From the Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021
Received for publication, April 17, 2003
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ABSTRACT |
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INTRODUCTION |
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The HCV RNA genome lacks a 5' cap structure, the signal used by most eukaryotic messages to initiate protein synthesis (1). HCV is nevertheless able to bind cellular ribosomes in the host and to initiate accurate protein translation due to the presence in the first 400 bases of the viral genome of an internal ribosome entry site (IRES). IRESs are found in certain other viral pathogens such as the picornavirus and pestivirus groups (2, 3), and even in rare cases in human cellular RNAs (4, 5). The mechanism by which IRESs function is unknown.
Studies in our laboratory and elsewhere (610) on HCV IRES ribosome binding sites have revealed that considerably more sequence is involved in IRES-directed protein synthesis initiation than in its cap-directed counterpart, up to 5-fold more in some cases (6). Jackson (2) has suggested, in light of limited sequence homology among IRES elements, that widely spaced structural elements may be the key to ribosome recognition thereof. For these reasons, Lytle et al. (6) considered several elements of higher order structure that could be responsible for IRES function. So far, however, no structural element with a known affinity for ribosomes has been shown to be a common feature within the ribosome recognition domains of more than one IRES element.
Probes of HCV IRES RNA higher order structure include the prediction of an RNA pseudo-knot involving sequences in stem-loop III (2, 7), our finding of a UV-sensitive tertiary structural element in stem-loop II (11), and the existence of tertiary structure in stem-loop IIId (12, 13). Surprisingly, one group of studies has also shown that human RNase P will recognize and cleave the HCV RNA genome in two places, one of which is in the IRES very close to the initiator AUG start triplet (14, 15). Although Nadal et al. (15) do not argue that this cleavage by a host RNA processing enzyme takes place as part of the HCV life cycle, they showed conservation of RNase P recognition by a number of HCV variants.
RNase P is an abundant cellular endoribonuclease that is responsible for processing tRNA precursors to produce mature 5' termini (1619). It is well known that RNase P works via structural, not sequence-dependent, recognition of tRNA domains (16, 17). In fact, the presence of an RNase P-cleavable site is one accepted test for the presence of tRNA-like structure (16, 20, 21) and has been used, along with other tests, to identify tRNA mimicry in various RNA species (2022).
Because the susceptibility to cleavage by RNase P is a strong indicator of tRNA-like structure, this property of the HCV IRES has caused us to suggest the existence of tRNA-like structure in or near stem-loop III (6, 20, 21). We have conducted the studies reported here to test whether such tRNA mimicry is unique to HCV or whether it might be a general property of IRES structure. In particular, we have compared the RNase P sensitivity of the HCV IRES with that of the IRES domains in two pestiviral RNA genomes related in structure and function to HCV, the classical swine fever virus (CSFV) and the bovine viral diarrhea virus (BVDV). Our findings show that tRNA-like structure occurs near the AUG start triplet in these three IRES domains. To generalize these findings further, we have also tested for RNase P cleavage in the IRESs of encephalomyocarditis virus, a picornavirus, and in the cricket paralysis virus, an insect virus. Both undergo RNase P cleavage at specific sites. These results strongly suggest that tRNA-like domains may be a general structural feature of IRESs, the first such IRES structure to be identified with a functional correlate. In fact, tRNA-like features could easily be recognized by ribosomal sites as a standard part of all IRES-directed protein synthesis initiation.
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EXPERIMENTAL PROCEDURES |
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All transcription reactions were based on earlier published work from this
laboratory (6,
10,
11).
[-32P]GTP (PerkinElmer Life Sciences) was used in all
labeled transcriptions unless noted otherwise. Transcripts were purified by
gel electrophoresis under denaturing conditions on 5% polyacrylamide gels with
7 M urea as described previously
(11,
15). Bands were visualized by
autoradiography, excised from the gel, and eluted in buffer (10 mM
Tris-HCl, pH 7.5, and 1 mM EDTA, pH 7.5).
