Detection of tRNA-like Structure through RNase P Cleavage of Viral Internal Ribosome Entry Site RNAs Near the AUG Start Triplet
2003; Elsevier BV; Volume: 278; Issue: 29 Linguagem: Inglês
10.1074/jbc.m304052200
ISSN1083-351X
AutoresAlita J. Lyons, Hugh D. Robertson,
Tópico(s)Animal Disease Management and Epidemiology
ResumoThe 9600-base RNA genome of hepatitis C virus (HCV) has an internal ribosome entry site (IRES) in its first 370 bases, including the AUG start triplet at bases 342–344. Structural elements of this and other IRES domains substitute for a 5′ terminal cap structure in protein synthesis. Recent work (Nadal, A., Martell, M., Lytle, J. R., Lyons, A. J., Robertson, H. D., Cabot, B., Esteban, J. I., Esteban, R., Guardia, J., and Gomez, J. (2002) J. Biol. Chem. 277, 30606–30613) has demonstrated that the host pre-tRNA processing enzyme, RNase P, can cleave the HCV RNA genome at a site in the IRES near the AUG initiator triplet. Although this step is unlikely to be part of the HCV life cycle, such a reaction could indicate the presence of a tRNA-like structure in this IRES. Because susceptibility to cleavage by mammalian RNase P is a strong indicator of tRNA-like structure, we have conducted the studies reported here to test whether such tRNA mimicry is unique to HCV or is a general property of IRES structure. We have assayed IRES domains of several viral RNA genomes: two pestiviruses related to HCV, classical swine fever virus and bovine viral diarrhea virus; and two unrelated viruses, encephalomyocarditis virus and cricket paralysis virus. We have found similarly placed RNase P cleavage sites in these IRESs. Thus a tRNA-like domain could be a general structural feature of IRESs, the first IRES structure to be identified with a functional correlate. Such tRNA-like features could be recognized by pre-existing ribosomal tRNA-binding sites as part of the IRES initiation cycle. The 9600-base RNA genome of hepatitis C virus (HCV) has an internal ribosome entry site (IRES) in its first 370 bases, including the AUG start triplet at bases 342–344. Structural elements of this and other IRES domains substitute for a 5′ terminal cap structure in protein synthesis. Recent work (Nadal, A., Martell, M., Lytle, J. R., Lyons, A. J., Robertson, H. D., Cabot, B., Esteban, J. I., Esteban, R., Guardia, J., and Gomez, J. (2002) J. Biol. Chem. 277, 30606–30613) has demonstrated that the host pre-tRNA processing enzyme, RNase P, can cleave the HCV RNA genome at a site in the IRES near the AUG initiator triplet. Although this step is unlikely to be part of the HCV life cycle, such a reaction could indicate the presence of a tRNA-like structure in this IRES. Because susceptibility to cleavage by mammalian RNase P is a strong indicator of tRNA-like structure, we have conducted the studies reported here to test whether such tRNA mimicry is unique to HCV or is a general property of IRES structure. We have assayed IRES domains of several viral RNA genomes: two pestiviruses related to HCV, classical swine fever virus and bovine viral diarrhea virus; and two unrelated viruses, encephalomyocarditis virus and cricket paralysis virus. We have found similarly placed RNase P cleavage sites in these IRESs. Thus a tRNA-like domain could be a general structural feature of IRESs, the first IRES structure to be identified with a functional correlate. Such tRNA-like features could be recognized by pre-existing ribosomal tRNA-binding sites as part of the IRES initiation cycle. The hepatitis C virus (HCV) 1The 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. is a flavivirus with a single-stranded RNA genome almost 9600 bases in length. HCV produces a chronic infection that can lead to cirrhosis and liver cancer and is the leading cause of liver transplants in the United States and elsewhere (1Houghton M. Fields B.N. Knipe D.M. Howley P.M. Fields' Virology. Lippincott-Raven, Philadelphia1996: 1035-1057Google Scholar). The HCV RNA genome lacks a 5′ cap structure, the signal used by most eukaryotic messages to initiate protein synthesis (1Houghton M. Fields B.N. Knipe D.M. Howley P.M. Fields' Virology. Lippincott-Raven, Philadelphia1996: 1035-1057Google Scholar). 