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• W2021983301 abstract "Saccharomyces cerevisiae strains carry single-stranded RNAs called 20 S RNA and 23 S RNA. These RNAs and their double-stranded counterparts, W and T dsRNAs, have been cloned and sequenced. A few nucleotides at both ends, however, remained unknown. These RNAs do not encode coat proteins but their own RNA-dependent RNA polymerases that share a high degree of conservation to each other. The polymerases are also similar to the replicases of RNA coliphages, such as Qβ. Here we have determined the nucleotide sequences of W and T dsRNAs at both ends using reverse transcriptase polymerase chain reaction-generated cDNA clones. We confirmed the terminal sequences by primer-extension and RNase protection experiments. Furthermore, these analyses demonstrated that W and T dsRNAs and their single-stranded RNA counterparts (i) are linear molecules, (ii) have identical nucleotide sequences at their ends, and (iii) have no poly(A) tails at their 3′ ends. Both 20 S and 23 S RNAs have GGGGC at the 5′ ends and the complementary 5-nucleotides sequence, GCCCC-OH, at their 3′ ends. S1 and V1 secondary structure-mapping of the 3′ ends of 20 S and 23 S RNAs shows the presence of a stem-loop structure that partially overlaps with the conserved 3′ end sequence. Nucleotide sequences and stem-loop structures similar to those described here have been found at the 3′ ends of RNA coliphages. These data, together with the similarity of the RNA-dependent RNA polymerases encoded among these RNAs and RNA coliphages, suggest that 20 S and 23 S RNAs are plus-strand single-stranded virus-like RNA replicons in yeast. Saccharomyces cerevisiae strains carry single-stranded RNAs called 20 S RNA and 23 S RNA. These RNAs and their double-stranded counterparts, W and T dsRNAs, have been cloned and sequenced. A few nucleotides at both ends, however, remained unknown. These RNAs do not encode coat proteins but their own RNA-dependent RNA polymerases that share a high degree of conservation to each other. The polymerases are also similar to the replicases of RNA coliphages, such as Qβ. Here we have determined the nucleotide sequences of W and T dsRNAs at both ends using reverse transcriptase polymerase chain reaction-generated cDNA clones. We confirmed the terminal sequences by primer-extension and RNase protection experiments. Furthermore, these analyses demonstrated that W and T dsRNAs and their single-stranded RNA counterparts (i) are linear molecules, (ii) have identical nucleotide sequences at their ends, and (iii) have no poly(A) tails at their 3′ ends. Both 20 S and 23 S RNAs have GGGGC at the 5′ ends and the complementary 5-nucleotides sequence, GCCCC-OH, at their 3′ ends. S1 and V1 secondary structure-mapping of the 3′ ends of 20 S and 23 S RNAs shows the presence of a stem-loop structure that partially overlaps with the conserved 3′ end sequence. Nucleotide sequences and stem-loop structures similar to those described here have been found at the 3′ ends of RNA coliphages. These data, together with the similarity of the RNA-dependent RNA polymerases encoded among these RNAs and RNA coliphages, suggest that 20 S and 23 S RNAs are plus-strand single-stranded virus-like RNA replicons in yeast. Many fungi carry viruses (mycoviruses), most often with double-stranded RNAs (dsRNAs) 1The abbreviations used are: dsRNAdouble-stranded RNARDRPRNA-dependent RNA polymerasentnucleotide(s)RACErapid amplification of cDNA endsPipespiperazine-N,N′-bis(2-ethanesulfonic acidPCRpolymerase chain reactionpCpcytidine 3′,5′-biphosphate. as genomes. Some of these viruses confer phenotypic changes in the host, but many others are maintained without any special properties associated. All of them are intracellular parasites with no extracellular stage. Transmission is mainly vertical or through mating or hyphal anastomosis. double-stranded RNA RNA-dependent RNA polymerase nucleotide(s) rapid amplification of cDNA ends