Dicistronic mRNA Research Articles

Internal initiation in Saccharomyces cerevisiae mediated by an initiator tRNA/eIF2-independent internal ribosome entry site element

Internal initiation of translation can be mediated by specific internal ribosome entry site (IRES) elements that are located in certain mammalian and viral mRNA molecules. Thus far, these mammalian cellular and viral IRES elements have not been shown to function in the yeast Saccharomyces cerevisiae. We report here that a recently discovered IRES located in the genome of cricket paralysis virus can direct the efficient translation of a second URA3 cistron in dicistronic mRNAs in S. cerevisiae, thereby conferring uracil-independent growth. Curiously, the IRES functions poorly in wild-type yeast but functions efficiently either in the presence of constitutive expression of the eIF2 kinase GCN2 or in cells that have two initiator tRNA(met) genes disrupted. Both of these conditions have been shown to lower the amounts of ternary eIF2-GTP/initiator tRNA(met) complexes. Furthermore, tRNA(met)-independent initiation was also observed in translation-competent extracts prepared from S. cerevisiae in the presence of edeine, a compound that has been shown to interfere with start codon recognition by ribosomal subunits carrying ternary complexes. Therefore, the cricket paralysis virus IRES is likely to recruit ribosomes by internal initiation in S. cerevisiae in the absence of eIF2 and initiator tRNA(met), by the same mechanism of factor-independent ribosome recruitment used in mammalian cells. These findings will allow the use of yeast genetics to determine the mechanism of internal ribosome entry.

A 5′ Leader of Rbm3, a Cold Stress-induced mRNA, Mediates Internal Initiation of Translation with Increased Efficiency under Conditions of Mild Hypothermia

Although mild hypothermia generally reduces protein synthesis in mammalian cells, the expression of a small number of proteins, including Rbm3, is induced under these conditions. In this study, we identify an Rbm3 mRNA with a complex 5' leader sequence containing multiple upstream open reading frames. Although these are potentially inhibitory to translation, monocistronic reporter mRNAs containing this leader were translated relatively efficiently. In addition, when tested in the intercistronic region of dicistronic mRNAs, this leader dramatically enhanced second cistron translation, both in transfected cells and in cell-free lysates, suggesting that the Rbm3 leader mediates cap-independent translation via an internal ribosome entry site (IRES). Inasmuch as Rbm3 mRNA and protein levels are both increased in cells exposed to mild hypothermia, the activity of this IRES was evaluated at a cooler temperature. Compared to 37 degrees C, IRES activity at 33 degrees C was enhanced up to 5-fold depending on the cell line. Moderate enhancements also occurred with constructs containing other viral and cellular IRESes. These effects of mild hypothermia on translation were not caused by decreased cell growth, as similar effects were not observed when cells were serum starved. The results suggest that cap-independent mechanisms may facilitate the translation of particular mRNAs during mild hypothermia.

EIF4G Functionally Differs from eIFiso4G in Promoting Internal Initiation, Cap-independent Translation, and Translation of Structured mRNAs

Eukaryotic initiation factor (eIF) 4G plays an important role in assembling the initiation complex required for ribosome binding to an mRNA. Plants, animals, and yeast each express two eIF4G homologs, which share only 30, 46, and 53% identity, respectively. We have examined the functional differences between plant eIF4G proteins, referred to as eIF4G and eIFiso4G, when present as subunits of eIF4F and eIFiso4F, respectively. The degree to which a 5'-cap stimulated translation was inversely correlated with the concentration of eIF4F or eIFiso4F and required the poly(A)-binding protein for optimal function. Although eIF4F and eIFiso4F directed translation of unstructured mRNAs, eIF4F supported translation of an mRNA containing 5'-proximal secondary structure substantially better than did eIFiso4F. Moreover, eIF4F stimulated translation from uncapped monocistronic or dicistronic mRNAs to a greater extent than did eIFiso4F. These data suggest that at least some functions of plant eIFiso4F and eIF4F have diverged in that eIFiso4F promotes translation preferentially from unstructured mRNAs, whereas eIF4F can promote translation also from mRNAs that contain a structured 5'-leader and that are uncapped or contain multiple cistrons. This ability may also enable eIF4F to promote translation from standard mRNAs under cellular conditions in which cap-dependent translation is inhibited.

