Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Cantoblanco 28049 Madrid, Spain1
Author for correspondence: Encarnación Martínez-Salas. Fax +34 91 3974799. e-mail emartinez{at}cbm.uam.es
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Abstract |
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Introduction |
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Experimental evidence in support of tertiary structure generated by RNARNA interactions is available for several IRES (Wang et al., 1995 ; Kanamori & Nakashima, 2001
). A pseudoknot structure in the HCV IRES was shown to be required for IRES activity, as mutations that destabilized tertiary interactions between residues of loop IIIf with complementary residues in domain IV were accompanied by a strong reduction in translation initiation (Honda et al., 1996
). Recently, interactions between distant residues of domain II have been claimed as another tertiary structure element of the HCV IRES (Lyons et al., 2001
).
Long-range RNARNA interactions have been shown to occur in vitro between functional domains of the foot-and-mouth disease virus (FMDV) IRES (Ramos & Martínez-Salas, 1999 ). These interactions are strand-specific and depend on RNA concentration, ionic conditions and temperature. The RNARNA interactions observed in vitro between separated domains of the FMDV IRES, in the absence of proteins, suggest that the IRES adopts a specific folding pattern depending upon environmental conditions. Notably, the central domain of the FMDV IRES (named domain I or 3) seems to play a key role in directing the folding of the molecule. Domain 3 is the only domain that interacts efficiently with all the other domains, suggesting that it holds the other domains of the IRES. Consistent with this essential role, mutations in conserved motifs in the distal loop of this domain impaired the activity of the element (López de Quinto & Martínez-Salas, 1997
; Robertson et al., 1999
). Furthermore, it is of note that the binding site for several eukaryotic initiation factors (eIFs) and other RNA-binding proteins are located outside of the central region, in the distal domains 2 and 45 (López de Quinto et al., 2001
).
The HCV IRES has been shown to adopt different folding patterns in response to different ionic concentrations (Kieft et al., 1999 ). Additionally, mutations in loop IIId cause a structural reorganization of the HCV IRES, as measured by RNase T1 sensitivity and Fe(II)EDTA cleavage, concomitantly with the reduction in IRES activity (Jubin et al., 2000
). In the absence of eIFs or the 40S ribosomal subunit, the HCV IRES seems to adopt a unique structure, which is stable at physiological salt concentrations. A structural element containing stemloops IIIa, b and c facilitates binding of eIF3 (Kieft et al., 2001
). On the other hand, subdomains IIId, e and f, together with the pseudoknot structural element, constitute the binding site for the 40S ribosomal subunit (Spahn et al., 2001
). Recently, it has been shown that discontinuous fragments in domains III and IV in the RNA isolated from 80S RNA complexes are protected from RNase A cleavage (Lytle et al., 2001
). Remarkably, sequences in domain II are required for IRES activity but the specific role of these sequences is still unknown.
Here we show that domain II of the HCV IRES can establish specific, long-range interactions with sequences located in domain IV, towards the 3' end of the IRES. In addition, stemloop IIId contributes to the interaction observed between domains II and IIIabcd, while the apical part of domain III seems to fold independently of the other domains. Consistent with this, interaction between domains IIIabcd and IV is barely detected by means of this assay. On the other hand, stemloop IIIef induces the formation of a compact structure with domain IV, which leads to a significant reduction in the ability of domain IV to interact with other IRES regions.
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Methods |
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The sequence of the entire length of the IRES under study was obtained using automatic sequencing (ABI PRIM Dye Terminator Cycle Sequencing Ready Reaction kit; Perkin Elmer). Additional nucleotides derived from the vector cloning sites do not seem to modify the predicted structure of the subcloned domains. No complementarity between the sense transcripts was observed in the additional residues.
In vitro transcription.
Sense RNAs were transcribed in vitro from 0·51 µg of the linearized plasmids using 50 U T7 RNA polymerase (New England Biolabs), 50 mM DTT, 0·5 mM rNTPs, 20 U RNasin (Promega) or using the MEGAshortscript kit (Ambion). Plasmids were linearized with HindIII, with the exception of the IIIabc transcript, which was obtained after NheI digestion. When needed, RNA transcripts were labelled to a specific activity of 3 µCi/µg using [32P]CTP (400 Ci/mmol). Reactions were incubated for 10 min with 1 U RQ1 DNase (Promega) and unincorporated [32P]CTP was eliminated by exclusion chromatography in TE (10 mM Tris and 1 mM EDTA, pH 8)-equilibrated Sephadex G 5080 (Sigma) columns. RNA was extracted with phenolchloroform, ethanol-precipitated and resuspended to an appropriate concentration in RNase-free water. The transcript of 136 nt, including a fragment of 80 nt from human 18S rRNA, was generated by T7 RNA polymerase-mediated transcription from pTRI-18S DNA template (Ambion).