piperazine-N,N′-bis(2-ethanesulfonic acid polymerase chain reaction cytidine 3′,5′-biphosphate. Yeast strains of Saccharomyces cerevisiae have been described to carry at least 5 types of double-stranded RNAs, L-A, L-BC, M, W, and T (1Wickner R.B. Microbiol. Rev. 1996; 60: 250-265Crossref PubMed Google Scholar). L-A, L-BC, and M are encapsidated into isometric viral particles. W and T are not encapsidated into viral coats (2Wesolowski M. Wickner R.B. Mol. Cell. Biol. 1984; 4: 181-187Crossref PubMed Scopus (53) Google Scholar). W (2.5 kilobases) and T (2.9 kilobases) have been cloned and sequenced almost entirely (3Rodrı́guez-Cousiño N. Esteban L.M. Esteban R. J. Biol. Chem. 1991; 266: 12772-12778Abstract Full Text PDF PubMed Google Scholar, 4Esteban L.M. Rodrı́guez-Cousiño N. Esteban R. J. Biol. Chem. 1992; 267: 10874-10881Abstract Full Text PDF PubMed Google Scholar). Both RNAs code for proteins with domains conserved among RNA-dependent RNA polymerases (RDRPs) of RNA viruses (5Kamer G. Argos P. Nucleic Acids Res. 1984; 12: 7269-7282Crossref PubMed Scopus (610) Google Scholar, 6Argos P. Nucleic Acids Res. 1988; 16: 9909-9916Crossref PubMed Scopus (297) Google Scholar, 7Poch O. Sauvaget I. Delarue M. Tordo N. EMBO J. 1989; 8: 3867-3874Crossref PubMed Scopus (983) Google Scholar, 8Koonin E.V. J. Gen. Virol. 1991; 72: 2197-2206Crossref PubMed Scopus (723) Google Scholar). The protein encoded by W (+) strands (p91) and the protein encoded by T (+) strands (p104) share a high degree of homology that extends beyond the RDRP consensus motifs, indicating a close evolutionary relationship between these RNAs (Fig.1). Comparison with other RDRPs suggests that these polymerases are more similar to the RNA coliphage replicases than to RDRPs from dsRNA viruses, including those present in the same host, namely L-A and L-BC viruses (3Rodrı́guez-Cousiño N. Esteban L.M. Esteban R. J. Biol. Chem. 1991; 266: 12772-12778Abstract Full Text PDF PubMed Google Scholar, 4Esteban L.M. Rodrı́guez-Cousiño N. Esteban R. J. Biol. Chem. 1992; 267: 10874-10881Abstract Full Text PDF PubMed Google Scholar, 9Esteban R. Rodrı́guez-Cousiño N. Esteban L.M. Prog. Nucleic Acid Res. Mol. Biol. 1993; 46: 155-182Crossref PubMed Scopus (19) Google Scholar, 10Koonin E.V. Semin. Virol. 1992; 3: 327-339Google Scholar, 11Bruenn J.A. Nucleic Acids Res. 1993; 21: 5667-5669Crossref PubMed Scopus (180) Google Scholar). All strains carrying W dsRNA also carry a single-stranded RNA called 20 S RNA, and all strains carrying T also have a single-stranded RNA called 23 S RNA. 20 S RNA and 23 S RNA have been proposed to be identical to the W and T (+) strands, respectively (3Rodrı́guez-Cousiño N. Esteban L.M. Esteban R. J. Biol. Chem. 1991; 266: 12772-12778Abstract Full Text PDF PubMed Google Scholar, 4Esteban L.M. Rodrı́guez-Cousiño N. Esteban R. J. Biol. Chem. 1992; 267: 10874-10881Abstract Full Text PDF PubMed Google Scholar, 12Matsumoto Y. Wickner R.B. J. Biol. Chem. 1991; 266: 12779-12783Abstract Full Text PDF PubMed Google Scholar). 20 S RNA and 23 S RNA copy number is highly induced under stress conditions such as growth under nitrogen starvation (4Esteban L.M. Rodrı́guez-Cousiño N. Esteban R. J. Biol. Chem. 1992; 267: 10874-10881Abstract Full Text PDF PubMed Google Scholar, 13Matsumoto Y. Fishel R. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7628-7632Crossref PubMed Scopus (65) Google Scholar), reaching up to 100,000 copies/cell. 20 S and 23 S RNAs are not encapsidated into viral particles (14Widner W.R. Matsumoto Y. Wickner R.B. Mol. Cell. Biol. 1991; 11: 2905-2908Crossref PubMed Scopus (22) Google Scholar, 15Esteban L.M. Fujimura T. Garcı́a-Cuéllar M.P. Esteban R. J. Biol. Chem. 1994; 269: 29771-29777Abstract Full Text PDF PubMed Google Scholar) but are associated with their own RNA polymerases, forming ribonucleoprotein complexes (15Esteban L.M. Fujimura T. Garcı́a-Cuéllar M.P. Esteban R. J. Biol. Chem. 1994; 269: 29771-29777Abstract Full Text PDF PubMed Google Scholar, 