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS)

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB Glc subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB Glc are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB Glc and IIA Glc were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA Glc and IICB Glc had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA Glc displayed increased stability against temperature and GuHCl-induced unfolding.

Genetic analysis of a poliovirus/hepatitis C virus chimera: new structure for domain II of the internal ribosomal entry site of hepatitis C virus.

Internal ribosomal entry sites (IRESs) of certain plus-strand RNA viruses direct cap-independent initiation of protein synthesis both in vitro and in vivo, as can be shown with artificial dicistronic mRNAs or with chimeric viral genomes in which IRES elements were exchanged from one virus to another. Whereas IRESs of picornaviruses can be readily analyzed in the context of their cognate genome by genetics, the IRES of hepatitis C virus (HCV), a Hepacivirus belonging to Flaviviridae, cannot as yet be subjected to such analyses because of difficulties in propagating HCV in tissue culture or in experimental animals. This enigma has been overcome by constructing a poliovirus (PV) whose translation is controled by the HCV IRES. Within the PV/HCV chimera, the HCV IRES has been subjected to systematic 5' deletion analyses to yield a virus (P/H710-d40) whose replication kinetics match that of the parental poliovirus type 1 (Mahoney). Genetic analyses of the HCV IRES in P/H710-d40 have confirmed that the 5' border maps to domain II, thereby supporting the validity of the experimental approach applied here. Additional genetic experiments have provided evidence for a novel structural region within domain II. Arguments that the phenotypes observed with the mutant chimera relate solely to impaired genome replication rather than deficiencies in translation have been dispelled by constructing novel dicistronic poliovirus replicons with the gene order [PV]cloverleaf-[HCV]IRES-Deltacore-R-Luc-[PV]IRES-F-Luc-P2,3-3'NTR, which have allowed the measurement of HCV IRES-dependent translation independently from the replication of the replicon RNA.

Internal initiation of translation of five dendritically localized neuronal mRNAs

In neurons, translation of dendritically localized mRNAs is thought to play a role in affecting synaptic efficacy. Inasmuch as components of the translation machinery may be limiting in dendrites, we investigated the mechanisms by which translation of five dendritically localized mRNAs is initiated. The 5' leader sequences of mRNAs encoding the activity-regulated cytoskeletal protein, the alpha subunit of calcium-calmodulin-dependent kinase II, dendrin, the microtubule-associated protein 2, and neurogranin (RC3) were evaluated for their ability to affect translation in the 5' untranslated region of a monocistronic reporter mRNA. In both neural and nonneural cell lines, the activity-regulated cytoskeletal protein, microtubule-associated protein 2, and alpha-CaM Kinase II leader sequences enhanced translation, whereas the dendrin and RC3 5' untranslated regions slightly inhibited translation as compared with controls. When cap-dependent translation of these constructs was suppressed by overexpression of a protein that binds the cap-binding protein eIF4E, it was revealed that translation of these mRNAs had both cap-dependent and cap-independent components. The cap-independent component was further analyzed by inserting the 5' leader sequences into the intercistronic region of dicistronic mRNAs. All five leader sequences mediated internal initiation via internal ribosome entry sites (IRESes). The RC3 IRES was most active and was further characterized after transfection in primary neurons. Although translation mediated by this IRES occurred throughout the cell, it was relatively more efficient in dendrites. These data suggest that IRESes may increase translation efficiency at postsynaptic sites after synaptic activation.

Identification of two short internal ribosome entry sites selected from libraries of random oligonucleotides.