RNARNA interaction assay.
RNA molecules were mixed in a volume of 4 µl, heated at 95 °C for 3 min before adding 1 µl of a fivefold-concentrated binding buffer to reach a final concentration of 50 mM sodium cacodylate, pH 7·5, 300 mM KCl and 10 mM MgCl2 (Ramos & Martínez-Salas, 1999 ; Ferrandon et al., 1997
). Independent RNA denaturation procedures did not modify the pattern of complex formation relative to mixed denaturation (R. Ramos & E. Martínez-Salas, unpublished data). Poly(I:C) (Pharmacia), yeast tRNA (Swartzmann) or a transcript including 80 nt from human 18S rRNA (transcribed from pTRI-18S) were used as nonspecific competitor molecules (800 nM each) in binding reactions, which also contained the specific interactor (800 nM) and the probe of interest (20 nM).
RNARNA complexes were allowed to form for 30 min at 37 °C and analysed immediately by electrophoresis in nondenaturing gels (Ramos & Martínez-Salas, 1999 ; Ferrandon et al., 1997
; Paillart et al., 1996
; Fedor & Uhlenbeck, 1990
). Gels were run at room temperature for 20 min at 23 V/cm in TBM buffer (45 mM Tris, pH 8·3, 43 mM boric acid and 0·1 mM MgCl2). RNA Century molecular mass markers (Ambion) (0·5 µg) were loaded in parallel. Prior to drying the gel, RNA was stained with ethidium bromide and photographed to record the mobility of each transcript under study. Dried gels were exposed for autoradiography, as well as to a phosphorImager plate, to quantify the intensity of the retarded bands. Data were represented as the percentage of the RNA complex of interest relative to the input probe, averaged from at least three independent experiments.
In competition- and oligonucleotide-binding assays, the molar ratio of primer to transcript ranged from 0·25 to 1. When required, oligonucleotides were labelled at the 5' end using [-32P]ATP (3000 Ci/mmol) and polynucleotide kinase (New England Biolabs) during 1 h at 37 °C. Unincorporated [
-32P]ATP was eliminated by exclusion chromatography in TE-equilibrated columns.
In vitro IRES activity.
HpaI-linearized plasmids were transcribed in vitro with T7 RNA polymerase to produce bicistronic transcripts of the form CATIRESluciferase. In vitro translations in nuclease-treated reticulocyte lysates (Promega) were programmed with p156-derived bicistronic transcript harbouring the HCV-1b IRES. The transcript derived from pBIC, which contains the FMDV IRES (López de Quinto & Martínez-Salas, 1999 ), was included for comparison. In both cases, 1 µg RNA, heated at 70 °C for 5 min, was translated for 1 h at 30 °C in 25 µl of 50% reticulocyte lysate in the presence of 25 µCi [35S]methionine (PRO-MIX L-35S; Amersham). Translation products were treated with 50 µg/ml RNase A, mixed with disruption buffer and resolved by 15% SDSPAGE.
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Results |
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Then, the RNA region corresponding to the HCV IRES was divided in five transcripts, named II, IIIabcd, IIIabc, IIIef-IV and IV (Fig. 1B). In order to preserve the structure of each domain, each transcript was designed to contain stable stemloop structures, according to mutational analysis carried out previously (Honda et al., 1996
, 1999
). The transcript corresponding to the 5' end of the IRES encompasses nt 43119 (stemloop II). The central region was cloned in two forms, one containing nt 134290 (stemloop IIIabcd) and a second one, IIIabc, devoid of stemloop IIId. The 3' region of the IRES was also designed in two forms. The first one, IIIef-IV, encompasses nt 290383, including stemloop IIIef and domain IV, fused to residues 125134. This transcript was designed to mimic the RNA structure that allows the formation of the pseudoknot shown by Wang et al. (1995)
. The second transcript contained domain IV alone (nt 125130 fused to nt 318383).
Following denaturation, pairs of RNA consisting of one 32P-labelled and one unlabelled transcript were incubated in binding buffer. Analysis of complex formation was carried out in native acrylamide gels, as described previously (Ramos & Martínez-Salas, 1999 ). Using domain II as probe, stable RNARNA complexes were detected with the different HCV transcripts tested, whose mobility changed according to the pair of RNA used in the assay (Fig. 2A
). A weak dimerization of probe II increased in intensity when the concentration of transcript II was 800 nM (Fig. 2A
, compare lanes 1 and 2), was always observed.
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The specificity of the interactions of the different transcripts with probe II was assessed by the lack of formation of retarded complexes with a transcript that included 80 nt of the 18S rRNA (pTRI-18S) or poly(I:C). Addition of these RNA molecules (800 nM) simultaneously with the probe and the specific interactor to the incubation mixture did not interfere with the formation of shifted bands observed in their absence (Fig. 3). In some experiments, the complex retarded with the full-length IRES led to the formation of a doublet rather than a single complex (Fig. 2A
, compare the lanes labelled IRES with those in Fig. 3
).
