV71 IRES-mediated translation at concentrations of 30μg/ml. We also found that MLB and RA inhibited EV71 infection when they were added to RD cells during the viral absorption stage. MLB had a low IC50 value of 0.09mM and a high TI value of 10.52. In contrast, RA had an IC50 value of 0.50mM with a TI value of 2.97. MLB and RA (100µg/ml) also reduced EV71 viral particle production and significantly decreased VP1 protein production. We propose that these two derivatives inhibit EV71 viral entry into cells and viral IRES activity, thereby reducing viral particle production and viral RNA expression and blocking viral VP1 protein translation. This study provides useful information for the development of anti-EV71 assays and reagents by demonstrating a convenient EV71 IRES-based bicistronic assay platform to screen for anti-EV71 IRES activity, and also reports 2 compounds, MLB and RA, which are responsible for the anti-EV71 activity of S. miltiorrhiza.

Apigenin inhibits enterovirus 71 replication through suppressing viral IRES activity and modulating cellular JNK pathway

Enterovirus 71 (EV71) is a member of genus Enterovirus in Picornaviridae family, which is one of the major causative agents for hand, foot and mouth disease (HFMD), and sometimes associated with severe central nervous system diseases in children. Currently there are no effective therapeutic medicines or vaccines for the disease. In this report, we found that apigenin and luteolin, two flavones that differ only in the number of hydroxyl groups could inhibit EV71-mediated cytopathogenic effect (CPE) and EV71 replication with low cytotoxicity. Both molecules also showed inhibitory effect on the viral polyprotein expression. They prevented EV71-induced cell apoptosis, intracellular reactive oxygen species (ROS) generation and cytokines up-regulation. Time-of-drug addition study demonstrated that apigenin and luteolin acted after viral entry. We examined the effect of apigenin and luteolin on 2Apro and 3Cpro activity, two viral proteases responsible for viral polyprotein processing, and found that they showed less inhibitory activity on 2Apro or 3Cpro. Further studies demonstrated that apigenin, but not luteolin could interfere with viral IRES activity. Also, apigenin inhibited EV71-induced c-Jun N-terminal kinase (JNK) activation which is critical for viral replication, in contrast to luteolin that did not. This study demonstrated that apigenin may inhibit EV71 replication through suppressing viral IRES activity and modulating cellular JNK pathway. It also provided evidence that one hydroxyl group difference in the B ring between apigenin and luteolin resulted in the distinct antiviral mechanisms. This study will provide the basis for better drug development and further identification of potential drug targets.

PTBP1 is required for glucose-stimulated cap-independent translation of insulin granule proteins and Coxsackieviruses in beta cells.

Glucose and GLP-1 stimulate not only insulin secretion, but also the post-transcriptional induction of insulin granule biogenesis. This process involves the nucleocytoplasmic translocation of the RNA binding protein PTBP1. Binding of PTBP1 to the 3′-UTRs of mRNAs for insulin and other cargoes of beta cell granules increases their stability. Here we show that glucose enhances also the binding of PTBP1 to the 5′-UTRs of these transcripts, which display IRES activity, and their translation exclusively in a cap-independent fashion. Accordingly, glucose-induced biosynthesis of granule cargoes was unaffected by pharmacological, genetic or Coxsackievirus-mediated inhibition of cap-dependent translation. Infection with Coxsackieviruses, which also depend on PTBP1 for their own cap-independent translation, reduced instead granule stores and insulin release. These findings provide insight into the mechanism for glucose-induction of insulin granule production and on how Coxsackieviruses, which have been implicated in the pathogenesis of type 1 diabetes, can foster beta cell failure.

