Cap-dependent Mechanism Research Articles

Retrovirus Translation Initiation: Issues and Hypotheses Derived from Study of HIV-1

Human immunodeficiency virus type 1 (HIV-1) has a small, multifunctional genome that encodes a relatively large and complex proteome. The virus has adopted specialized post-transcriptional control mechanisms to maximize its coding capacity while economically maintaining the information stored in cis-acting replication sequences. The conserved features of the 5' untranslated region of all viral transcripts suggest they are poor substrates for cap-dependent ribosome scanning and provide a compelling rationale for internal initiation of translation. This article summarizes key experimental results of studies that have evaluated HIV-1 translation initiation. A model is discussed in which cap-dependent and cap-independent initiation mechanisms of HIV-1 co-exist to ensure viral protein production in the context of 1) structured replication motifs that inhibit ribosome scanning, and 2) alterations in host translation machinery in response to HIV-1 infection or other cellular stresses. We discuss key issues that remain to be understood and suggest parameters to validate internal initiation activity in HIV-1 and other retroviruses.

Expression of RUNX2 isoforms: Involvement of cap-dependent and cap-independent mechanisms of translation

RUNX2, a major regulator of skeletogenesis, is expressed as type-I and type-II isoforms. Whereas most eukaryotic mRNAs are translated by the cap-dependent scanning mechanism, translation of many mRNAs including type-I and type-II RUNX2 mRNAs has been reported to be initiated by a cap independent internal ribosomal entry site (IRES). Since the dicistronic plasmid assay used to demonstrate IRES has been questioned, we investigated the presence of IRES in RUNX2 mRNAs using dicistronic plasmid and mRNA assays. Our results show that the dicistronic plasmid assay cannot be used to demonstrate IRES in RUNX2 mRNAs because the intercistronic region of dicistronic plasmids containing the 5'-UTRs of both RUNX2 mRNAs operates as a cryptic promoter. In dicistronic mRNA transfection studies the 5'-UTRs of both RUNX2 mRNAs exhibited no IRES activity. When transfected into osteoblastic cells, monocistronic reporter mRNA preceded by the 5'-UTR of type-II RUNX2 (Type-II-FLuc-A100) was translated to a high degree only in the presence of a functional cap (m(7)GpppG); in contrast, luciferase mRNA preceded by the 5'-UTR of type-I RUNX2 mRNA (Type-I-FLuc-A100) was translated poorly in the presence of either m(7)GpppG or a nonfunctional cap (ApppG). Notably, in transfected cells inhibitors of cap-dependent translation suppressed the translation of m(7)GpppG-capped Type-II-FLuc-A100, but not ApppG-capped reporter mRNA preceded by the IRES-containing hepatitis C virus (HCV) 5'-UTR. Our study demonstrates that type-II RUNX2 mRNA is translated by the cap-dependent mechanism. Although efficient translation of type-I RUNX2 mRNA appears to require a process other than cap-dependent, the mechanism of type-I RUNX2 mRNA translation remains to be resolved.

Control of MYEOV Protein Synthesis by Upstream Open Reading Frames

The myeov gene has been isolated by the tumorigenicity assay and is localized at chromosome 11q13, a frequent site for chromosomal rearrangements in various carcinomas and B-cell neoplasms. In addition, myeov is coamplified with cyclin D1 and overexpressed in carcinomas of various organs. The mechanisms of myeov regulation remain enigmatic. The 5'-untranslated region (5'-UTR) of the myeov gene is long, encompasses several upstream AUGs, and is predicted to fold in a strong secondary structure, suggesting that its translation might be regulated by an internal ribosomal entry site. Here we show that initial experiments using monocistronic and dicistronic reporter constructs supported this assumption. However, the application of in vitro transcription/translation assays, Northern blot analysis, and promoterless dicistronic constructs revealed promoter activity of the myeov 5'-UTR. DNA transfection of dicistronic DNA constructs, normal and mutated forms of myeov cDNA fragments cloned in a eukaryotic expression vector, and direct RNA transfection analysis revealed that upstream AUG triplets in the 5'-UTR of the myeov transcript abrogate translation. Alternative splicing mechanisms in specific cell types and/or developmental stage may evade this translation control. Control experiments suggest that the 5'-UTR from encephalomyocarditis virus, when inserted at the midpoint of a dicistronic vector, is also able to function as a cryptic promoter.

