IRES Trans-acting Factors Research Articles

The different pathways of HIV genomic RNA translation

Lentiviruses, the prototype of which is HIV-1, can initiate translation either by the classical cap-dependent mechanism or by internal recruitment of the ribosome through RNA domains called IRESs (internal ribosome entry sites). Depending on the virus considered, the mechanism of IRES-dependent translation differs widely. It can occur by direct binding of the 40S subunit to the mRNA, necessitating a subset or most of the canonical initiation factors and/or ITAF (IRES trans-acting factors). Nonetheless, a common feature of IRESs is that ribosomal recruitment relies, at least in part, on IRES structural determinants. Lentiviral genomic RNAs present an additional level of complexity, as, in addition to the 5'-UTR (untranslated region) IRES, the presence of a new type of IRES, embedded within Gag coding region was described recently. This IRES, conserved in all three lentiviruses examined, presents conserved structural motifs that are crucial for its activity, thus reinforcing the link between RNA structure and function. However, there are still important gaps in our understanding of the molecular mechanism underlying IRES-dependent translation initiation of HIV, including the determination of the initiation factors required, the dynamics of initiation complex assembly and the dynamics of the RNA structure during initiation complex formation. Finally, the ability of HIV genomic RNA to initiate translation through different pathways questions the possible mechanisms of regulation and their correlation to the viral paradigm, i.e. translation versus encapsidation of its genomic RNA.

The role of IRES trans-acting factors in regulating translation initiation

The majority of mRNAs in eukaryotic cells are translated via a method that is dependent upon the recognition of, and binding to, the methylguanosine cap at the 5' end of the mRNA, by a set of protein factors termed eIFs (eukaryotic initiation factors). However, many of the eIFs involved in this process are modified and become less active under a number of pathophysiological stress conditions, including amino acid starvation, heat shock, hypoxia and apoptosis. During these conditions, the continued synthesis of proteins essential to recovery from stress or maintenance of a cellular programme is mediated via an alternative form of translation initiation termed IRES (internal ribosome entry site)-mediated translation. This relies on the mRNA containing a complex cis-acting structural element in its 5'-UTR (untranslated region) that is able to recruit the ribosome independently of the cap, and is often dependent upon additional factors termed ITAFs (IRES trans-acting factors). A limited number of ITAFs have been identified to date, particularly for cellular IRESs, and it is not yet fully understood how they exert their control and which cellular pathways are involved in their regulation.

Occult infection of peripheral B cells by hepatitis C variants which have low translational efficiency in cultured hepatocytes

BackgroundPlasma hepatitis C virus (HCV) originates from hepatocytes. However, in certain subjects, B cells may harbour both plasma strains and occult HCV strains tha t are not detected in the...

Polypyrimidine tract-binding protein interacts with coxsackievirus B3 RNA and influences its translation

We have investigated the possible role of trans-acting factors interacting with the untranslated regions (UTRs) of coxsackievirus B3 (CVB3) RNA. We show here that polypyrimidine tract-binding protein (PTB) binds specifically to both 5' and 3' UTRs, but with different affinity. We have demonstrated that PTB is a bona fide internal ribosome entry site (IRES) trans-acting factor (ITAF) for CVB3 RNA by characterizing the effect of partial silencing of PTB ex vivo in HeLa cells. Furthermore, IRES activity in BSC-1 cells, which are reported to have a very low level of endogenous PTB, was found to be significantly lower than that in HeLa cells. Additionally, we have mapped the putative contact points of PTB on the 5' and 3' UTRs by an RNA toe-printing assay. We have shown that the 3' UTR is able to stimulate CVB3 IRES-mediated translation. Interestingly, a deletion of 15 nt at the 5' end or 14 nt at the 3' end of the CVB3 3' UTR reduced the 3' UTR-mediated enhancement of IRES activity ex vivo significantly, and a reduced interaction was shown with PTB. It appears that the PTB protein might help in circularization of the CVB3 RNA by bridging the ends necessary for efficient translation of the viral RNA.