The multi-targeted kinase inhibitor Sorafenib inhibits Enterovirus 71 IRES activity

The multi-targeted kinase inhibitor Sorafenib inhibits Enterovirus 71 IRES activity

Landes Highlights

Promoterless transcription of DNA coligo templates by RNA polymerase IIIEpigenetic loss of the PIWI/piRNA machinery in testicular tumorigenesisA common exon is important for IRES activity in different HIV-1 transcriptsNematode endogenous small RNA pathways

Modulation of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3 by Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) is highly dependent on cellular proteins for its own propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assays using the HCV core protein as a probe. Of ~9,000 host proteins immobilized in a microarray, approximately 100 cellular proteins were identified as HCV core-interacting partners. Of these candidates, mitogen-activated protein kinase-activated protein kinase 3 (MAPKAPK3) was selected for further characterization. MAPKAPK3 is a serine/threonine protein kinase that is activated by stress and growth inducers. Binding of HCV core to MAPKAPK3 was confirmed by in vitro pulldown assay and further verified by coimmunoprecipitation assay. HCV core protein interacted with MAPKAPK3 through amino acid residues 41 to 75 of core and the N-terminal half of kinase domain of MAPKAPK3. In addition, both RNA and protein levels of MAPKAPK3 were elevated in both HCV subgenomic replicon cells and cell culture-derived HCV (HCVcc)-infected cells. Silencing of MAPKAPK3 expression resulted in decreases in both protein and HCV infectivity levels but not in the intracellular HCV RNA level. We showed that MAPKAPK3 increased HCV IRES-mediated translation and MAPKAPK3-dependent HCV IRES activity was further increased by core protein. These data suggest that HCV core may modulate MAPKAPK3 to facilitate its own propagation.

Molecular Analysis of RNA-RNA Interactions between 5’ and 3’ Untranslated Regions during the Initiation of Translation of a Cardiovirulent and a Live-Attenuated Coxsackievirus B3 Strains

Coxsackievirus B3 (CVB3) is a causative agent of viral myocarditis, meningitis and pancreatitis. CVB3 overcome their host cells by usurping the translation machinery to benefit viral gene expression. This is accomplished through alternative translation initiation in a cap independent manner at the viral internal ribosomal entry site. The 5′ untranslated region (5′UTR) of CVB3 genomic RNA is highly structured. It is the site of multiple RNA-protein and RNA-RNA interactions and it plays a critical role during translation initiation. Similar to the 5′UTR, CVB3 3′ untranslated region (3′UTR) also contains secondary structural elements consisting of three stem-loops followed by a poly (A) tail sequence. Long-range RNA-RNA interactions between 5′ and 3′ ends of some viral genomes have been observed. Because of their dual role in translation and replication, the 5′ and 3′UTRs represent promising candidates for the study of CVB3 cardiovirulence. Taking into account that efficient initiation of mRNA translation depends on a temporally and spatially orchestrated sequence of protein-protein, protein-RNA and RNA-RNA interactions, and that, at present, little is known about RNA-RNA interactions between CVB3 5′ and 3′UTRs, we aimed in the present study, to assess a possible RNA-RNA interaction between 5′ and 3′UTRs during the initiation of translation of a wild-type and a previously characterized mutant (Sabin3-like) CVB3 strains and to investigate the effect of the Sabin3-like mutation on these potential interactions. For this purpose, “Electrophoretic Mobility Shift” assays were carried out. Data obtained did not show any RNA-RNA direct interactions between the 5′- and 3′- ends. Therefore, we can suggest that the possible mechanism by which 3′UTR enhances CVB3 IRES activity may be by bridging the 5′ to the 3′ end through RNA-protein interaction and not through RNA-RNA direct contact. However, these findings need to be confirmed by carrying out further experiments.

Inhibition of enterovirus 71 infections and viral IRES activity by Fructus gardeniae and geniposide

Fructus gardeniae has long been used by traditional Chinese medical practitioners for its anti-inflammatory, anti-oxidant, anti-tumor and anti-hyperlipidemic characteristics. Here we describe our finding that F. gardeniae greatly reduces anti-enterovirus 71 (EV71) activity, resulting in significant decreases in EV71 virus yields, EV71 infections, and internal ribosome entry site activity. We also found that geniposide, a primary F. gardeniae component, inhibited both EV71 replication and viral IRES activity. Our data suggest the presence of a mechanism that blocks viral protein translation. According to our findings, F. gardeniae and geniposide deserve a closer look as potential chemopreventive agents against EV71 infections.