An internal ribosome entry site mediates translation of lymphoid enhancer factor-1

The lymphoid enhancer factor-1 LEF1 locus produces multiple mRNAs via alternative promoters. Full-length LEF-1 protein is produced via translation of an mRNA with a 1.2-kb, GC-rich 5'-untranslated region (UTR), whereas a truncated LEF-1 isoform is produced by an mRNA with a short, 60-nucleotide (nt) 5'-UTR. Full-length LEF-1 promotes cell growth via its interaction with the WNT signaling mediator beta-catenin. Truncated LEF-1 lacks the beta-catenin binding domain and opposes WNT signaling as a competitive inhibitor for WNT response elements. In this study we tested the hypothesis that the long, GC-rich 5'-UTR within the full-length LEF1 mRNA contains an internal ribosome entry site (IRES). Using a dicistronic vector in transient DNA transfections, we show that the LEF1 5'-UTR mediates cap-independent translation. Additional experiments involving a promoter-less dicistronic vector, Northern blot analysis, and transient transfections of dicistronic mRNAs into cultured mammalian cells compromised for cap-dependent translation demonstrate that the 5'-UTR of full-length LEF1 mRNA contains a bona fide IRES. Deletion analysis of the 5'-UTR shows that maximal IRES activity requires the majority of the 5'-UTR, consistent with the notion that cellular IRESs require multiple modules for efficient activity. This study demonstrates that full-length LEF1 mRNA has evolved to utilize a cap-independent mechanism for translation of full-length LEF-1, whereas the truncated isoform is produced via the canonical cap-dependent ribosome scanning mechanism.

Reconstitution of eukaryotic translation elongation in vitro following initiation by internal ribosomal entry

The ability to reconstitute different stages of eukaryotic translation process in vitro is a prerequisite for detailed biochemical analysis of their mechanisms. Reconstitution of elongation and subsequent processes such as termination and recycling on natural mRNAs translated by the cap-dependent mechanism is very complicated, and has not so far been done because of the necessity to first reconstitute the process of translation initiation, which is the most complex stage of eukaryotic translation, which requires at least nine initiation factors. The recent discovery of internal ribosomal entry sites (IRESs) in the intergenic region (IGR) of the genomes of dicistroviruses such as cricket paralysis virus (CrPV) and Plautia stali intestine virus (PSIV) that mediate initiation of translation by a mechanism that does not involve aminoacylated initiator tRNA ( Met - tRNA i Met ) or any initiation factors has provided a simple means to assemble active ribosomes on an mRNA that can be used to investigate these downstream stages in the translation process. Here we describe the methods for the assembly of active mammalian ribosomes on the CrPV IGR IRES and for reconstitution and analysis of subsequent steps in the elongation process. The composition of the reconstituted in vitro translation system can be fully controlled, and we therefore suggest that the methods described here could in future be adapted to permit template-dependent synthesis of peptidomimetics by eukaryotic ribosomes, by reassigning individual codons in an mRNA to non-natural amino acids using tRNAs that have been appropriately mischarged either chemically or enzymatically.

Internal Translation Initiation Mediated by the Angiogenic Factor Tie2

Tie2 is an endothelium-specific receptor tyrosine kinase required for normal blood vessel maturation. We report that Tie2 mRNA translation is maintained under hypoxic conditions. To identify the mechanism responsible for this, we undertook structure/function analysis of the Tie2 5'-untranslated region (UTR). Transcription start site mapping indicates the existence of a several mRNA isoforms containing unusually long 5'-UTRs (>350 nucleotides) with five upstream open reading frames. We find internal ribosome binding activity that allows the Tie2 mRNA to initiate in a cap-independent fashion. Our data provide a framework for understanding how Tie2 mRNA is translated despite a cumbersome structured 5'-UTR and how its production is secured under unfavorable environmental conditions.

Internal ribosome entry segment-mediated translation during apoptosis: the role of IRES-trans-acting factors

During apoptosis, there is a reduction in translation initiation caused by caspase cleavage of several of the factors required for the cap-dependent scanning mechanism. Under these circumstances, many proteins that are required for apoptosis are instead translated by the alternative method of internal ribosome entry. This mechanism requires the formation of a complex RNA structural element and in the presence of internal ribosome entry segment (IRES)-trans-acting factors (ITAFs), the ribosome is recruited to the RNA. The interactions of several ITAFs with IRESs have been investigated in detail, and several mechanisms of action have been noted, including acting as chaperones, stabilising and remodelling the RNA structure. Structural remodelling by PTB in particular will be discussed, and how this protein is able to facilitate recruitment of the ribosome to several IRESs by causing previously occluded sites to become more accessible.

