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1 Department of Genetics and Microbiology, Faculty of Science, Charles University in Prague, Vinicna 5, 128 44 Prague, Czech Republic
2 Institute of Molecular Genetics, Laboratory of Leukocyte Antigens, AS CR, Videnska 1083, 140 00 Prague, Czech Republic
Correspondence
Martin Pospisek
martin{at}natur.cuni.cz
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Supplementary data and sequences can be found with the online version of this paper and in IRESite, the Database of Experimentally Verified IRES Structures. http://www.iresite.org/; IRESite ID 86, 88, 90–99, 126–128 (Mokrejs et al., 2006
).
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Hwang et al., 1998; Reynolds et al., 1995; Rijnbrand et al., 1995, 2001; Tsukiyama-Kohara et al., 1992). The HCV IRES is unique in its low requirement for host translation initiation factors and its ability to bind directly to the small 40S ribosomal subunit (Fukushi et al., 2001; Kolupaeva et al., 2000; Lytle et al., 2001; Otto et al., 2002). The translation initiation complex is further stabilized by translation initiation factor 3, which interacts physically with both the ribosome and loop III of the HCV IRES (Buratti et al., 1998; Collier et al., 2002; Kieft et al., 2001; Sizova et al., 1998). The resulting complex associates with the eukaryotic initiation factor 2 (eIF2)–GTP–met-tRNAi ternary complex and recruits the 60S ribosomal subunit to begin viral protein synthesis (Sizova et al., 1998). The HCV IRES contains several initiation and termination codons; however, the ribosome does not utilize them and, due to the structural and sequence features of the IRES, is positioned internally and initiates protein synthesis from the sixth – i.e. the last – 342AUG codon (Reynolds et al., 1996; Rijnbrand et al., 1996). The cellular translation initiation machinery can, to some extent, tolerate both mutations of the authentic initiation codon to non-AUG codons and minor changes of its position within loop IV (Honda et al., 1996; Hwang et al., 1998; Reynolds et al., 1995). Curiously, inclusion of a markedly longer part of the core protein coding sequence significantly suppressed the translation measured by bicistronic mRNAs both in vivo and in vitro (Wang et al., 2000).
The yeast Saccharomyces cerevisiae has been shown to be a powerful model organism for studies of basic molecular biology and genetic processes, including translation initiation. Nevertheless, there is little known about the ability of yeast cells to utilize viral IRES elements for internal translation initiation (Altmann et al., 1990; Iizuka et al., 1994; Paz et al., 1999). Several viral IRESs, including those from poliovirus, coxsackievirus, HCV, tobamovirus and cricket paralysis virus, have been reported to be functional in translation-competent yeast-cell extracts (Altmann et al., 1990; Dorokhov et al., 2002; Iizuka & Sarnow, 1997; Iizuka et al., 1994). Among them, only the function of the cricket paralysis virus IRES has been plausibly demonstrated in living yeast cells (Thompson et al., 2001). Rosenfeld & Racaniello (2005) recently presented the first attempt to demonstrate HCV IRES activity in yeast.
To analyse HCV IRES activity and cap-independent translation in yeast generally, we developed a sensitive system based upon the new pFGAL4 bicistronic vectors and engineered yeast strains. These generate an amplified signal in vivo by the primary reporter (GAL4)-mediated transcription activation of secondary reporters bearing measurable enzymic (lacZ) and physiological (ADE2, HIS3) activities. Presented data suggest that, in yeast, the HCV IRES is fully functional, translation initiates at the authentic initiation codon at position 342 and mutations that abrogate IRES translational activity in mammals act similarly in yeast.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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library was created by ligation of HindIII/TasI- or TasI-digested bacteriophage DNA (Fermentas) into pFGAL4 cut by HindIII/EcoRI or EcoRI, respectively. The whole-length IRES sequence from HCV genotype 1a (nt 1–385) was PCR-amplified from the p90HCVFL vector (courtesy of Charles Rice, Rockefeller University, New York, USA; forward primer, 5'-AAAGGATCCGCCAGCCCCCTGATGGGGGCGACAC-3'; reverse primer, 5'-AGGAATTCGTGTTACGTTTGGTTTTTCTTTGAGGTTTAGG-3') and inserted behind the stem–loop between the reporter genes via EcoRI and BamHI sites (pFGAL4h-HCV1). XmaJI/BamHI digestion of pFGAL4h-HCV1 and subsequent circularization of the blunt-ended vector was used to create a control vector (pFGAL4-HCV1) lacking the 13G : C hairpin. The tricistronic vector pFGAL4-L270-HCV1 and its derivative containing the inverted HCV IRES sequence (pFGAL4-L270-HCV1rc) were prepared by ligation of the full-length HCV IRES amplicon into the single EcoRI site behind the 270 insert. The mammalian bicistronic vector pRG is a derivative of the pDsRed2-C1 vector (pUC ori, KanR, CMV promoter, DsRed2; Clontech) containing the enhanced green fluorescent protein (EGFP) gene from the pEGFP-N1 vector (Clontech) inserted into the HincII site. A new stop codon was introduced at the end of the DsRed2 gene by ligation of the 5'-GATCCAGCAAGATATCCTAA-3'/5'-GATCTTAGGATATCTTGCTG-3' adaptor into the BglII site.