RNase P CleavageAll substrates for RNase P cleavage, derived from transcription reactions described above, were preheated at 90 °C for 1 min, added to reaction buffer (10 mM HEPES-KOH, pH 7.5, 10 mM MgOAc, 100 mM NH4OAc), and left to cool to room temperature. Cleavage reactions were performed with 4% polyethylene glycol, 20 units of RNasin and RNase P and carried out at 30 °C in a volume of 10 µl for 1 h. The final concentration of the substrates in these reactions was 1.8 nM. Human RNase P (glycerol gradient fraction, the kind gift of Dr. S. Altman) was used with an excess of enzyme units over substrate (18). At the end of the reactions, samples were subjected to treatment with SDS and proteinase K at 50 °C as described previously (22), and cleavage products were then separated on 4% denaturing polyacrylamide gels and visualized by autoradiography as described (15). For kinetic studies, samples were treated as above and incubated in scaled-up 30-µl reaction volumes prepared as above. Aliquots of 5 µl were withdrawn at 0, 30, 60, 90, and 120 min, subjected to SDS-proteinase K treatment, and analyzed on polyacrylamide gels as described above. Starting material and product bands were quantified as follows using an Amersham Biosciences Storm PhosphorImager: percent RNase P cleavage = (products)/(starting material + products).
RNA FingerprintingRNA fingerprinting, a technique in which RNA molecules are digested to completion with RNase T1 (Calbiochem) and the digestion products are separated in two dimensions by charge and size, has been described previously (23, 24).
Determination of Cleavage SitesTo carry out precise characterization and mapping, it is necessary to use direct sequencing techniques involving internally labeled RNA molecules when the normal internal sequence pattern of an RNA is interrupted. Examples of such events include mapping of RNA splice junctions and lariats; UV-induced covalent RNA-RNA cross-links; RNA editing to produce noncanonical bases, such as inosine; tRNA modification of pre-existing bases; and cleavage by enzymes (such as the RNase P used here) that produce 5' phosphate and 3'-hydroxyl termini rather than the usual 5'-hydroxyl and 3'-phosphate ends. Thus, direct methods were employed here rather than indirect approaches such as DNA primer extension with reverse transcriptase or partial digestion of end-labeled transcripts.
Oligonucleotides derived from RNase T1 digestions were fractionated to yield two-dimensional fingerprints, from which individual oligonucleotides were located by autoradiography and eluted by standard techniques (24). Standard conditions for secondary analysis of the T1 digestion products (with pancreatic RNase A, RNase U2, RNase T2 or 0.4 M NaOH, followed by high voltage electrophoresis on Whatman DE81 or 3MM papers (23)) were used, permitting the oligonucleotides to be identified. Digestion by calf alkaline phosphatase (Promega) was used to identify oligonucleotides containing 5' terminal phosphate residues produced by RNase P cleavage.
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RESULTS |
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Mapping the RNase P Cleavage Site in the CSFV IRESIn the case of CSFV, one larger and two smaller product bands are recovered (Fig. 1) following RNase P cleavage. Enumerated bands were eluted for further analysis. To map the exact location of the HCV and CSFV cleavages, transcripts were digested with RNase P under conditions outlined above and then fractionated on polyacrylamide denaturing gels. Both control transcripts and all digestion products labeled in Fig. 1 were then eluted from the gels, digested with RNase T1, and subjected to two-dimensional RNA fingerprinting analysis (23). Direct characterization of the internally labeled RNA fragments produced by RNase P cleavage in the HCV IRES (Fig. 1) showed that cleavage occurs just beyond the AUG initiator, centering on bases 361362 (data not shown) as found previously (15).