Sequences that control translation of mRNA may play critical roles in regulating protein levels. One such element is the internal ribosome entry site (IRES). We previously showed that a 9-nt segment in the 5' leader sequence of the mRNA encoding Gtx homeodomain protein could function as an IRES. To identify other short sequences with similar properties, we designed a selection procedure that uses a retroviral vector to express dicistronic mRNAs encoding enhanced green and cyan fluorescent proteins as the first and second cistrons, respectively. Expression of the second cistron was dependent upon the intercistronic sequences and was indicative of IRES activity. B104 cells were infected with two retroviral libraries that contained random sequences of 9 or 18 nt in the intercistronic region. Cells expressing both cistrons were sorted, and sequences recovered from selected cells were reassayed for IRES activity in a dual luciferase dicistronic mRNA. Two novel IRESes were identified by this procedure, and both contained segments with complementarity to 18S rRNA. When multiple copies of either segment were linked together, IRES activities were dramatically enhanced. Moreover, these synthetic IRESes were differentially active in various cell types. These properties are similar to those of the previously identified 9-nt IRES module from Gtx mRNA. These results provide further evidence that short nucleotide sequences can function as IRESes and support the idea that some cellular IRESes may be composed of shorter functional modules. The ability to identify IRES modules with specific expression properties may be useful in the design of vectors for biotechnology and gene therapy.

Transcript leader regions of two Saccharomyces cerevisiae mRNAs contain internal ribosome entry sites that function in living cells.

In higher eukaryotes, translation of some mRNAs occurs by internal initiation. It is not known, however, whether this mechanism is used to initiate the translation of any yeast mRNAs. In this report, we identify naturally occurring nucleotide sequences that function as internal ribosome entry sites (IRESes) within the 5' leader sequences of Saccharomyces cerevisiae YAP1 and p150 mRNAs. When tested in the 5' untranslated regions of monocistronic reporter genes, both leader sequences enhanced translation efficiency in vegetatively growing yeast cells. Moreover, when tested in the intercistronic region of dicistronic mRNAs, both sequences were shown to contain IRESes that functioned in living cells. The activity of the p150 leader was much greater than that of the YAP1 leader. The second cistron was not expressed in control dicistronic constructs that lacked these sequences or contained the 5' leader sequence of the CLN3 mRNA in the intercistronic region. Further analyses of the p150 IRES revealed that it contained several nonoverlapping segments that were able independently to mediate internal initiation. These results suggested a modular composition for the p150 IRES that resembled the composition of IRESes contained within some cellular mRNAs of higher eukaryotes. Both YAP1 and p150 leaders contain several complementary sequence matches to yeast 18S rRNA. The findings are discussed in terms of our understanding of internal initiation in higher eukaryotes.

Demonstration of Functional Requirement of Polypyrimidine Tract-binding Protein by SELEX RNA during Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation Initiation

Polypyrimidine tract-binding protein (PTB) has been previously shown to physically interact with the hepatitis C virus (HCV) RNA genome at its 5'- and 3'-noncoding regions. Using high affinity SELEX RNA molecules, we present evidence for the functional requirement of PTB during HCV internal ribosome entry site (IRES)-controlled translation initiation. This study was carried out in rabbit reticulocyte translation lysates in which the HCV IRES-driven reporter RNA was introduced along with the PTB-specific SELEX RNA molecules. The SELEX RNAs specifically inhibited the HCV IRES function in the context of mono- and dicistronic mRNAs. The cap-dependent translation of a reporter (chloramphenicol acetyltransferase) RNA or naturally capped brome mosaic virus RNA, however, was not affected by the presence of SELEX during in vitro translation assays. The SELEX-mediated inhibition of the HCV IRES is shown to be relieved by the addition of recombinant human PTB in an add-back experiment. The in vivo requirement of PTB was further confirmed by cotransfection of Huh7 cells with reporter RNA and PTB-specific SELEX RNA. The HCV IRES activity was inhibited by the SELEX RNA in these cells, but not by an unrelated control RNA. Together, these results demonstrate the functional requirement of cellular PTB in HCV translation and further support the feasible use of SELEX RNA strategy in demonstrating the functional relevance of cellular protein(s) in complex biological processes.

High-level synthesis of human prolactin in Chinese-Hamster ovary cells.