Interleukin-6 (IL-6) Enhances C-MYC Translation IN MULTIPLE MYELOMA (MM) CELLS: ROLE of IL-6-INDUCED EFFECTS On the C-MYC RNA-Binding PROTEIN, HNRNPA1.

Abstract 3839Poster Board III-775Our previous work (Cancer Research 68:10215, 2008) demonstrated that IL-6 enhanced c-myc protein expression in MM cells and function of the RNA-binding protein, hnRNPA1 (A1), was required. This occurred by way of enhanced cap-independent translation mediated via the internal ribosome entry site (IRES) in the 5'UTR of the myc RNA. IRES-dependent translation is the fail safe mechanism for protein expression when cap-dependent translation is suppressed by mTOR inhibition (with curtailed RNA cap-ribosome binding) and is especially important when an mRNA leader is relatively long and highly structured, such that scanning ribosomes are unlikely to efficiently initiate translation. The IRES directly recruits the ribosome to within close proximity to the start codon, bypassing the need for cap binding and ribosome scanning. HNRNPA1 (A1) is a documented IRES trans-acting factor (ITAF) for the myc IRES, facilitating translation but how its effects are enhanced by IL-6 is unknown. HNRNPA1 is a shuttling protein which binds the myc RNA in the nucleus and transports it to the cytoplasm. Initial experiments demonstrated that IL-6 stimulation of ANBL-6 and OCI-My5 MM cell lines, as well as several primary MM specimens, significantly increased the cytoplasmic localization of A1. To test if this enhanced cytoplasmic localization was critical, we stably expressed a dominant negative (DN) A1 gene that is incapable of nuclear-to-cytoplasmic shuttling and which also prevents endogenous hnRNPA1 shuttling. The DN also prevented A1-mediated transport of the shuttling protein, FUS. Expression of the DN in ANBL-6 cells prevented IL-6-induced effects on myc expression and on ANBL-6 cell growth. We also tested if IL-6 treatment affected A1 binding to the myc RNA by immunoprecipitating A1 and performing real time PCR on the immunoprecipitate for myc RNA levels. A significant increase in A1-myc RNA binding was confirmed. Mass spectroscopy demonstrated that IL-6 induced phosphorylation of A1 in its RNA-binding domain, which possibly mediated the enhanced binding. These results demonstrate that, by enhanced binding of the myc ITAF, hnRNPA1, to the myc IRES, and by enhanced transport of the complex to the translationally active cytoplasmic subcellular site, IL-6 may stimulate c-myc translation and subsequent MM cell growth. Disclosures:No relevant conflicts of interest to declare.

IRESite--a tool for the examination of viral and cellular internal ribosome entry sites.

The IRESite (http://www.iresite.org) presents carefully curated experimental evidence of many eukaryotic viral and cellular internal ribosome entry site (IRES) regions. At the time of submission, IRESite stored >600 records. The IRESite gradually evolved into a robust tool providing (i) biologically meaningful information regarding the IRESs and their experimental background (including annotation of IRES secondary structures and IRES trans-acting factors) as well as (ii) thorough concluding remarks to stored database entries and regularly updated evaluation of the reported IRES function. A substantial portion of the IRESite data results purely from in-house bioinformatic analyses of currently available sequences, in silico attempts to repeat published cloning experiments, DNA sequencing and restriction endonuclease verification of received plasmid DNA. We also present a newly implemented tool for displaying RNA secondary structures and for searching through the structures currently stored in the database. The supplementary material contains an updated list of reported IRESs.

NF45 functions as an IRES trans-acting factor that is required for translation of cIAP1 during the unfolded protein response

Expression of the cellular inhibitor of apoptosis protein 1 (cIAP1) is unexpectedly repressed at the level of translation under normal physiological conditions in many cell lines. We have previously shown that the 5' untranslated region of cIAP1 mRNA contains a stress-inducible internal ribosome entry site (IRES) that governs expression of cIAP1 protein. Although inactive in unstressed cells, the IRES supports cap-independent translation of cIAP1 in response to endoplasmic reticulum stress. To gain an insight into the mechanism of cIAP1 IRES function, we empirically derived the minimal free energy secondary structure of the cIAP1 IRES using enzymatic cleavage mapping. We subsequently used RNA affinity chromatography to identify several cellular proteins, including nuclear factor 45 (NF45) as cIAP1 IRES binding proteins. In this report we show that NF45 is a novel RNA binding protein that enhances IRES-dependent translation of endogenous cIAP1. Further, we show that NF45 is required for IRES-mediated induction of cIAP1 protein during the unfolded protein response. The data presented are consistent with a model in which translation of cIAP1 is governed, at least in part, by NF45, a novel cellular IRES trans-acting factor.