Cells and media.
S. cerevisiae strain PJ69-4A (MATa, trp1-901, leu2-3,112, ura3-52, his3-200, gal4
, gal80, GAL2-ADE2, LYS2 : : GAL1-HIS3, met2 : : GAL7-lacZ) was used throughout this study (James et al., 1996). Yeast transformation was performed by the one-step LiCl method (Gietz & Woods, 2002) and obtained clones were grown in drop-out synthetic minimal medium (SD) without uracil to ensure maintenance of plasmids. Selection for IRES activity was performed on SD plates lacking uracil, adenine and histidine and supplemented with 0–120 mM 3-aminotriazole (3-AT; Sigma), a competitive inhibitor of histidine biosynthesis.
Luciferase, 
-galactosidase (-gal) and flow-cytometry assays.
Yeast cell cultures were grown in SD medium without uracil and harvested in the exponential-growth phase (OD595, 0.5–0.7). Cell extracts were prepared by homogenization with glass beads in a Mixer Mill apparatus MM301 (Retsch) in ice-cold 100 mM potassium phosphate lysis buffer (pH 7.8), containing 0.2 % Triton X-100 and 0.5 mM dithiothreitol. Firefly luciferase activity was quantified in quadruplicate by the Luciferase Assay system (Promega) according the manufacturer's instructions in a Microlite TLX2 Dynatech luminometer. -Gal assays were done in triplicate by using a Galacto-Light Plus -galactosidase assay kit (Tropix) following the manufacturer's instructions. All luminescence measurements were performed within 1 min of starting the respective reactions. The results are expressed as relative normalized luminescence units of -gal activity (RGU), calculated as a quotient of -gal luminescence units/5x105 luciferase luminescence units. Mean values and SD are shown. For flow-cytometry analyses, human liver epithelial CCL-13 cells were transfected in ExGene transfection reagent dissolved in 100 mM NaCl solution according the manufacturer's instructions (Fermentas). After 2 days, cells were harvested by trypsinization and resuspended in Dulbecco's modified Eagle's medium (Sigma-Aldrich). At least fifty thousand cells were analysed by flow cytometry in each experiment, using an LSRII machine (BD Biosciences).
RNA analysis.
Five hundred millilitres of yeast cells (OD595, 0.65) was sedimented and washed twice in 10 ml 10 mM Tris/HCl (pH 7), 5 mM MgCl2, 140 mM KCl. Cell lysis was carried out by using RNA–DNA stabilization reagent (Roche). An mRNA Isolation Kit for Blood and Bone Marrow (Roche) was used to isolate mRNA. DNase treatment and inactivation were carried out by using a DNA-free kit (Ambion) following the manufacturer's instructions. cDNA was synthesized with a gene-specific primer and 200 U SuperScript II RNaseH– reverse transcriptase (Invitrogen). One microlitre of reverse transcriptase reaction was subjected to PCR amplification [95 °C, 1 min (initial template denaturation); 24 cycles of 94 °C, 30 s (denaturation); 54 °C, 30 s (primer annealing); and 68 °C ,1 min (extension); 72 °C, 4 min (final extension)] by using the High Fidelity PCR Master system according to the manufacturer's instructions (Roche). Aliquots (10 µl) of the PCR samples were run on TAE/1 % agarose gels and visualized by ethidium bromide (EtBr) staining. For real-time PCR analysis, a LightCycler 1.5 and QuantiTect SYBR Green PCR kit (Qiagen) were used with essentially the same protocol as described above, with exception that the initial denaturation step was extended to 15 min.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The pFGAL4 vector can be used in any yeast strain lacking the wild-type GAL4 and GAL80 alleles and containing either episomally or chromosomally encoded Gal4-inducible reporter genes, typical in yeast strains that are used routinely for two-hybrid screens and other similar approaches. Throughout this study, we used the PJ69A strain (James et al., 1996), which contains bacterial -gal (LacZ) and yeast ADE2 and HIS3 reporter genes under the control of the Gal4-inducible GAL1 promoter. Data were normalized to the activity of the firefly luciferase encoded by the first cistron of the bicistronic Fluc-IRES-GAL4 mRNA. Employment of the Gal4 transcription factor as a natural signal amplifier in the PJ69A yeast strain thus allows simultaneous determination of even tiny changes in IRES activity, both in lysates by measuring the ratio of the -gal/luciferase (-gal/luc) enzymic activities and in living cells by analysing colony-growth rates on selection SD plates lacking adenine and histidine and containing various concentrations of 3-AT, a competitive inhibitor of the histidine biosynthetic pathway (Fig. 1a). For most experiments, ribosome read-through beyond the end of the first cistron was reduced by inserting a stable 13G : C hairpin loop just behind the firefly luciferase stop codon, thus giving rise to the pFGAL4h vector.