Two eukaryotic human prolactin (hPRL) expression vectors, based on a selectable dihydrofolate reductase (dhfr) marker, were used to transfect dhfr(-) Chinese- hamster ovary (CHO) cells. One vector, p658-hPRL, contains the hepatitis-B virus-X cDNA coding for a viral transactivator and sequences mediating dhfr mRNA degradation. The other, pEDdc-hPRL, carries the encephalomyocarditis virus leader sequence coupled to hPRL cDNA to provide high-level protein expression, possibly via a mechanism of internal translation initiation in dicistronic mRNA. Without methotrexate (MTX) amplification, p658-hPRL-transfected stable cell lines, secreting up to approximately 10 microg of hPRL/10(6) cells per day, could be rapidly obtained; production by pEDdc-hPRL-transfected cells was about 10-fold lower. However, a three-step MTX amplification of the latter led to clones secreting up to approximately 30 microg of hPRL/10(6) cells per day. A pilot production using a hollow-fibre bioreactor indicated that highly concentrated hormone levels in the medium could be obtained, with a production of up to 150 microg of hPRL/ml per day. SDS/PAGE analysis indicated that recombinant hPRL contained approximately 10% glycosylated PRL. Chromatographically purified non-glycosylated and glycosylated recombinant hPRL had bioactivities of 35 and 16 i.u./mg, respectively (Nb2 cell bioassay). This appears to be the first report describing production and purification of recombinant hPRL from CHO cells, secreted at levels higher than reported thus far in eukaryotic systems.

Human La Antigen Is Required for the Hepatitis C Virus Internal Ribosome Entry Site-mediated Translation

The 5'-noncoding region (5'-NCR) of the hepatitis C virus (HCV) RNA genome serves as an internal ribosome entry site (IRES) and mediates translation initiation in a cap-independent manner. Previously, we reported the interaction between La antigen and the HCV IRES, which appeared to occur in the context of initiator AUG. It was further shown that HCV IRES-mediated translation was stimulated in the presence of human La antigen. In this study, we have defined the cis- and trans-acting elements responsible for La-5'-NCR interactions and established the dependence of the HCV IRES efficiency on cellular La antigen. During the La-IRES interaction, initiator AUG but not the neighboring codons was found to be the direct target of La binding. The C terminus effector domain-dependent modulation of La binding to the HCV IRES is demonstrated by deletion and substitution mutagenesis of the protein. An RNA systematic evolution of ligands by exponential enrichment (SELEX), generated against La protein that selectively binds La in HeLa lysates and competes for the protein binding to the 5'-NCR, was used to demonstrate the requirement of La for the HCV IRES function in the context of mono- and dicistronic mRNAs. Sequestration of La antigen by the RNA SELEX in HeLa translation lysates blocked the HCV and poliovirus IRES-mediated translation in vitro. The functional requirement of La protein for the HCV IRES activity was further established in a liver-derived cell line and in an add-back experiment in which the inhibited IRES was rescued by recombinant human La. These results strongly argue for the novel role of La protein during selection of the initiator AUG and its participation during internal initiation of translation of the HCV RNA genome.

Duplication, dicistronic transcription, and subsequent evolution of the Alcohol dehydrogenase and Alcohol dehydrogenase-related genes in Drosophila.

It has recently been discovered that the Alcohol dehydrogenase and Alcohol dehydrogenase-related genes of Drosophila melanogaster and closely related species constitute a single transcription unit and that the Alcohol dehydrogenase-related gene is exclusively expressed from a dicistronic mRNA. Here, we show that in Drosophila lebanonensis, subgenus Scaptodrosophila, Adhr: is also transcribed as a dicistronic transcript with Adh Using degenerate primers designed on the sequence of the known Adhr proteins, we have been able to amplify and sequence a partial sequence of Adhr: in species representative of the whole subgenus Drosophila. This has allowed the study of the organization and expression of Adhr: in Drosophila buzzatii. We find that in D. buzzatii Adhr is transcribed as a monocistronic transcript. Adh and Adhr are believed to originate by duplication, and our data suggest that the cotranscription of these two genes was the primitive state, and that their independent transcription in the subgenus Drosophila is derived. We can rationalize the D. buzzatii condition as being correlated with the two genes evolving independent transcriptional control. However, why these two genes with clear divergence in the functions of their proteins should remain cotranscribed in groups as divergent as the subgenus Sophophora and the subgenus Scaptodrosophila remains a mystery.