A cell-permeable peptide inhibits hepatitis C virus replication by sequestering IRES transacting factors

Hepatitis C virus (HCV) infection frequently leads to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. There is no effective therapy or vaccine available to HCV-infected patients other than interferon-ribavarin combination, which is effective in a relatively small percentage of infected patients. Our previous results have shown that a synthetic peptide (LAP) corresponding to the N-terminal 18 amino acids of the Lupus autoantigen (La) was a potent inhibitor of HCV IRES-mediated translation. We demonstrate here that LAP efficiently blocks HCV replication of infectious JFH1 virus in cell culture. Our data suggest that LAP forms complexes with IRES-transacting factors (ITAFs) PTB and PCBP2. LAP-mediated inhibition of HCV IRES-mediated translation in vitro could be fully rescued by recombinant PCB and PCBP2. Also transient expression of PTB / PCBP2 combination significantly restores HCV replication in LAP-inhibited cultures. These results suggest that ITAFs could be potential targets to block HCV replication.

The Polypyrimidine Tract-binding Protein Is Required for Efficient Dengue Virus Propagation and Associates with the Viral Replication Machinery

The polypyrimidine tract-binding protein (PTB) functions primarily as an IRES trans-acting factor in the propagation of hepatitis C virus and picornaviruses. PTB interacts with secondary structures within the 3'- and 5'-untranslated regions of these viral genomes to mediate efficient IRES-mediated viral translation. PTB has also been reported to bind to the untranslated region of the single-stranded RNA dengue virus (DENV), suggesting a similar function for PTB in flaviviruses. Indeed small interfering RNA-mediated PTB knockdown inhibited the production of infectious DENV, and this inhibition was specific to PTB knockdown and not due to a nonspecific anti-viral state. In fact, PTB depletion did not inhibit the production infectious yellow fever virus, another flavivirus. Nevertheless, whereas PTB knockdown led to a significant decrease in the accumulation of DENV viral RNAs, it did not impair translation. Moreover, PTB was shown to interact with the DENV nonstructural 4A protein, a known component of the viral replication complex, and with the DENV genome during infection. These data suggest that PTB interacts with the replication complex of DENV and is acting at the level of viral RNA replication.

Far upstream element binding protein 2 interacts with enterovirus 71 internal ribosomal entry site and negatively regulates viral translation

An internal ribosomal entry site (IRES) that directs the initiation of viral protein translation is a potential drug target for enterovirus 71 (EV71). Regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the internal ribosomal entry site. Biotinylated RNA-affinity chromatography and proteomic approaches were employed to identify far upstream element (FUSE) binding protein 2 (FBP2) as an ITAF for EV71. The interactions of FBP2 with EV71 IRES were confirmed by competition assay and by mapping the association sites in both viral IRES and FBP2 protein. During EV71 infection, FBP2 was enriched in cytoplasm where viral replication occurs, whereas FBP2 was localized in the nucleus in mock-infected cells. The synthesis of viral proteins increased in FBP2-knockdown cells that were infected by EV71. IRES activity in FBP2-knockdown cells exceeded that in the negative control (NC) siRNA-treated cells. On the other hand, IRES activity decreased when FBP2 was over-expressed in the cells. Results of this study suggest that FBP2 is a novel ITAF that interacts with EV71 IRES and negatively regulates viral translation.