Protein synthesis in eukaryotes: The growing biological relevance of cap-independent translation initiation

Ribosome recruitment to eukaryotic mRNAs is generally thought to occur by a scanning mechanism, whereby the 40S ribosomal subunit binds in the vicinity of the 5'cap structure of the mRNA and scans until an AUG codon is encountered in an appropriate sequence context. Study of the picornaviruses allowed the characterization of an alternative mechanism of translation initiation. Picornaviruses can initiate translation via an internal ribosome entry segment (IRES), an RNA structure that directly recruits the 40S ribosomal subunits in a cap and 5' end independent fashion. Since its discovery, the notion of IRESs has extended to a number of different virus families and cellular RNAs. This review summarizes features of both cap-dependent and IRES-dependent mechanisms of translation initiation and discusses the role of cis-acting elements, which include the 5' cap, the 5'-untranslated region (UTR) and the poly(A) tail as well as the possible roles of IRESs as part of a cellular stress response mechanism and in the virus replication cycle.

Structural characterization of the gene encoding the large zinc finger protein ZAS3: implication to the origin of multiple promoters in eukaryotic genes

ZAS3 is a large zinc finger protein that regulates κB-mediated transcription and TNF-driven signal transduction pathway. Herein, we have characterized the mouse ZAS3 gene that spans 400 kb and splits into 16 exons. Four ZAS3 exons, ranging from 676 to 3956 nucleotides, are significantly larger than the average size of mammalian internal exons. Intron 10, when retained in mRNAs, encodes N-terminal DNA binding domain, called ZASN. As predicted from cDNAs, 5′ untranslated region composed of the 2317 nucleotides is extremely long and contains upstream open reading frames, suggesting that translation initiation of ZAS3 transcripts by conventional cap-dependent ribosome scanning mechanism may be inefficient. Additionally, cDNA data analysis followed by reporter gene assays shows that the ZAS3 locus harbors two promoters that are 80 kb apart. The data suggest that the expression of ZAS3 is controlled by a combination of differential promoter usage, alternative splicing, and possible intergenic splicing. The distribution and degree of conservation of exons within the ZAS3 locus, together with the complex alternative splicing events and upstream open reading frame in 5′ untranslated exons, lead us to speculate that multiple promoters of an eukaryotic gene might be residual traces of regulatory regions of other genes lost in evolution.

Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo.

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase-polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc-IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.

Epstein-Barr virus U leader exon contains an internal ribosome entry site.

Eukaryotic translation can be initiated either by a cap-dependent mechanism or by internal ribosome entry, a process by which ribosomes are directly recruited to structured regions of mRNA upstream of the initiation codon. Here we report the finding of an internal ribosome entry site (IRES) in the untranslated region of the Epstein-Barr nuclear antigen 1 (EBNA1) gene. EBNA1 is the only nuclear protein expressed in all known states of Epstein-Barr virus (EBV) latency and in the virus lytic cycle, and is required for the maintenance of the EBV episome. Using cDNA reporter constructs and in vitro transfection assays, we found that sequences contained in the 5' untranslated region (UTR) of the Fp and Qp initiated EBNA1 mRNA increased the expression level 4-14- fold in different Burkitt lymphoma cell lines. The U leader exon, located within the 5' UTR, included in all known EBNA1 transcripts and also contained in the EBNA3, 4 and 6 mRNAs, was demonstrated by bicistronic expression analyses to contain an IRES. The EBNA IRES initiates translation more efficiently than the encephalomyocarditis virus IRES in EBV-positive lymphoma cells. We propose that the EBNA IRES constitute a novel mechanism, whereby EBV regulates latent gene expression.

Polypyrimidine tract binding protein and poly r(C) binding protein 1 interact with the BAG-1 IRES and stimulate its activity in vitro and in vivo.

The 5'-untranslated region of Bag-1 mRNA contains an internal ribosome entry segment (IRES) and the translation of Bag-1 protein can be initiated by both cap-dependent and cap-independent mechanisms. In general, cellular IRESs require non-canonical trans-acting factors for their activity, however, very few of the proteins that act on cellular IRESs have been identified. Proteins that interact with viral IRESs have also been shown to stimulate the activity of cellular IRESs and therefore the ability of a range of known viral trans-acting factors to stimulate the Bag-1 IRES was tested. Two proteins, poly r(C) binding protein 1 (PCBP1) and polypyrimidine tract binding protein (PTB), were found to increase the activity of the Bag-1 IRES in vitro and in vivo. The regions of the Bag-1 IRES RNA to which they bind have been determined, and it was shown that PCBP1 binds to a short 66 nt section of RNA, whilst PTB interacts with a number of sites over a larger area. The minimum section of the RNA that still retained activity was determined and both PCBP1 and PTB interacted with this region suggesting that these proteins are essential for Bag-1 IRES function.

Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons.

Eukaryotic translation initiation factor 4GI (eIF4GI) is an essential protein that is the target for translational regulation in many cellular processes and viral systems. It has been shown to function in both cap-dependent and cap-independent translation initiation by recruiting the 40S ribosomal subunit to the mRNA cap structure or internal ribosome entry site (IRES) element, respectively. Interestingly eIF4GI mRNA itself has been reported to contain an IRES element in its 5' end that facilitates eIF4GI protein synthesis via a cap-independent mechanism. In HeLa cells, eIF4GI exists as several isoforms that differ in their migration in sodium dodecyl sulfate (SDS) gels; however, the nature of these isoforms was unclear. Here, we report a new cDNA clone for eIF4GI that extends the 5' sequence 340 nucleotides beyond the previously published sequence. The new extended sequence of eIF4GI is located on chromosome 3, within two additional exons immediately upstream of the previously published eIF4GI sequence. When mRNA transcribed from this cDNA clone was translated in vitro, five eIF4GI polypeptides were generated that comigrated in SDS-polyacrylamide gels with the five isoforms of native eIF4GI. Furthermore, translation of eIF4GI-enhanced green fluorescent protein fusion constructs in vitro or in vivo generated five isoforms of fusion polypeptides, suggesting that multiple isoforms of eIF4GI are generated by alternative translation initiation in vitro and in vivo. Mutation of two of the five in-frame AUG residues in the eIF4GI cDNA sequence resulted in loss of corresponding polypeptides after translation in vitro, confirming alternate use of AUGs as the source of the multiple polypeptides. The 5' untranslated region of eIF4GI mRNA also contains an out-of-frame open reading frame (ORF) that may down-regulate expression of eIF4GI. Further, data are presented to suggest that a proposed IRES embedded in the eIF4GI ORF is able to catalyze synthesis of multiple eIF4GI isoforms as well. Our data suggest that expression of the eIF4GI isoforms is partly controlled by a complex translation strategy involving both cap-dependent and cap-independent mechanisms.

Translational control of gene expression: Role of IRESs and consequences for cell transformation and angiogenesis

Translational control of gene expression has, over the last 10 years, become appreciated as an important process in its regulation in eukaryotes. Among a series of control mechanisms exerted at the translational level, the use of alternative codons provides a very subtle means of increasing gene diversity by expressing several proteins from a single mRNA. The internal ribosome entry sites (IRESs) act as specific translational enhancers that allow translation initiation to occur independently of the classic cap-dependent mechanism, in response to specific stimuli and under the control of different trans-acting factors. It is striking to observe that the two processes mostly concern genes coding for control proteins such as growth factors, protooncogenes, angiogenesis factors, and apoptosis regulators. Here, we focus on the translational regulation of four mRNAs, with both IRESs and alternative initiation codons, which are the messengers of retroviral murine leukemia virus, fibroblast growth factor 2, vascular endothelial growth factor, and protooncogene c-myc. Four of them are involved in cell transformation and/or angiogenesis, with important consequences for such translation regulations in these pathophysiological processes.

Internal ribosome entry segment-mediated initiation of c-Myc protein synthesis following genotoxic stress

Initiation of translation of the proto-oncogene c-myc can occur by either the cap-dependent scanning mechanism or by internal ribosome entry. The latter mechanism requires a complex RNA structural element that is located in the 5′ untranslated region of c-myc, termed an internal ribosome entry segment (IRES). Recent work has shown that IRESs are used to maintain protein expression under conditions when cap-dependent translation initiation is compromised; for example, during mitosis, apoptosis and under conditions of cell stress, such as hypoxia or heat shock. Induction of genotoxic stress also results in a large reduction in global protein synthesis rates and therefore we investigated whether the c-myc IRES was active following DNA damage. As expected, in cells treated with either ethylmethane sulphonate or mitomycin C there was a large reduction in protein synthesis, although this was brought about by two different mechanisms. However, in each case the c-myc IRES was active and c-Myc protein expression was maintained. Finally we showed that the proteins required for this process are downstream of the p38 mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase (ERK)/MEK(MAPK/ERK kinase) signalling pathways, since pre-treatment of cells with inhibitors of these pathways before DNA damage is initiated inhibits both c-myc IRES activity and expression of c-Myc protein.