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 (2Jackson R.J. Sonnenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 127-183Google Scholar, 3Jackson R.J. Wickens M. Curr. Opin. Genet. Dev. 1997; 7: 233-241Crossref PubMed Scopus (69) Google Scholar), and even in rare cases in human cellular RNAs (4Carter M.S. Kuhn K.M. Sarnow P. Sonnenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 615-635Google Scholar, 5Bernstein, J., Shefler, I., and Elroy-Stein, O. (l995) J. Biol. Chem., 270, 10559–10565Google Scholar). The mechanism by which IRESs function is unknown. Studies in our laboratory and elsewhere (6Lytle J.R. Wu L. Robertson H.D. J. Virol. 2001; 75: 7629-7636Crossref PubMed Scopus (41) Google Scholar, 7Wang, C., Le, S. Y., and Siddiqui, A. (1995) RNA, 1, 526–537Google Scholar, 8Pestova T.V. Shatsky I.N. Fletcher S.P. Jackson R.J. Hellen C.U.T. Genes Dev. 1998; 12: 67-83Crossref PubMed Scopus (634) Google Scholar, 9Spahn C.M.T. Kieft J.S. Grassucci R.A. Penczek P.A. Zhou K. Doudna J.A. Frank J. Science. 2001; 291: 1959-1962Crossref PubMed Scopus (444) Google Scholar, 10Lytle J.R. Wu L. Robertson H.D. RNA. 2002; 8: 1045-1055Crossref PubMed Scopus (65) Google Scholar) 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 (6Lytle J.R. Wu L. Robertson H.D. J. Virol. 2001; 75: 7629-7636Crossref PubMed Scopus (41) Google Scholar). Jackson (2Jackson R.J. Sonnenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 127-183Google Scholar) 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. (6Lytle J.R. Wu L. Robertson H.D. J. Virol. 2001; 75: 7629-7636Crossref PubMed Scopus (41) Google Scholar) 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 (2Jackson R.J. Sonnenberg N. Hershey J. Mathews M.B. Translational Control of Gene Expression. Cold Spring Harbor Press, Cold Spring Harbor, NY2000: 127-183Google Scholar, 7Wang, C., Le, S. Y., and Siddiqui, A. (1995) RNA, 1, 526–537Google Scholar), our finding of a UV-sensitive tertiary structural element in stem-loop II (11Lyons A.J. Lytle J.R. Gomez J. Robertson H.D. Nucleic Acids Res. 2001; 29: 2535-2541Crossref PubMed Scopus (38) Google Scholar), and the existence of tertiary structure in stem-loop IIId (12Jubin R. Vantuno N.E. Kieft J.S. Murray M.G. Doudna J.A. Johnson Y.N.L. Baroudy B.M. J. Virol. 2000; 74: 10430-10437Crossref PubMed Scopus (103) Google Scholar, 13Klinck R. Westhof E. Walker S. Afshar M. Collier A. Aboul-Ela F. RNA. 2000; 6: 1423-1431Crossref PubMed Scopus (100) Google Scholar). 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 (14Nadal A. Martell M. Lytle J.R. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. Buti M. Esteban R. Guardia J. Viral Hepatitis. Accion Medicia, S.A., Madrid2000: 93-99Google Scholar, 15Nadal A. Martell M. Lytle J.R. Lyons A.J. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. J. Biol. Chem. 2002; 277: 30606-30613Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Although Nadal et al. (15Nadal A. Martell M. Lytle J.R. Lyons A.J. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. J. Biol. Chem. 2002; 277: 30606-30613Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) 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 (16Robertson H.D. Altman S. Smith J.D. J. Biol. Chem. 1972; 247: 5243-5251Abstract Full Text PDF PubMed Google Scholar, 17Altman S. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 1-36PubMed Google Scholar, 18Jarrous N. Altman S. Methods Enzymol. 2001; 342: 93-100Crossref PubMed Scopus (52) Google Scholar, 19Altman S. Bio/Technology. 1995; 13: 327-329Crossref PubMed Scopus (28) Google Scholar). It is well known that RNase P works via structural, not sequence-dependent, recognition of tRNA domains (16Robertson H.D. Altman S. Smith J.D. J. Biol. Chem. 1972; 247: 5243-5251Abstract Full Text PDF PubMed Google Scholar, 17Altman S. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 1-36PubMed Google Scholar). In fact, the presence of an RNase P-cleavable site is one accepted test for the presence of tRNA-like structure (16Robertson H.D. Altman S. Smith J.D. J. Biol. Chem. 1972; 247: 5243-5251Abstract Full Text PDF PubMed Google Scholar, 20Komine Y. Kitabatake M. Yokogawa T. Nishikawa K. Proc. Natl. Acad. Sci. 1994; 91: 9223-9277Crossref PubMed Scopus (366) Google Scholar, 21Joshi S. Chapeville F. Haenni A.L. Nucleic Acids Res. 1982; 10: 1947-1962Crossref PubMed Scopus (35) Google Scholar) and has been used, along with other tests, to identify tRNA mimicry in various RNA species (20Komine Y. Kitabatake M. Yokogawa T. Nishikawa K. Proc. Natl. Acad. Sci. 1994; 91: 9223-9277Crossref PubMed Scopus (366) Google Scholar, 21Joshi S. Chapeville F. Haenni A.L. Nucleic Acids Res. 1982; 10: 1947-1962Crossref PubMed Scopus (35) Google Scholar, 22Branch A.D. Robertson H.D. Greer C. Gegenheimer P. Peebles C. Abelson J. Science. 1982; 217: 1147-1149Crossref PubMed Scopus (58) Google Scholar). 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 (6Lytle J.R. Wu L. Robertson H.D. J. Virol. 2001; 75: 7629-7636Crossref PubMed Scopus (41) Google Scholar, 20Komine Y. Kitabatake M. Yokogawa T. Nishikawa K. Proc. Natl. Acad. Sci. 1994; 91: 9223-9277Crossref PubMed Scopus (366) Google Scholar, 21Joshi S. Chapeville F. Haenni A.L. Nucleic Acids Res. 1982; 10: 1947-1962Crossref PubMed Scopus (35) Google Scholar). 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. Preparation of RNA Transcripts—HCV IRES RNAs were transcribed from Bluescript plasmid pN(1–4728), which contains nucleotides 1–4728 of hepatitis C virus under the T7 promoter (a gift from Dr. Stanley Lemon, University of Texas, Galveston). This DNA template was cleaved by the SacII restriction enzyme. When transcribed in vitro, a 32P-labeled RNA spanning bases 1–641 is produced. RNA transcripts of CSFV are from a pGEMII plasmid with an insert containing the CSFV IRES fused to influenza protein NS1, (a gift from Dr. R. J. Jackson, Cambridge University). This plasmid, when linearized with the NcoI restriction enzyme, serves as template for a 32P-labeled RNA containing bases 1–450 of the CSFV genome. BVDV RNA was obtained from transcription of pACNR plasmid BVDV NADL 5′Δ 2702, containing the BVDV RNA sequence with a deletion of nucleotides 3094–12147 (a gift from Dr. C. Rice, Rockefeller University, New York). Linearization with SacI yields a DNA that serves as template for a transcript spanning bases 1–695 of the BVDV genome upon transcription. EMCV IRES RNAs were transcribed from a pGEM plasmid with the EMCV IRES sequence fused to that encoding the N-terminal viral polyprotein of the virus. Restriction with Bst4C1 yields the template for RNA spanning bases 262–834 of the EMCV IRES. This plasmid was a generous gift of Dr. A. Kaminski, University of Cambridge. RNA transcripts of cricket paralysis virus (CrPV) were made from the pEJ4 construct, a kind gift from Dr. E. Jan, Stanford University. This plasmid, when linearized with the NcoI restriction enzyme, encodes an RNA containing bases 1–220 of the CrPV IGR IRES. All transcription reactions were based on earlier published work from this laboratory (6Lytle J.R. Wu L. Robertson H.D. J. Virol. 2001; 75: 7629-7636Crossref PubMed Scopus (41) Google Scholar, 10Lytle J.R. Wu L. Robertson H.D. RNA. 2002; 8: 1045-1055Crossref PubMed Scopus (65) Google Scholar, 11Lyons A.J. Lytle J.R. Gomez J. Robertson H.D. Nucleic Acids Res. 2001; 29: 2535-2541Crossref PubMed Scopus (38) Google Scholar). [α-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 (11Lyons A.J. Lytle J.R. Gomez J. Robertson H.D. Nucleic Acids Res. 2001; 29: 2535-2541Crossref PubMed Scopus (38) Google Scholar, 15Nadal A. Martell M. Lytle J.R. Lyons A.J. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. J. Biol. Chem. 2002; 277: 30606-30613Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 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 Cleavage—All 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 (18Jarrous N. Altman S. Methods Enzymol. 2001; 342: 93-100Crossref PubMed Scopus (52) Google Scholar). At the end of the reactions, samples were subjected to treatment with SDS and proteinase K at 50 °C as described previously (22Branch A.D. Robertson H.D. Greer C. Gegenheimer P. Peebles C. Abelson J. Science. 1982; 217: 1147-1149Crossref PubMed Scopus (58) Google Scholar), and cleavage products were then separated on 4% denaturing polyacrylamide gels and visualized by autoradiography as described (15Nadal A. Martell M. Lytle J.R. Lyons A.J. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. J. Biol. Chem. 2002; 277: 30606-30613Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 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 Fingerprinting—RNA 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 (23Branch A.D. Benenfeld B.J. Robertson H.D. Methods Enzymol. 1989; 180: 130-154Crossref PubMed Scopus (28) Google Scholar, 24Barrell B.G. Cantoni G.L. Davies D.R. Procedures in Nucleic Acid Research. Harper and Row, New York1971: 751-779Google Scholar). Determination of Cleavage Sites—To 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 (24Barrell B.G. Cantoni G.L. Davies D.R. Procedures in Nucleic Acid Research. Harper and Row, New York1971: 751-779Google Scholar). 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 (23Branch A.D. Benenfeld B.J. Robertson H.D. Methods Enzymol. 1989; 180: 130-154Crossref PubMed Scopus (28) Google Scholar)) 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. RNase P Cleavage of Pestiviral Transcripts—The 5′ terminal region of the genomic RNA for each of the HCV-type viral RNAs was transcribed as described under "Experimental Procedures," purified, and digested with RNase P. These reactions yield the results shown in Fig. 1, a 5% sequencing gel electrophoretic profile of HCV (HC), CSFV (CS), and BVDV (BV) IRES substrates without (–) or with (+) treatment by highly purified human RNase P. An early time point in the RNase P cleavage kinetics for HCV was chosen for all substrates to highlight the primary cleavage event. As shown in Fig. 1, the three HCV-type IRES RNAs are cleaved, CSFV more efficiently than HCV and BVDV less so. Principal cleavage products are indicated by numerals to the right of the lanes showing RNase P-treated RNAs. Fig. 2 depicts kinetics of RNase P cleavage of the HCV IRES transcript, showing >50% cleavage over a 90-min time course.Fig. 2Kinetics of HCV IRES cleavage by RNase P. The HCV SacII IRES transcript shown in Fig. 1, left panel, was incubated with RNase P for 120 min with aliquots withdrawn and analyzed at time points from 0 to 120 min. Left panel, autoradiograph of a 4% polyacrylamide gel showing HCV IRES starting material and principal RNase P cleavage products (1 and 2) at the times indicated. "O" indicates the origin of electrophoresis; "XC" indicates the position of the xylene cyanol dye marker. Right panel, kinetic plot of the cleavage reaction shown in the left panel, with the extent of cleavage at each time point determined by PhosphorImager analysis as described under "Experimental Procedures."View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mapping the RNase P Cleavage Site in the CSFV IRES—In 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 (23Branch A.D. Benenfeld B.J. Robertson H.D. Methods Enzymol. 1989; 180: 130-154Crossref PubMed Scopus (28) Google Scholar). 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 361–362 (data not shown) as found previously (15Nadal A. Martell M. Lytle J.R. Lyons A.J. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. J. Biol. Chem. 2002; 277: 30606-30613Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). 