Heterogeneous Nuclear Ribonucleoprotein A1 Regulates Cyclin D1 and c-myc Internal Ribosome Entry Site Function through Akt Signaling

The translation of the cyclin D1 and c-myc mRNAs occurs via internal ribosome entry site (IRES)-mediated initiation under conditions of reduced eIF-4F complex formation and Akt activity. Here we identify hnRNP A1 as an IRES trans-acting factor that regulates cyclin D1 and c-myc IRES activity, depending on the Akt status of the cell. hnRNP A1 binds both IRESs in vitro and in intact cells and enhances in vitro IRES-dependent reporter expression. Akt regulates this IRES activity by inducing phosphorylation of hnRNP A1 on serine 199. Serine 199-phosphorylated hnRNP A1 binds to the IRESs normally but is unable to support IRES activity in vitro. Reducing expression levels of hnRNP A1 or overexpressing a dominant negative version of the protein markedly inhibits rapamycin-stimulated IRES activity in cells and correlated with redistribution of cyclin D1 and c-myc transcripts from heavy polysomes to monosomes. Importantly, knockdown of hnRNP A1 also renders quiescent Akt-containing cells sensitive to rapamycin-induced G(1) arrest. These results support a role for hnRNP A1 in mediating rapamycin-induced alterations of cyclin D1 and c-myc IRES activity in an Akt-dependent manner and provide the first direct link between Akt and the regulation of IRES activity.

The IRES‐Mediated Translation of Cytoplasmic Serine Hydroxymethyltransferase (cSHMT) is Stimulated by UV‐Induced DNA Damage

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a key regulator of folate‐mediated one‐carbon metabolism. By preferentially directing one‐carbon units to thymidylate synthase (TS), cSHMT gives purine synthesis priority over methionine synthesis, resulting in elevated levels of thymidylate for DNA replication and repair. The 5′ untranslated region (UTR) of the cSHMT transcript contains an internal ribosome entry site (IRES) that regulates cSHMT expression through interactions with the IRES trans‐acting factors BrunoL2 and heavy chain ferritin (HCF). In this study, we investigate the physiological function of the cSHMT IRES. We show that cSHMT IRES activity is stimulated in response to a non‐lethal dose of UV irradiation, resulting in a four‐fold increase in cSHMT protein levels despite a global inhibition of cap‐dependent translation. Furthermore, we show that the mechanism of IRES stimulation involves the UV‐induced activation of HCF transcription and the relocalization of BrunoL2 to the cytoplasm. This response appears to be specific to DNA damage‐induced cellular stress, as nocodazole‐induced cell cycle arrest does not produce the same results. The UV‐induced increase in cSHMT expression is accompanied by an increase in the nuclear localization of cSHMT and TS, suggesting that cSHMT IRES activity functions to provide thymidylate for the repair of DNA lesions arising from UV irradiation.

IRES-mediated pathways to polysomes: nuclear versus cytoplasmic routes

Eukaryotic mRNA initiates translation by cap-dependent scanning, ribosome shunting and cap-independent internal ribosome entry. Internal ribosome entry was first discovered for cytoplasmic RNA viruses but has also been identified for DNA viruses and cellular mRNAs. An internal ribosome entry site (IRES) directs internal binding of ribosomes and nucleates the formation of a translation initiation complex. Current research is aimed at identifying interactions between IRES elements and RNA-binding proteins known as ITAFs (IRES trans-acting factors). Here we compare IRES elements from cytoplasmic RNA viruses with those of cellular mRNAs and DNA viruses with nuclear mRNA synthesis, and suggest that ITAF composition and IRES function directly reflect the site of synthesis of mRNA and the history of its pathway to polysomes.

For IRES trans-acting factors, it is all about location

The translation of many proteins involved in transcription, cell cycle progression, apoptosis and cell survival is mediated by internal ribosome entry sites (IRESs) present within the 5'-untranslated regions (5'-UTRs) of their messenger RNA molecules (mRNAs). Several recent reports now demonstrate that the proteins controlling IRES-dependent translation initiation are regulated by their subcellular localization.