Fig. 3 shows RNA fingerprinting analysis of CSFV IRES RNase P cleavage products. RNAs eluted from the gel depicted in Fig. 1, center panel, were subjected to exhaustive RNase T1 digestion and two-dimensional RNA fingerprinting. Panel A shows a control fingerprint of the entire 450-base CSFV IRES transcript, whereas Panels BD show fingerprints of the three RNase P digestion products, CSFV bands 1, 2, and 3, respectively (Fig. 1). Spots numbered 17 (Fig. 3) are RNase T1-resistant fragments, characterized by direct methods (23, 24), that are present in the control sequence, absent in band 1, and present in bands 2 and 3 (Table I). It is noteworthy that spot 4, 370CACAUG375, contains the CSFV IRES AUG initiator triplet.
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CSFV band 1 (Fig. 3B) contains all T1-resistant RNA oligonucleotides from the control CSFV transcript sequence up to base 359, including the 5' end of CSFV (see legend to Fig. 3). This suggests that cleavage takes place downstream of that position in the IRES sequence. Bands 2 and 3 (Fig. 3, C and D) were found to contain all T1-resistant fragments between bases 370 and 450. Notably, the unique T1-resistant oligonucleotide 363UACAUG368 was missing or significantly reduced in the fingerprint patterns of all of the CSFV RNase P digestion products. Furthermore, bands 2 and 3 (Fig. 3, C and D) were found to contain a novel oligonucleotide, designated spot "X." Upon further secondary analyses, including alkaline phosphatase treatment, spot X was found to have the sequence 5'-pACAUG-3'. RNase P cleavage is well known to yield 5'-phosphate and 3'-hydroxyl termini (1618); thus it is likely that spot X represents the new 5'-ends created by RNase P.
With respect to band 2, the presence of spot 4 along with spot X suggests that cleavage must have occurred within the "missing" oligonucleotide, 363UACAUG368, precisely between bases 363U and 364A, yielding the T1-resistant oligonucleotide X (364pACAUG368). This cleavage event is shown schematically in Fig. 4 (circled "P" on left).
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RNase P Cleavage of the BVDV IRESRNA fingerprinting analysis was carried out on control and RNase P-treated BVDV IRES-containing RNAs transcribed from SacI-treated DNA templates (Fig. 1). Comparison of the resulting patterns and knowledge of the identity of the control T1-resistant fragments (data not shown) allowed us to conclude that all of the T1-resistant oligonucleotides included in this assay (7 bases or longer) from the 5'-end of the BVDV IRES up to base 372 (a total of 8 fragments) were present in BVDV product 1 (Fig. 1, right lane, BV). BVDV product 2 (Fig. 1) was not recovered in sufficient quantities to be included in this T1 mapping survey. Nonetheless, although the BVDV IRES region spanning bases 373394 (which includes the AUG initiation codon at 386388) does not contain any RNase T1-resistant fragments of 7 bases or longer, the T1-resistant fragment spanning bases 395404 was absent from the BVDV product 1 fingerprint pattern, as were all other such T1-resistant oligonucleotides through the 3'-end of the BVDV SacI transcript at base 695 (a total of 11 fragments). We therefore conclude that the primary RNase P cleavage site within the BVDV IRES maps between bases 373 and 394. Because of the similarity of the BVDV and CSFV IRES sequences in this region (see below), we predict that the BVDV IRES RNase P cleavage sites will closely resemble those in CSFV.
IRES RNase P Cleavage Map PositionsFig. 5 summarizes RNase P cleavage locations for all three viral IRES-containing transcripts as identified by RNA fingerprinting and secondary analysis or oligonucleotide survey techniques. The HCV IRES RNase P cleavage site, shown as a circled letter P in Fig. 5, maps at bases 361362 (15). The CSFV RNase P cleavage sites map at positions 363364 and 370371 whereas BVDV RNase P cleavage occurs between bases 373394, part of an extensive region of identity between the CSFV and BVDV IRESs (see below). The possible occurrence of a dual cleavage event in the BVDV IRES is indicated. It is clear that, in all three of these viral IRES RNAs, RNase P cleavages map very closely to the initiator AUG, suggesting the presence of a tRNA-like structural element nearby.