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Competition studies between different HCV domains were carried out to determine whether formation of retarded complexes could be diminished by the presence of the other HCV IRES domains. Using domain IV as probe, increasing amounts of transcript IIIef-IV competed out efficiently the interaction between transcripts IIIabcd and IV (Fig. 4B). These results were fully consistent with those shown in Table 2
, indicating not only that the interaction between transcripts IIIabcd and IV was weak but also that it was rapidly displaced by the interaction between transcripts IIIef-IV and IV.
Mixtures of transcripts II, IIIabcd and IV, representing most of the IRES domains, did not give raise to trimeric complexes (Fig. 4C) even under conditions of high concentrations of transcripts II and IIIabcd. This result suggested that formation of each heterodimer complex, IIIV and IIIabcdIV, occurred independently of the third component in the mixture. Moreover, increasing concentrations of domain II reduced only slightly the intensity of the IIIabcdIV complex, indicating that domain II was not an efficient competitor of the interaction between domains IV and IIIabcd. It has to be noticed that domain IV formed a homodimer that comigrated with complex IIIV.
The HCV IRES transcripts were able to self-dimerize to a lower extent than the FMDV IRES transcripts (Ramos & Martínez-Salas, 1999 ). The exception was domain IV, which reached values of about 30% (Table 2
), although still below the self-dimerization capacity observed for the FMDV IRES domain 3 under similar conditions of RNA concentration and ionic strength. Remarkably, dimerization of domain IV was efficiently competed out by the presence of transcript IIIef-IV (Fig. 4B
).
Hairpin IIId can interact with domain II
As shown in Table 2, a significant decrease in the interactions between the two versions of domain III, IIIabcd and IIIabc, and domain II was observed in the transcript devoid of stemloop IIId. This result suggested that stemloop IIId could contribute to the interactions mediated by domain IIIabcd with domain II. To test this possibility, a IIId sense RNA oligonucleotide (sIIId) was used to determine whether this short structure was able to interact with any of the HCV transcripts. Remarkably, using equimolar amounts of RNA to oligonucleotide, the 5' end-labelled sIIId interacted with domain II (Fig. 5
). Two discrete bands appeared to be labelled after incubation of this RNA oligonucleotide with domain II, which may correspond to the monomer and the dimer of this transcript bound to the sIIId oligonucleotide, according to ethidium bromide-staining data.
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In addition, interaction of the sIIId oligonucleotide with transcript IIIabc was observed (Fig. 5), suggesting the involvement of this hairpin in the interactions detected between the apical and basal part of domain III. The specificity of this contact was confirmed by the lack of binding with transcripts IIIef-IV and IV. Furthermore, labelled sIIId barely interacted with transcript IIIabcd (Fig. 5
). This result could be interpreted as a direct involvement of IIId residues in RNA interactions between IIIabc and IIId or as a modified structure of IIIabcd transcript relative to IIIabc, which precluded IIId from binding.
Then, an antisense version of the IIId stemloop sequence (asIIId; Table 1) was used to study the contribution of stemloop IIId to the interactions between the apical and basal part of domain III. As expected, the asIIId oligoribonucleotide interacted very efficiently with transcript IIIabcd but not with transcript IIIabc (Fig. 6A
). Additionally, asIIId interacted with transcript IIIef-IV, albeit to a lower extent.
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These results were consistent with the weak interaction between domains IIIabcd and IV, indicating that the apical part of domain III folds independently of stemloops IIIef and IV. In agreement with this result, a mixture of three transcripts, domains II, IIIabcd and IV, did not yield trimeric complexes (Fig. 4C). Taking the results of binding and competition assays together, we concluded that the apical part of domain III folds independently of stemloops IIIef and IV. Notably, stemloop IIId was responsible for the interaction observed between domains II and IIIabcd but it did not seem to interact with domain IV or IIIef-IV.
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Discussion |
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The interactions shown here between domains II and IV in the absence of proteins provide support for the essential role played by domain II in HCV IRES activity. It is possible that these interactions are transient, being displaced by RNA-binding proteins or the small ribosomal subunit. However, domain IIIabc barely interacted with the rest of the IRES, indicating that it folds independently of domains II, IIIef-IV and IV, which is in agreement with data recently reported (Spahn et al., 2001 ; Beales et al., 2001
).