Hepatitis C virus internal ribosome entry site initiates protein synthesis at the authentic initiation codon in yeast

Hepatitis C virus (HCV) is an important pathogen causing both acute and chronic infections in humans. The HCV polyprotein is synthesized by cap-independent translation initiation after ribosome binding to the highly structured internal ribosome entry site (IRES). The HCV IRES has been shown to have a low requirement for translation initiation factors and the ability to bind directly to the 40S ribosomal subunit. A novel yeast bicistronic reporter system, suitable for sensitive and accurate analysis of IRES activity, has been developed. It employs signal amplification based on the Gal4p transcription factor-mediated activation of a variety of secondary reporter genes. The system has a broad dynamic range and, depending on the nature of the particular secondary reporter, can be used both for precise measurements of IRES activity and for selection and screening for novel IRES variants and IRES trans-acting factors. By using this novel bicistronic system, it was shown that the HCV IRES is functional in yeast cells. Mutational analysis of the IRES loop IV and the adjacent region revealed that, in yeast, as in mammalian cells, translation initiates preferentially at the authentic (342)AUG codon and that disruption of the HCV IRES loop IV abrogates its function, whilst minor positional changes or substitutions of the initiation codon within loop IV are largely tolerated. These findings bring more general insights to translation initiation, but also open the door for utilization of yeast and its sophisticated genetics for searching for new antiviral drugs and HCV IRES trans-acting proteins.

The Myotonic Dystrophy Type 2 Protein ZNF9 Is Part of an ITAF Complex That Promotes Cap-independent Translation

The 5'-untranslated region of the ornithine decarboxylase (ODC) mRNA contains an internal ribosomal entry site (IRES). Mutational analysis of the ODC IRES has led to the identification of sequences necessary for cap-independent translation of the ODC mRNA. To discover novel IRES trans-acting factors (ITAFs), we performed a proteomics screen for proteins that regulate ODC translation using the wild-type ODC mRNA and a mutant version with an inactive IRES. We identified two RNA-binding proteins that associate with the wild-type ODC IRES but not the mutant IRES. One of these RNA-binding proteins, PCBP2, is an established activator of viral and cellular IRESs. The second protein, ZNF9 (myotonic dystrophy type 2 protein), has not been shown previously to bind IRES-like elements. Using a series of biochemical assays, we validated the interaction of these proteins with ODC mRNA. Interestingly ZNF9 and PCBP2 biochemically associated with each other and appeared to function as part of a larger holo-ITAF ribonucleoprotein complex. Our functional studies showed that PCBP2 and ZNF9 stimulate translation of the ODC IRES. Importantly these results may provide insight into the normal role of ZNF9 and why ZNF9 mutations cause myotonic dystrophy.

Subcellular relocalization of a trans-acting factor regulates XIAP IRES-dependent translation.

Translation of the X-linked inhibitor of apoptosis (XIAP) proceeds by internal ribosome entry site (IRES)-mediated initiation, a process that is physiologically important because XIAP expression is essential for cell survival under conditions of compromised cap-dependent translation, such as cellular stress. The regulation of internal initiation requires the interaction of IRES trans-acting factors (ITAFs) with the IRES element. We used RNA-affinity chromatography to identify XIAP ITAFs and isolated the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1). We find that hnRNP A1 interacts with XIAP IRES RNA both in vitro and in vivo and that hnRNP A1 negatively regulates XIAP IRES activity. Moreover, XIAP IRES-dependent translation is significantly reduced when hnRNP A1 accumulates in the cytoplasm. Osmotic shock, a cellular stress that causes cytoplasmic accumulation of hnRNP A1, also leads to a decrease in XIAP levels that is abrogated by knockdown of hnRNP A1 expression. These results suggest that the subcellular localization of hnRNP A1 is an important determinant of its ability to negatively regulate XIAP IRES activity, suggesting that the subcellular distribution of ITAFs plays a critical role in regulating IRES-dependent translation. Our findings demonstrate that cytoplasmic hnRNP A1 is a negative regulator of XIAP IRES-dependent translation, indicating a novel function for the cytoplasmic form of this protein.