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Exploratory RNase P Treatment of Other Viral IRES RNAsTo test whether tRNA-like regions recognizable by RNase P could be a general feature of IRES RNAs, we tested IRES transcripts from two unrelated viruses. As shown in Fig. 6, RNase P both recognizes and cleaves RNA transcripts of the CrPV IRES. It is evident from the widely differing sizes of the two RNase P cleavage products in Fig. 6, lane 3, that cleavage occurs near one end of this IRES sequence. Furthermore, in similar tests, specific RNase P cleavage of transcripts from the EMCV IRES, similar to that for CrPV shown in Fig. 6, was also obtained (data not shown).
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DISCUSSION |
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IRES Sites for RNase P RecognitionA number of earlier studies have shown that RNase P can often exhibit noncanonical behavior. For example, both bacterial and eukaryotic RNase P enzymes cleave substrates other than pre-tRNAs (1719). Furthermore, RNase P can cleave the same pre-tRNA simultaneously at two sites near each other (25, 26). Conditions inducing this behavior include substrate mutations as well as ionic or pH variation. The IRES cleavage reactions studied here show signs of noncanonical substrate behavior both by variable cleavage efficiencies (Fig. 1) and, in at least one case, by the presence of two RNase P cleavage sites close together (the CSFV IRES, Fig. 4).
In practice, RNase P is known to rely principally on the recognition of tRNA secondary and tertiary structure for its cleavage specificity rather than on particular sequence elements at the point of cleavage (1719). Fig. 7 shows the schematic diagrams of the HCV, CSFV, and BVDV IRES RNAs represented in their predicted secondary structural folding patterns (11). The RNase P cleavage sites are indicated in context, with the exact bases cleaved in the BVDV IRES still to be mapped. The major IRES structural domains, stem-loops II and III, are also indicated. If there is tRNA-like structure in these IRES RNAs, it might be more highly conserved if it occurred in noncoding sequences upstream from the cleavage sites. In seeking tRNA-like structure in the vicinity of HCV, CSFV, and BVDV RNase P cleavage, the cloverleaf-like regions in stem-loop III, highlighted by a thick black outline in Fig. 7, are obvious candidates. Furthermore, these domains in CSFV and BVDV represent identical 27-base sequences (bases 309335 in CSFV and bases 324350 in BVDV; Ref. 27). A third pestivirus, border disease virus, has 26 of these 27 bases conserved as well (28). In addition, the primary sequence of the 5'-proximal portion of this region is highly conserved in a large number of HCV and pestiviral isolates, including the 14 bases of HCV spanning residues 292305 (Fig. 7, black highlighting in the HCV IRES; Refs. 27 and 29). Future studies correlating RNase P cleavage and protein synthesis in IRES RNAs mutagenized to affect translation in these and other domains will be needed to pinpoint the structural elements involved in RNase P recognition and their relationship to IRES function.
Although Fig. 7 also shows that the two pestiviral IRESs, CSFV and BVDV, contain regions of homology in the initiator AUG-containing region cleaved by RNase P, it is unlikely that these could specify RNase P cleavage (1719). As shown by a thick gray line in Fig. 7, the 35 bases spanning the start triplet and the RNase P sites are identical in these two IRESs (27). The CSFV version of this sequence, bases 348382, is depicted in Fig. 4 in italic letters. The equivalent domain of BVDV includes bases 361395. However, this sequence is unlikely to be a general IRES feature signaling RNase P cleavage because the HCV IRES, which has no significant homology to this sequence (27, 29), is cleaved in a fashion similar to CSFV and BVDV.
In the well studied cases of tRNA mimicry in other viral RNAs (including the 3' terminal (21) and internal (30) sites of action of tRNA-modifying enzymes), the tRNA elements can usually be recognized by more than one tRNA-specific system. In the case of RNase P cleavage of IRES elements presented here, studies on recognition by appropriate tRNA-binding initiation and elongation factors of protein synthesis, along with tRNA nucleotidyl transferase and appropriate tRNA-modifying activities, may shed additional light (15, 3133).