IRES elements: fine-tuning of gene expression at the translational level

Some viral mRNAs contain internal ribosome entry sites (IRESs), which are cis elements that allow the binding of the translational apparatus directly within the mRNA. IRESs therefore offer an alternative to the general mechanism of the ribosome scanning from the 5′-end cap structure to the initiator codon that prevails for most cellular mRNAs. Interestingly, IRESs have also been identified in a few cellular messengers. Whereas viral IRESs provide the advantage of initiating translation in the case of uncapped mRNAs (e.g. picornaviruses mRNAs) or under conditions where the global host protein synthesis is inhibited (e.g. during infection), the biological function of cellular IRESs is still unclear. Recent ex vivo data have shown that certain cellular IRESs could mediate a cell-cycle-dependent translational control. In the July issue of the Journal of Cell Biology, Creancier and co-workers describe the first in vivo study of the activity of a mammalian cellular IRES, the human fibroblast growth factor 2 (FGF2) IRES (Ref. 1xFibroblast growth factor 2 internal ribosome entry site (IRES) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation. Creancier, L. et al. J. Cell Biol. 2000; 150: 275–281Crossref | PubMed | Scopus (103)See all ReferencesRef. 1).The authors have created transgenic mice that express a dicistronic mRNA bearing two open reading frames (ORFs) encoding two distinct luciferases. In this standard assay, the upstream ORF is translated via the cap-dependent scanning mechanism, whereas translation of the second ORF is driven by the IRES located between them. By this method they could measure the IRES activity of the FGF2 mRNA specifically. Strikingly, they observed a stage- and tissue-specific modulation of the FGF2 IRES activity. It was high in the embryo (especially in the brain and the heart at E16) in comparison with the adult, in which this strong IRES activity appeared to be restricted mainly to the brain. This contrasted with the widespread strong activity of picornavirus IRESs. How does the tropism of FGF2 IRES activity fit with the broad expression of this growth factor all over the organism? The fact that FGF2 translation can be initiated via the IRES but also by a cap-dependent mechanism could explain such a discrepancy. Nevertheless, the poor IRES activity in organs that otherwise express FGF2 raises the question of the inducibility of IRESs. Indeed, stimulation of the FGF2 IRES could represent a means to adapt the level of FGF2 expression selectively in response to a given stimulus. This could involve potential as yet unidentified trans-acting factors that would be active in the adult brain while silent in other organs (in the steady state).The stringent in vivo regulation of the FGF2 IRES activity described in this paper emphasizes the role of translation in the control of gene expression, and the strategy applied here can be used for the in vivo study of other cellular IRESs. This could allow to address the question whether IRES elements coordinately regulate the translation of a subclass of mRNAs. Furthermore, the understanding of the molecular mechanisms underlying IRES regulation in vivo might lead to strategies designed to control the expression of endogenous factors artificially, as well as to target the expression of trans genes of interest. The animal model described here will represent an indispensable tool to validate such strategies.

The role of 5'-leader length, secondary structure and PABP concentration on cap and poly(A) tail function during translation in Xenopus oocytes.

The 5'-cap structure and poly(A) tail of eukaryotic mRNAs function synergistically to promote translation initiation through a physical interaction between the proteins that bind to these regulatory elements. In this study, we have examined the effect of leader length and the presence of secondary structure on the translational competence and the function of the cap and poly(A) tail for mRNAs microinjected into Xenopus oocytes. Increasing the length of the 5'-leader from 17 to 144 nt resulted in a 2- to 4-fold increase in expression from an mRNA containing an unstructured leader but increased expression up to 20-fold for an mRNA containing 5'-proximal structure. Consequently, the presence of secondary structure was less inhibitory for those mRNAs with a longer 5'-leader. Co-injection of poly(A)-binding protein (PABP) mRNA increased the function of the cap and poly(A) tail in promoting translation from poly(A)(+) but not poly(A)(-) mRNAs, particularly for mRNAs containing secondary structure. In the absence of an internal ribosome entry site, expression from the distal cistron of a dicistronic mRNA increased as a function of the length of the intercistronic region and the concentration of PABP. The inhibitory effect of intercistronic located secondary structure on translation was position-dependent. Indeed, the effect of secondary structure was abolished if positioned 134 nt upstream of the distal cistron. These data suggest that the length of a leader, the presence of secondary structure and the concentration of PABP determine the extent to which the cap and poly(A) tail regulate translation.