The Double-Stranded RNA Binding Protein 76:NF45 Heterodimer Inhibits Translation Initiation at the Rhinovirus Type 2 Internal Ribosome Entry Site

Poliovirus (PV) plus-strand RNA genomes initiate translation in a cap-independent manner via an internal ribosome entry site (IRES) in their 5' untranslated region. Viral translation is codetermined by cellular IRES trans-acting factors, which can influence viral propagation in a cell-type-specific manner. Engineering of a poliovirus recombinant devoid of neuropathogenic properties but highly lytic in malignant glioma cells was accomplished by exchange of the cognate poliovirus IRES with its counterpart from human rhinovirus type 2 (HRV2), generating PV-RIPO. Neuroblast:glioma heterokaryon analyses revealed that loss of neurovirulence is due to trans-dominant repression of PV-RIPO propagation in neuronal cells. The double-stranded RNA binding protein 76 (DRBP76) was previously identified to bind to the HRV2 IRES in neuronal cells and to inhibit PV-RIPO translation and propagation (M. Merrill, E. Dobrikova, and M. Gromeier, J. Virol. 80:3347-3356, 2006). The results of size exclusion chromatography indicate that DRBP76 heterodimerizes with nuclear factor of activated T cells, 45 kDa (NF45), in neuronal but not in glioma cells. The DRBP76:NF45 heterodimer binds to the HRV2 IRES in neuronal but not in glioma cells. Ribosomal profile analyses show that the heterodimer preferentially associates with the translation apparatus in neuronal cells and arrests translation at the HRV2 IRES, preventing PV-RIPO RNA assembly into polysomes. Results of this study suggest that the DRBP76:NF45 heterodimer selectively blocks HRV2 IRES-driven translation initiation in neuron-derived cells.

IRES-dependent regulation of FGF-2 mRNA translation in pathophysiological conditions in the mouse

The mRNA coding for FGF-2 (fibroblast growth factor 2), a major angiogenic factor, is translated by an IRES (internal ribosome entry site)-dependent mechanism. We have studied the role of the IRES in the regulation of FGF-2 expression in vivo, under pathophysiological conditions, by creating transgenic mice lines expressing bioluminescent bicistronic transgenes. Analysis of FGF-2 IRES activity indicates strong tissue specificity in adult brain and testis, suggesting a role of the IRES in the activation of FGF-2 expression in testis maturation and brain function. We have explored translational control of FGF-2 mRNA under diabetic hyperglycaemic conditions, as FGF-2 is implied in diabetes-related vascular complications. FGF-2 IRES is specifically activated in the aorta wall in streptozotocin-induced diabetic mice, in correlation with increased expression of endogenous FGF-2. Thus, under hyperglycaemic conditions, where cap-dependent translation is blocked, IRES activation participates in FGF-2 overexpression, which is one of the keys of diabetes-linked atherosclerosis aggravation. IRES activation under such pathophysiological conditions may involve ITAFs (IRES trans-acting factors), such as p53 or hnRNP AI (heterogeneous nuclear ribonucleoprotein AI), recently identified as inhibitory or activatory ITAFs respectively for FGF-2 IRES.

IRES-dependent regulation of FGF-2 mRNA translation in pathophysiological conditions in the mouse

The mRNA coding for FGF-2 (fibroblast growth factor 2), a major angiogenic factor, is translated by an IRES (internal ribosome entry site)-dependent mechanism. We have studied the role of the IRES in the regulation of FGF-2 expression in vivo, under pathophysiological conditions, by creating transgenic mice lines expressing bioluminescent bicistronic transgenes. Analysis of FGF-2 IRES activity indicates strong tissue specificity in adult brain and testis, suggesting a role of the IRES in the activation of FGF-2 expression in testis maturation and brain function. We have explored translational control of FGF-2 mRNA under diabetic hyperglycaemic conditions, as FGF-2 is implied in diabetes-related vascular complications. FGF-2 IRES is specifically activated in the aorta wall in streptozotocin-induced diabetic mice, in correlation with increased expression of endogenous FGF-2. Thus, under hyperglycaemic conditions, where cap-dependent translation is blocked, IRES activation participates in FGF-2 overexpression, which is one of the keys of diabetes-linked atherosclerosis aggravation. IRES activation under such pathophysiological conditions may involve ITAFs (IRES trans-acting factors), such as p53 or hnRNP AI (heterogeneous nuclear ribonucleoprotein AI), recently identified as inhibitory or activatory ITAFs respectively for FGF-2 IRES.