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 B–D show fingerprints of the three RNase P digestion products, CSFV bands 1, 2, and 3, respectively (Fig. 1). Spots numbered 1–7 (Fig. 3) are RNase T1-resistant fragments, characterized by direct methods (23Branch A.D. Benenfeld B.J. Robertson H.D. Methods Enzymol. 1989; 180: 130-154Crossref PubMed Scopus (28) Google Scholar, 24Barrell B.G. Cantoni G.L. Davies D.R. Procedures in Nucleic Acid Research. Harper and Row, New York1971: 751-779Google Scholar), 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.Table IRNase T1-resistant oligonucleotides in the 3′-proximal CSFV IRES domainSpotSequence and position1392AAUUAUUAUACAAAACAAG4102411CAAACAAAAACCAG4243382AAUCAUUUUG3914370CACAUG3755437AACCG4416444UACCAUGoh4507379UUG381X364pACAUG368aFig. 3C. or 371pACAUG375bFig. 3D.a Fig. 3C.b Fig. 3D. Open table in a new tab 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 (16Robertson H.D. Altman S. Smith J.D. J. Biol. Chem. 1972; 247: 5243-5251Abstract Full Text PDF PubMed Google Scholar, 17Altman S. Adv. Enzymol. Relat. Areas Mol. Biol. 1989; 62: 1-36PubMed Google Scholar, 18Jarrous N. Altman S. Methods Enzymol. 2001; 342: 93-100Crossref PubMed Scopus (52) Google Scholar); 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). With respect to band 3 (Fig. 3D), it was found to contain the same T1-resistant fragments as band 2 (Fig. 3C), with the notable exception of spot 4, which is missing from band 3. As shown in Fig. 4, we conclude that CSFV band 3 is the product of a second RNase P cleavage, which occurs near the first one, this time between bases 370C and 371A (see Fig. 4, circled letter P on right). This second cleavage would also produce a T1-resistant oligonucleotide with the sequence pACAUG (spot X in Table I). However, in band 3 (Fig. 3D) we conclude that the sequence identity of the spot labeled X is 371pACAUG375. Thus it appears that RNase P makes two cuts, the second seven bases away from the first, at sites near the CSFV IRES initiator AUG triplet that have a very close sequence homology to each other (G(U/C)ACAUGG in each case). This series of events also explains why the fingerprint of CSFV band 1 (Fig. 3B) contains reduced levels of the oligonucleotide 363UACAUG368; the band is actually an unresolved mixture of two sequences, CSFV bases 1–363 and 1–370. RNase P Cleavage of the BVDV IRES—RNA 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 373–394 (which includes the AUG initiation codon at 386–388) does not contain any RNase T1-resistant fragments of 7 bases or longer, the T1-resistant fragment spanning bases 395–404 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 Positions— Fig. 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 361–362 (15Nadal A. Martell M. Lytle J.R. Lyons A.J. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. J. Biol. Chem. 2002; 277: 30606-30613Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The CSFV RNase P cleavage sites map at positions 363–364 and 370–371 whereas BVDV RNase P cleavage occurs between bases 373–394, 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. Exploratory RNase P Treatment of Other Viral IRES RNAs—To 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). In this paper we have demonstrated that there are similarly placed recognition elements for the human pre-tRNA processing enzyme, RNase P, in several viral IRES domains including HCV, CSFV, BVDV, CrPV, and EMCV. In the cases of HCV, CSFV, and BVDV, the RNase P-sensitive elements map in close proximity to the AUG initiator triplet. As first pointed out by Nadal et al. (15Nadal A. Martell M. Lytle J.R. Lyons A.J. Robertson H.D. Cabot B. Esteban J.I. Esteban R. Guardia J. Gomez J. J. Biol. Chem. 2002; 277: 30606-30613Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) concerning human RNase P cleavage of the HCV IRES, it is likely that these events reflect neither the replication cycle of these viruses nor the canonical action of RNase P. Rather, because of the presence of pseudo-knot elements and other structural motifs in IRES RNAs like t
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