If recognition elements are located, as suggested above, in the upstream "cloverleaf" IRES domain in stem-loop III, RNase P would be cleaving to their right or 3' side. RNase P usually cleaves to the left, on the 5' or upstream side, of its recognition elements, forming the mature 5'-end of the tRNA involved (1619). With regard to the polarity of cleavage it is important to note that RNase P activities can cleave in the interior of multimeric tRNA precursor molecules (34) to the left and right of the pre-tRNA elements. In addition, in some cases, the tRNA elements of larger molecules that are RNase P substrates, such as bacterial tmRNA (20), are composed of sequences from both the 5'- and 3'-ends of molecules over 300 bases long. On the other hand, it remains possible that some specificity determinants for the IRES RNase P cleavage reactions reported here could be located downstream from the cleavage sites mapped, although recent studies appear to favor the idea that the region immediately downstream from the HCV IRES initiator AUG triplet must be relatively unstructured (35). Further studies focusing on selected elements in IRES stem-loops II and III should narrow the choice of potential RNase P specificity determinants.
Potential Function of tRNA-like IRES ElementsRegardless of which domains near the IRES AUG start triplets are found to specify the RNase P cleavages mapped here in three viral RNAs, we need to know why such elements are present and whether they have a function. If tRNA-like elements are required for the initiation of translation, for example, we can begin correlating the retention of RNase P cleavability with the IRES function in translation in various mutant IRESs. There is no question that recent studies on IRES domains protected from RNase digestion by various initiation complexes containing ribosomes or ribosomal subunits (6, 10) have shown that extensive additional domains beyond the immediate vicinity of AUG-containing sequences are involved. These results, along with recent studies on the 40 S ribosomal subunit·HCV IRES complexes by cryo-electron microscopy (9), have suggested the potential involvement of the ribosomal E-site.
The initiation pathway for these IRESs, as determined by various published studies, involves the initial formation of a binary complex with 40 S subunits through a 48 S complex with added factors to a full initiation complex with 80 S ribosomes. In our hypothesis, as initiation progresses, the cloverleaf structure shown in Fig. 7, HCV IRES stem-loop III bases 291323, or their pestiviral equivalents, will move near to the unoccupied ribosomal E-site as the nearby AUG-containing region occupies the P-site. Recognition of the tRNA-like domain of the IRESs by the E-sites of the ribosomes could help to specify accurate initiation by bringing the P-site and the AUG start triplet into close and stable proximity. The IRES domain would then be ejected from the E-site as normal initiation and translocation progressed.
Our preliminary studies also suggest that this scheme for recognition of tRNA-like elements in HCV and pestiviral IRESs could be extended to other unrelated IRES elements; and thus this step could be a general part of IRES-based translation initiation. In Fig. 6, we showed that the CrPV IGR IRES contains an RNase P-cleavable element. This IRES has recently been found to contain a highly structured RNA domain that maps, like similar elements in HCV-like viral IRESs, immediately upstream from the AUG initiator triplet (36). This CrPV element may bind to a ribosomal site as the first peptide bond forms between amino acids in the P- and A-sites. Furthermore, our preliminary results (not shown) indicate that the IRES structure of EMCV, a representative of picornaviral IRESs, which have a number of protein recognition elements within them (37), harbors a tRNA-like structure. Future work with RNase P and other probes for tRNA-like structures should tell how widespread these structures are in IRES and elucidate their role in IRES function.
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FOOTNOTES |
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To whom correspondence should be addressed: Dept. of Biochemistry, Weill
Medical College of Cornell University, Room E-012A, 1300 York Ave., New York,
NY 10021. Tel.: 212-746-6400; Fax: 212-746-8144; E-mail:
hdrober{at}mail.med.cornell.edu.
1 The abbreviations used are: HCV, hepatitis C virus; IRES, internal ribosome
entry site; CSFV, classical swine fever virus; BVDV, bovine viral diarrhea
virus; EMCV, encephalomyocarditis virus; CrPV, cricket paralysis virus; IGR,
intergenic region.
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ACKNOWLEDGMENTS |
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REFERENCES |
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