Internal ribosome entry segment-mediated initiation of c-Myc protein synthesis following genotoxic stress.

Initiation of translation of the proto-oncogene c-myc can occur by either the cap-dependent scanning mechanism or by internal ribosome entry. The latter mechanism requires a complex RNA structural element that is located in the 5' untranslated region of c-myc, termed an internal ribosome entry segment (IRES). Recent work has shown that IRESs are used to maintain protein expression under conditions when cap-dependent translation initiation is compromised; for example, during mitosis, apoptosis and under conditions of cell stress, such as hypoxia or heat shock. Induction of genotoxic stress also results in a large reduction in global protein synthesis rates and therefore we investigated whether the c-myc IRES was active following DNA damage. As expected, in cells treated with either ethylmethane sulphonate or mitomycin C there was a large reduction in protein synthesis, although this was brought about by two different mechanisms. However, in each case the c-myc IRES was active and c-Myc protein expression was maintained. Finally we showed that the proteins required for this process are downstream of the p38 mitogen-activated protein kinase (MAPK)/extracellular-signal-regulated protein kinase (ERK)/MEK(MAPK/ERK kinase) signalling pathways, since pre-treatment of cells with inhibitors of these pathways before DNA damage is initiated inhibits both c-myc IRES activity and expression of c-Myc protein.

Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs.

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.

N-myc translation is initiated via an internal ribosome entry segment that displays enhanced activity in neuronal cells.

Eukaryotic translation can be initiated either by a cap-dependent mechanism or by internal ribosome entry, a process by which ribosomes are directly recruited to structured regions of mRNA upstream of the initiation codon. We analysed the 5' untranslated region (UTR) of the proto-oncogene N-myc, and demonstrated by transfections in a dicistronic vector system that it contains a potent internal ribosome entry segment (IRES). The IRES is similar in length to the c-myc IRES and the activities of these IRESs are comparable in non-neuronal cells. Transfections were also carried out in cell lines derived from neuroblastomas, in which N-myc is expressed, and in a neuronal precursor cell line. In these cells the N-myc IRES is up to seven times more active than that of c-myc, suggesting that neuronal-specific non-canonical trans-acting factors are used by the N-myc but not the c-myc IRES. N-myc expression is increased by gene amplification in many neuroblastomas, but this is the first example of a translational mechanism by which N-myc expression could be further increased. The discovery of an IRES that displays enhanced activity in neuronal cell lines has important potential as a tool for protein expression in neural tissue.

Vitamin B12 and hepatitis C: molecular biology and human pathology.

Cobalamins are stored in high concentrations in the human liver and thus are available to participate in the regulation of hepatotropic virus functions. We show that cyanocobalamin (vitamin B12) inhibited the HCV internal ribosome entry site (IRES)-dependent translation of a reporter gene in vitro in a dose-dependent manner without significantly affecting the cap-dependent mechanism. Vitamin B12 failed to inhibit translation by IRES elements from encephalomyocarditis virus (EMCV) or classical swine fever virus (CSFV). We also demonstrate a relationship between the total cobalamin concentration in human sera and HCV viral load (a measure of viral replication in the host). The mean viral load was two orders of magnitude greater when the serum cobalamin concentration was above 200 pM (P < 0.003), suggesting that the total cobalamin concentration in an HCV-infected liver is biologically significant in HCV replication.

C-myc Internal ribosome entry site activity is developmentally controlled and subjected to a strong translational repression in adult transgenic mice.

The expression of c-myc proto-oncogene, a key regulator of cell proliferation and apoptosis, is controlled at different transcriptional and posttranscriptional levels. In particular, the c-myc mRNA contains an internal ribosome entry site (IRES) able to promote translation initiation independently from the classical cap-dependent mechanism. We analyzed the variations of c-myc IRES activity ex vivo in different proliferating cell types, and in vivo in transgenic mice expressing a bicistronic dual luciferase construct. c-myc IRES efficiency was compared to that of encephalomyocarditis virus (EMCV) IRES under the same conditions. The c-myc IRES was active but with variable efficiency in all transiently transfected cell types; it was also active in the 11-day- old (E11) embryo and in some tissues of the E16 embryo. Strikingly, its activity was undetected or very low in all adult organs tested. In contrast, EMCV IRES was very active in most cell types ex vivo, as well as in embryonic and adult tissues. These data suggest a crucial role of IRES in the control of c-myc gene expression throughout development, either during embryogenesis where its activity might participate in cell proliferation or later on, where its silencing could contribute to the downregulation of c-myc expression, whose deregulation leads to tumor formation.