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sequences. Despite the fact that we used random DNA sequences, which therefore contained an unpredictable number of ORFs, multiple translation initiation codons and cryptic promoters, for mini-library construction, we obtained data similar to those reported by Rajkowitsch et al. (2004), who reported that the elongating yeast ribosomes maintain competence to reinitiate only over short distances. This phenomenon is seen clearly in our results, where ribosome reinitiation 100 nt downstream of the luciferase gene stop codon declines by two orders of magnitude (Fig. 1b
). Insertion of the 13G : C stable loop, of minimum free-energy change (G) 177.5 kJ mol–1, substantially lowers the ribosome read-through of the intercistronic region of pFGAL4h bicistronic mRNA and reduces the normalized relative -gal activity by approximately 90 % (Fig. 1b).
Yeast cells can utilize the HCV IRES
Reported observations of moderate activity of the HCV IRES in translation-competent yeast-cell lysates led us to test the functionality of the HCV IRES in living yeast cells. We cloned the full-length 5' UTR of HCV genomic RNA, together with the first 45 nt of the viral polyprotein gene, into the bicistronic pFGAL4h vector in frame with the second reporter, thus making an N-terminal fusion of the first 15 aa from the HCV polyprotein with the yeast transcriptional activator Gal4p. In the PJ69A yeast tester strain, the resulting plasmid, pFGAL4h-HCV1, produced about 42 % of normalized -gal activity compared with the empty bicistronic pFGAL4h vector, but gave rise to approximately 26 times higher relative activity of the -gal reporter than control vectors containing similarly sized pieces of phage DNA inserted between the firefly luciferase and GAL4 genes (Fig. 2a). As well as the lacZ gene, encoding bacterial -gal, the yeast PJ69A tester strain contains also HIS3 and ADE2 genes under the transcriptional control of the Gal4p activator. As shown clearly in Fig. 2(b), results similar to the biochemical -gal/luc assays can be obtained by careful monitoring of yeast-colony growth on selection agar medium containing various concentrations of the competitive inhibitor of the yeast imidazoleglycerol-phosphate dehydratase, encoded by the reporter HIS3 gene.
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). This point-mutated clone differs from standard pFGAL4h-HCV1 by a 4 nt insertion between the authentic IRES initiation codon and the inherent Gal4p initiation codon. The difference of 82 % between the relative -gal activity of HCV1 and that of the mutated HCV3 clone points clearly to the preferential usage of the authentic HCV initiation codon.
Translational activity of the HCV IRES is not influenced by cryptic transcription, splicing or mRNA breakage
We decided to measure the effect of insertion of another ‘blocking’ sequence between the luciferase gene and the HCV IRES on IRES activity in order to exclude an influence of ribosome reinitiation or of a possible artificial promoter lying within the bicistronic part of the vector. To obtain such blocking sequences, we used the pFGAL4 : : library described herein for in vivo selection of clones giving the lowest GAL4 expression in a plate assay as monitored by growth retardation on SD drop-out medium. To prevent instability of the vector and the bicistronic reporter mRNA, we chose among the selected clones those containing the shortest DNA inserts. It is not surprising that such clones usually reveal other short ORFs within the DNA insert. For further work, we picked the pFGAL4-L270 vector, containing a 270 nt long DNA insert, which includes a short ORF located between nt 91 and 147 of the intercistronic region. The HCV IRES was inserted in both orientations just behind the L270 DNA fragment. The resulting vectors, pFGAL4-L270, pFGAL4-L270-HCV1 and pFGAL4-L270-HCV1rc, are thus tricistronic. The HCV IRES activity in these tricistronic vectors was compared with that in standard bicistronic pFGAL4h-HCV1 and pFGAL4h-HCV1rc plasmids, where ‘rc’ means the inverted HCV IRES (reverse complement; Fig. 3). Careful analysis of HCV IRES activity in tri- and bicistronic vectors revealed that the ratio between the activities of the HCV IRES in the correct and inverted orientations is exactly the same in both kinds of vector (RGUHCV1/RGUHCV1rc=2.45). If shortening of the bicistronic mRNA by cryptic transcription, splicing or mRNA breakage within the intercistronic region occurred, then the measured IRES activity would be the same for both the bi- and tricistronic systems, which is not the case. On the other hand, the results show clearly that some kind of ribosome reinitiation at the last cistron – the second or the third – exists and that the HCV IRES-mediated translation is at least partly dependent on ribosomes supplied from the first cistron of the bi(tri)cistronic mRNA.