Circadian clock-protein expression in cyanobacteria: rhythms and phase setting.

The cyanobacterial gene cluster kaiABC encodes three essential circadian clock proteins: KaiA, KaiB and KaiC. The KaiB and KaiC protein levels are robustly rhythmical, whereas the KaiA protein abundance undergoes little if any circadian oscillation in constant light. The level of the KaiC protein is crucial for correct functioning of the clock because induction of the protein at phases when the protein level is normally low elicits phase resetting. Titration of the effects of the inducer upon phase resetting versus KaiC level shows a direct correlation between induction of the KaiC protein within the physiological range and significant phase shifting. The protein synthesis inhibitor chloramphenicol prevents the induction of KaiC and blocks phase shifting. When the metabolism is repressed by either translational inhibition or constant darkness, the rhythm of KaiC abundance persists; therefore, clock protein expression has a preferred status under a variety of conditions. These data indicate that rhythmic expression of KaiC appears to be a crucial component of clock precession in cyanobacteria.

Naturally occurring dicistronic cricket paralysis virus RNA is regulated by two internal ribosome entry sites.

Cricket paralysis virus is a member of a group of insect picorna-like viruses. Cloning and sequencing of the single plus-strand RNA genome revealed the presence of two nonoverlapping open reading frames, ORF1 and ORF2, that encode the nonstructural and structural proteins, respectively. We show that each ORF is preceded by one internal ribosome entry site (IRES). The intergenic IRES is located 6,024 nucleotides from the 5' end of the viral RNA and is more active than the IRES located at the 5' end of the RNA, providing a mechanistic explanation for the increased abundance of structural proteins relative to nonstructural proteins in infected cells. Mutational analysis of this intergenic-region IRES revealed that ORF2 begins with a noncognate CCU triplet. Complementarity of this CCU triplet with sequences in the IRES is important for IRES function, pointing to an involvement of RNA-RNA interactions in translation initiation. Thus, the cricket paralysis virus genome is an example of a naturally occurring, functionally dicistronic eukaryotic mRNA whose translation is controlled by two IRES elements located at the 5' end and in the middle of the mRNA. This finding argues that eukaryotic mRNAs can express multiple proteins not only by polyprotein processing, reinitiation and frameshifting but also by using multiple IRES elements.

Genetic analysis of a poliovirus/hepatitis C virus (HCV) chimera: interaction between the poliovirus cloverleaf and a sequence in the HCV 5' nontranslated region results in a replication phenotype.

Internal ribosomal entry sites (IRESs) can function in foreign viral genomes or in artificial dicistronic mRNAs. We describe an interaction between the wild-type hepatitis C virus (HCV)-specific sequence and the poliovirus (PV) 5'-terminal cloverleaf in a PV/HCV chimeric virus (containing the HCV IRES), resulting in a replication phenotype. Either a point mutation at nucleotide (nt) 29 or a deletion up to nt 40 in the HCV 5' nontranslated region relieved the replication block, yielding PV/HCV variants replicating to high titers. Fortuitous yet crippling interactions between an IRES and surrounding heterologous RNA must be considered when IRES-based dicistronic expression vectors are being constructed.

A 9-nt segment of a cellular mRNA can function as an internal ribosome entry site (IRES) and when present in linked multiple copies greatly enhances IRES activity.