Insights into Factorless Translational Initiation by the tRNA-Like Pseudoknot Domain of a Viral IRES

The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae family adopts an overlapping triple pseudoknot structure to directly recruit the 80S ribosome in the absence of initiation factors. The pseudoknot I (PKI) domain of the IRES mimics a tRNA-like codon:anticodon interaction in the ribosomal P site to direct translation initiation from a non-AUG initiation codon in the A site. In this study, we have performed a comprehensive mutational analysis of this region to delineate the molecular parameters that drive IRES translation. We demonstrate that IRES-mediated translation can initiate at an alternate adjacent and overlapping start site, provided that basepairing interactions within PKI remain intact. Consistent with this, IGR IRES translation tolerates increases in the variable loop region that connects the anticodon- and codon-like elements within the PKI domain, as IRES activity remains relatively robust up to a 4-nucleotide insertion in this region. Finally, elements from an authentic tRNA anticodon stem-loop can functionally supplant corresponding regions within PKI. These results verify the importance of the codon:anticodon interaction of the PKI domain and further define the specific elements within the tRNA-like domain that contribute to optimal initiator Met-tRNAi-independent IRES translation.

The Internal Ribosome Entry Site-Medaited Expression of VEGF Rescues HS Sultan Tumor Cells From Mammalian Target of Rapamycin (mTOR)-Inhibitors Induced Apoptosis in Vivo

Abstract 1659An important molecular target of mTOR inhibitors in cancer therapy is VEGF expression and neo-angiogenesis. In prior studies, we demonstrated that, although rapalog mTOR inhibitors only induce G1 arrest in B-cell tumor lines, their administration in vivo in xenograft models resulted in tumor cell apoptosis that correlated with inhibition of neo-angiogenesis and VEGF expression within the tumor bed. Other in vitro studies have shown that IRES-dependent translation of myc and D-cyclins can provide a fail-safe mechanism for expression when mTOR inhibitors prevent cap-dependent translation and may act as a resistance mechanism to induction of G1 arrest in vitro. We, thus, tested if VEGF IRES activity could likewise regulate induction of anti-tumor effects and apoptosis in vivo. To test if VEGF IRES activity can regulate anti-tumor responses in vivo, we utilized the HS-Sultan B cell lymphoma line that is PTEN null. Its heightened AKT activity disarms the VEGF IRES, preventing this fail-safe mechanism and sensitizing to mTOR inhibitors. We ectopically expressed a version of the VEGF ORF in these cells fused to the p27 IRES (p27-VEGF), an IRES which is insensitive to AKT and effective in PTEN-null tumors. p27-VEGF transfected tumor cells were used as subcutaneous challenges in immunodeficient mice and results of mTOR inhibitor treatment compared to control mice challenged with tumor cells transfected with the VEGF ORF but without an AKT-resistant IRES. The anti-tumor responses were enumerated by assessing tumor size and tumor apoptosis, neo-angiogenesis and VEGF expression assessed by immunohistochemistry, ELISA, and Western blot analysis of tumor lysate. Ectopic expression of VEGF fused to the p27 IRES specifically enhanced VEGF expression and neo-angiogenesis in tumors of mice treated with the rapalog temsirolimus or active site mTOR inhibitor pp242 and significantly reduced tumor cell apoptosis and anti-tumor responses. The results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and, furthermore, underscore the importance of IRES activity as a resistance mechanism to such targeted therapy. Disclosures:No relevant conflicts of interest to declare.

Methods for studying IRES-mediated translation of positive-strand RNA viruses

Internal ribosome entry sites are RNA elements that mediate translation in a cap-independent manner. A subset of positive strand RNA viruses utilize an IRES mechanism as a viral strategy to ensure efficient viral protein synthesis. IRES elements vary in sequence, structure, and factor requirements between virus families. Here, we describe methods to determine IRES activity and approaches to study the regulation and function of IRES-mediated translation both in vitro and in vivo. Finally, we describe a new IRES-directed reporter system which exploits the 2A ‘self-cleavage’ or ‘stop–go’ peptide for optimal detection of IRES activity.