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). In all cases, we obtained amplification products of the expected size, thus there is no evidence for cryptic splicing or for any kind of mRNA shortening (Fig. 4b). Also, the amplification curves of the L and G fragments, which correspond to the first and the second cistron, respectively, fit each other for both IRES-containing and empty vectors. This clearly points to no cryptic promoter activity residing in the HCV IRES segment. The quantification of I fragment, corresponding to the intercistronic regions, was affected by the differing sizes of the amplicons (Fig. 4c).
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; Table 1). Disruption of loop IV and removal of the polyprotein coding sequence (pFGAL4h-HCV11–15AA) or alteration of the authentic HCV and inherent GAL4 initiation codons to the UUG codon (pFGAL4A/UUGh-HCV1A/UUG) eliminates HCV IRES activity almost completely. About three times higher, but still very low, IRES activities are exhibited by clones either bearing a frameshift mutation between the HCV IRES and GAL4 initiation codons (pFGAL4h-HCV3) or bearing the HCV IRES authentic AUG initiation codon changed to UUG (pFGAL4h-HCV1A/UUG). Also, the change of the inherent AUG codon of the GAL4 reporter gene to the UUG codon alone (pFGAL4A/UUGh-HCV1) led to a substantial decrease of IRES activity, whilst a slight change of AUG position within loop IV produced only a moderate decrease in the measured IRES activity (pFGAL4h-HCV1ACCAUG/AUGACC). The presence of the stable hairpin loop preceding the HCV IRES in the pFGAL4h-HCV1 vector series had almost no influence on IRES activity [compare pFGAL4h-HCV1 and pFGAL4-HCV1 columns in Fig. 5(a)].
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). We obtained mutually comparable results that are, in the case of the mammalian system, in good agreement with data presented elsewhere (Hwang et al., 1998). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Overall identity between the mammalian and yeast 40S ribosomal proteins is as high as 60 % (Wool et al., 1996), yet the human HCV IRES-binding ribosomal proteins share on average an even higher degree of identity (65 %) and similarity (79 %) with their yeast counterparts (Csank et al., 2002). Less striking is the homology between the yeast Nip1 and TIF34, the subunits of eIF3 implicated in HCV IRES binding, and their mammalian counterparts (Csank et al., 2002; Hershey & Merrick, 2000) (see also Supplementary Table S2, available in JGV Online). The potency of the yeast system can also be implicated from the recent results of Sarnow's group, who detected HCV IRES-mediated translation in a reconstituted system containing no translation initiation factors, but HeLa ribosomes, yeast elongation factors 1A and 2 and bulk aminoacylated tRNAs (Lancaster et al., 2006).
We present herein a novel bicistronic system that enables use of the yeast model to study HCV IRES function and facilitates the use of various detection and selection methods (Fig. 1a). This system is very sensitive, has a high dynamic range and, due to intrinsic features of the natural transcription regulatory network of the GAL gene family, reproducibly allows transcriptional amplification of the target signal and estimated quantification of probably just a few molecules of the primary reporter – Gal4p (Fig. 1b). The pFGAL4 vector can be employed as general tool for the search for new IRESs, as well as for the precise characterization of any particular IRES, including the search for IRES trans-acting proteins and compounds.