This study addresses the properties of a newly identified internal ribosome entry site (IRES) contained within the mRNA of the homeodomain protein Gtx. Sequential deletions of the 5' untranslated region (UTR) from either end did not define distinct IRES boundaries; when five nonoverlapping UTR fragments were tested, four had IRES activity. These observations are consistent with other cellular IRES analyses suggesting that some cellular IRESes are composed of segments (IRES modules) that independently and combinatorially contribute to overall IRES activity. We characterize a 9-nt IRES module from the Gtx 5' UTR that is 100% complementary to the 18S rRNA at nucleotides 1132-1124. In previous work, we demonstrated that this mRNA segment could be crosslinked to its complement within intact 40S subunits. Here we show that increasing the number of copies of this IRES module in the intercistronic region of a dicistronic mRNA strongly enhances IRES activity in various cell lines. Ten linked copies increased IRES activity up to 570-fold in Neuro 2a cells. This level of IRES activity is up to 63-fold greater than that obtained by using the well characterized encephalomyocarditis virus IRES when tested in the same assay system. When the number of nucleotides between two of the 9-nt Gtx IRES modules was increased, the synergy between them decreased. In light of these findings, we discuss possible mechanisms of ribosome recruitment by cellular mRNAs, address the proposed role of higher order RNA structures on cellular IRES activity, and suggest parallels between IRES modules and transcriptional enhancer elements.

Expression of the Totivirus Helminthosporium victoriae 190S Virus RNA-Dependent RNA Polymerase from Its Downstream Open Reading Frame in Dicistronic Constructs

The undivided double-stranded RNA (dsRNA) genome of Helminthosporium victoriae 190S virus (Hv190SV) (genus Totivirus) consists of two large overlapping open reading frames (ORFs). The 5'-proximal ORF encodes a capsid protein (CP), and the downstream, 3'-proximal ORF encodes an RNA-dependent RNA polymerase (RDRP). Unlike the RDRPs of some other totiviruses, which are expressed as a CP-RDRP (Gag-Pol-like) fusion protein, the Hv190SV RDRP is detected only as a separate, nonfused polypeptide. In this study, we examined the expression of the RDRP ORF fused in frame to the coding sequence of the green fluorescent protein (GFP) in bacteria and Schizosaccharomyces pombe cells. The GFP fusions were readily detected in bacteria transformed with the monocistronic construct RDRP:GFP; expression of the downstream RDRP:GFP from the dicistronic construct CP-RDRP:GFP could not be detected. However, fluorescence microscopy and Western blot analysis indicated that RDRP:GFP was expressed at low levels from its downstream ORF in the dicistronic construct in S. pombe cells. No evidence that the RDRP ORF was expressed from a transcript shorter than the full-length dicistronic mRNA was found. A coupled termination-reinitiation mechanism that requires host or eukaryotic cell factors is proposed for the expression of Hv190SV RDRP.

Identification and characterization of the functional elements within the tobacco etch virus 5' leader required for cap-independent translation.

Translation in plants is highly cap dependent, and the only plant mRNAs known to naturally lack a cap structure (m(7)GpppN) are viral in origin. The genomic RNA of tobacco etch virus (TEV), a potyvirus that belongs to the picornavirus superfamily, is a polyadenylated mRNA that is naturally uncapped and yet is a highly competitive mRNA during translation. The 143-nucleotide 5' leader is responsible for conferring cap-independent translation even on reporter mRNAs. We have carried out a deletion analysis of the TEV 5' leader to identify the elements responsible for its regulatory function and have identified two centrally located cap-independent regulatory elements (CIREs) that promote cap-independent translation. The introduction of a stable stem-loop structure upstream of each element demonstrated that CIRE-1 is less 5' end dependent in function than CIRE-2. In a dicistronic mRNA, the presence of the TEV 5' leader sequence in the intercistronic region increased expression of the second cistron, suggesting that the viral sequence can function in a 5'-distal position. Interestingly, the introduction of a stable stem-loop upstream of the TEV leader sequence or upstream of either CIRE in dicistronic constructs markedly increased their regulatory function. These data suggest that the TEV 5' leader contains two elements that together promote internal initiation but that the function of one element, in particular, is facilitated by proximity to the 5' end.