On the other hand, the 40S ribosomal subunit produces an RNase T1 footprint in a guanine residue of the apical loop of domain II as well as three guanine residues of stemloop IIId (Kieft et al., 2001 ). Thus, some proximity or association between these RNA regions is likely. Consistent with this, the reduction observed when stemloop IIId was removed from transcript IIIabcd suggested that stemloop IIId was able to interact with domain II. We tested this hypothesis by incubating a labelled RNA oligonucleotide carrying the IIId sense sequence with domain II. Remarkably, efficient binding was observed with sequences of domain II but not with domain IV or IIIef-IV, which is in agreement with the possibility mentioned above. These results were also consistent with the lack of competition shown by the antisense oligonucleotide asIIId. Binding of asIIId to its target sequence did not interfere with the interaction between transcripts IIIabcd and IIIef-IV. The IIId antisense oligonucleotide, which binds very efficiently to domain IIIabcd, remained bound to its target sequence when the latter was forming a complex with transcript IIIef-IV, which is in agreement with the conclusion that IIIabcd folds independently of stemloop IIIef.
Cross-interaction was observed between domains II and IV, including the stemloop that contains the initiator codon and the unstructured region at the beginning of the coding sequence. However, in some instances, the efficiency, but not the pattern, of the interactions observed display a lack of symmetry. This asymmetry is only observed with interactions exerted by probes IIIabc and IIIabcd, where the interactions are weaker. The reason for this asymmetry in the intensity of the interactions is not known but it can be attributed to the difference in concentration of probe and interactor RNA used in the assay.
The absence of stemloop IIIef from transcript IV resulted in a significant increase in the interaction of transcript IV with the other RNAs. Therefore, stemloop IIIef induced the formation of a compact RNA structure with domain IV, leading to a strong reduction in interactions with residues from outside the stemloop. Although the exact residues involved in the interaction between domains IIIef and IV are not known yet, formation of a pseudoknot structure (Wang et al., 1995 ) may contribute to the significant reduction in binding between domain IV and the rest of the IRES.
Compared to FMDV IRES domain 3, the HCV transcripts were able to weakly self-interact. Of note is that domain IIIabcd is unable to self-dimerize, in spite of having a very long stemloop at its base. Under our experimental conditions, the best self-interactor is domain IV, which could reach values of about 30%, close to those obtained with the whole HCV IRES transcript but far below the strong self-dimerization shown by domain 3 of FMDV (Ramos & Martínez-Salas, 1999 ). It is not known yet whether self-dimerization has something to do with IRES trans-complementation. However, it is interesting to note that there are published data reporting the inability of HCV IRES mutants to complement in trans, as opposed to FMDV IRES (Tang et al., 1999
).
We have shown here cross-interactions between domains II and IV, corresponding to the 5' and 3' distal regions of the HCV IRES. This is also in contrast to the FMDV IRES, where the central domain was the one interacting strongly with all the others, at least in the absence of proteins (Ramos & Martínez-Salas, 1999 ). Therefore, a very different structural organization adopted by FMDV and HCV IRES allows efficient internal initiation of translation.
The IRES of FMDV and HCV adopt different structural organizations that reflect a different manner to promote internal initiation. Accordingly, a different pattern of RNAprotein interaction is observed for each of these IRES elements (López de Quinto et al., 2001 ; Kieft et al., 2001
; Pestova et al., 1998
; López de Quinto & Martínez-Salas, 2000
; Buratti et al., 1998
; Sizova et al., 1998
; Kolupaeva et al., 2000
; Pilipenko et al., 2000
). Results of toe-print analysis indicated that 48S initiation complex formation driven by the HCV and CSFV IRES required eIF2-GTP/Met-tRNAi, eIF3 and 40S subunits (Pestova et al., 1998
). As a consequence of the use of different mechanisms to initiate translation, HCV IRES does not require eIF4G to assemble a 48S initiation complex, whereas FMDV does (reviewed by Martínez-Salas et al., 2001
). This observation poses the question of how the RNA present in the HCV IRES recognizes the translational machinery. A direct contact between the HCV IRES and the 40S ribosomal subunit has been demonstrated recently (Spahn et al., 2001
). In agreement with the IRES structural model derived from the latter study, the apical part of domain III, which binds eIF3 (Kieft et al., 2001
; Buratti et al., 1998
; Sizova et al., 1998
), folds independently of the rest of the IRES.
In summary, the results shown here indicate that the apical part of domain III forms a structural element separate from domains II and IV. While domains II and IV can interact, at least in the absence of proteins, stemloop IIIef induces the formation of a compact structure with domain IV, which strongly reduces its binding to other regions of the IRES. Furthermore, we have also shown that the interaction observed between domains IIIabcd and II is facilitated by stemloop IIId. It has been shown that IIId sequences form part of the structural motif involved in the recognition of the ribosomal subunit (Jubin et al., 2000 ; Kieft et al., 2001
; Spahn et al., 2001
; Lytle et al., 2001
). Thus, it is likely that the interactions we have observed between domains II and IIId may play a regulatory role in the activity of this IRES element, modulating its capacity to interact with the ribosome.
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Acknowledgments |
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Received 25 October 2001;
accepted 18 January 2002.