The first successful attempt to test functionality of the HCV IRES in yeast has been published recently (Rosenfeld & Racaniello, 2005). However, the authors had serious difficulty detecting IRES activity and had to extend the IRES sequence with 360 nt of the HCV polyprotein coding region, thus almost doubling the length of the HCV IRES. Aside from the increased risk of measuring artefacts in the yeast system, the IRES extended by another 120 codons of the viral polyprotein almost reaches the length that has been reported to suppress translation in mammalian cells (Wang et al., 2000). The extended length of the HCV IRES, along with probable low sensitivity and reproducibility of the strain/vector system used, could be one of the reasons for the observed high experiment-to-experiment variations of measured Miller units (Rosenfeld & Racaniello, 2005). In comparison to that system, the HCV IRES sequence used herein is extended by only 15 codons of the HCV polyprotein, which have been shown to be important for loop IV stability and for efficient HCV expression in mammals (Hwang et al., 1998; Jackson, 2000; Reynolds et al., 1995). Moreover, our system takes advantage of sensitive normalization to intracellular bicistronic transcript level by assaying activity of the firefly luciferase encoded by the first cistron of the bicistronic Fluc-IRES-GAL4 mRNA.
HCV IRES activity measured by the pFGAL4-based yeast system has been compared with the activity of various sets of controls, including several DNA fragments of similar length and inverted and mutated versions of the HCV IRES. HCV IRES activity was 26 times higher than the almost basal activity of phage DNA sequences spanning a window of similar length (Fig. 2). Initiation and termination codons appear very frequently in the randomized or naturally occurring non-coding nucleotide sequences. Indeed, the full-length HCV 5' UTR contains five AUG codons and numerous stop codons in all three reading frames. This is also the case with the intercistronic regions derived from phage DNA. These data, together with the knowledge that yeast cap-dependent translation initiation starts almost exclusively at the first initiation codon and is sensitive to extensive RNA secondary structures such as the 13G : C hairpin or the HCV IRES itself, led us to the conclusion that yeast cells are able to support cap-independent translation initiation at the HCV IRES.
To dissect HCV IRES-dependent translation in yeast in more detail and to prove that the HCV IRES performs similarly in yeast and mammals, we mutated the authentic 342AUG to UUG, which reduced the HCV IRES activity to 22 % of that of the wild-type control (pFGAL4h-HCV1) in yeast (Fig. 5a). This result is comparable with our analysis of essentially the same mutation in Chang CCL-13 liver cells (Fig. 5b) and shows an even higher reduction of IRES activity than has been already described by Hwang et al. (1998) for the same mutation in Huh-7 hepatocytes. This result, together with the effect of the frameshift mutation in the HCV3 clone, further demonstrates the ability of the HCV IRES to mediate translation initiation in yeast cells and points to the importance of the HCV authentic 342AUG.
We also show that the disruption of stem–loop IV by deletion of the first 15 codons of the HCV polyprotein almost eliminates HCV IRES activity (Fig. 5a). However, we cannot distinguish in this case whether the elimination of HCV IRES activity is due to the disruption of stem–loop IV or whether there is some more general requirement for HCV polyprotein sequence. It is presumed that the permissiveness of reporter sequences to the HCV IRES-driven translation lies in the A-rich, unstructured character of the 5'-proximal region (Jackson, 2000). We performed an alignment of coding sequences for the first 15 aa of those reporters that were used most commonly to study HCV IRES function in mammalian systems and compared them with the HCV polyprotein sequence and with the sequences of those reporters used in yeast (Fig. 6). The yeast reporters share a higher degree of sequence similarity with the reporters that do not support translation mediated by the HCV IRES without the N-terminal fusion with HCV polyprotein sequence [summarized in detail by Fletcher et al. (2002)]. This analysis also explains why Rosenfeld & Racaniello (2005), using LacZ as a second reporter, failed to detect any activity of the HCV IRES extended by only five codons.
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; Rijnbrand et al., 1996).
In another set of control experiments, we focused on detection of Fluc-IRES-GAL4 mRNA integrity. Under certain circumstances, the yeast S. cerevisiae can utilize very short and simple sequences to initiate transcription from both regular and cryptic promoters (Hecht et al., 2002; Hellen & Sarnow, 2001; Robinson & Lopes, 2000). As a control, we compared the translational activities of the wild-type and inverted HCV IRESs in the bi- and tricistronic vectors (Fig. 3). We demonstrate here that inserting another short ORF in front of the HCV IRES or its inverted form shifted the activities proportionally down. This result shows that translation driven by the HCV IRES is not influenced significantly by cryptic transcription from the intercistronic region, because in the case of a cryptic promoter occurrence in this region, the measured IRES activities would not change by insertion of any other ORF upstream from the IRES, as has been done in tricistronic vectors. These functional in vivo assays are in good agreement with the result of quantification of bicistronic mRNA in yeast cells.
The observed similarities of HCV IRES-mediated translation between the mammalian and yeast systems promise future prospects for the use of yeast genetics for functional studies of the HCV IRES.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 11 December 2006;
accepted 23 February 2007.
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