16Garcı́a-Cuéllar M.P. Esteban L.M. Fujimura T. Rodrı́guez-Cousiño N. Esteban R. J. Biol. Chem. 1995; 270: 20084-20089Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Recently we have shown that the p91/20 S RNA complexes have in vitro RNA polymerase activity that synthesizes 20 S RNA (17Garcı́a-Cuéllar M.P. Esteban R. Fujimura T. RNA. 1997; 3: 27-36PubMed Google Scholar). p104/23 S RNA complexes have similar activity. Since cis-acting signals at the ends of the RNA viral genomes often play critical roles in the template specificity of viral RNA polymerases (18Miller W.A. Bujarski J.J. Dreher T.W. Hall T.C. J. Mol. Biol. 1986; 187: 537-546Crossref PubMed Scopus (141) Google Scholar, 19Esteban R. Fujimura T. Wickner R.B. EMBO J. 1989; 8: 947-954Crossref PubMed Scopus (69) Google Scholar, 20Buck K.W. Adv. Virus Res. 1996; 47: 159-251Crossref PubMed Google Scholar, 21Patton J.T. Wentz M. Xiaobo J. Ramig R.F. J. Virol. 1996; 70: 3961-3971Crossref PubMed Google Scholar), we decided to determine the nucleotide sequences at the ends of W and T dsRNAs. Here we report the cloning and analysis of the nucleotide sequences at the 5′ and 3′ ends of W and T dsRNAs. Both (+) strands have conserved 5′ end GGGGC and 3′ end GCCCC-OH sequences. Primer extension analysis and RNase protection experiments confirmed that the single-stranded forms (20 S RNA and 23 S RNA) are identical to the (+) strands of the corresponding double-stranded forms (W and T) and that all these RNAs are linear molecules. S1 and V1 secondary structure mapping of the 3′ ends confirm that not only 20 S RNA and 23 S RNA share similar sequences at their ends but 3′ end secondary structures as well. These sequences and secondary structures are similar to those found at the 3′ ends of the genomic RNAs in (+) strand single-stranded RNA coliphages. Based on the available data we believe that 20 S and 23 S RNAs are similar to positive-stranded RNA viruses. Yeast strain used was strain 37-4C (a leu, kar1–1, 20 S RNA, 23 S RNA, W, T, L-A-0, L-BC-0) (2Wesolowski M. Wickner R.B. Mol. Cell. Biol. 1984; 4: 181-187Crossref PubMed Scopus (53) Google Scholar). W and T dsRNAs from strain 37-4C were purified by CF-11 cellulose chromatography as described previously (22Toh-e A. Guerry P. Wickner R.B. J. Bacteriol. 1978; 136: 1002-1007Crossref PubMed Google Scholar). Then, W and T dsRNAs were separated on an agarose gel, electroeluted from the gel, and further passed through Elutip columns (Schleicher & Schuell). 20 S RNA and 23 S RNA were purified from strain 37-4C grown under induction conditions as described (3Rodrı́guez-Cousiño N. Esteban L.M. Esteban R. J. Biol. Chem. 1991; 266: 12772-12778Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were grown for 48 h to stationary phase, washed, and incubated in the presence of 1% potassium acetate for 14–16 h to achieve induction of 20 S RNA and 23 S RNA (23Wejksnora P. Haber J.E. J. Bacteriol. 1978; 134: 246-260Crossref PubMed Google Scholar). Total nucleic acids were separated on 1.5% agarose gels, and 23 S RNA or 20 S RNA were electroeluted from the gel, extracted once with phenol:chloroform, and precipitated with ethanol. To isolate the + and − strands of W dsRNA, the dsRNA was first denatured in the presence of 7 m urea at 90 °C for 1 min and then loaded onto a 5% polyacrylamide strand separation gel (24Rodrı́guez-Cousiño N. Esteban R. Nucleic Acids Res. 1992; 20: 2761-2766Crossref PubMed Scopus (16) Google Scholar). Both strands were located by ethidium bromide staining, excised from the gel, and purified. To clone the 3′ ends of (+) and (−) strands of W and T dsRNAs, we used 3′ RACE (25Frohman M.A. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols. A Guide to Methods and Applications. Academic Press, Inc., New York1990: 28-38Google Scholar). The 3′ ends of W and T dsRNAs were first A-tailed using poly(A) polymerase (Life Technologies, Inc.) in a buffer that contained 50 mm Tris-HCl, pH 7.9, 10 mm MgCl2, 2.5 mm MnCl2, 125 mm NaCl, 0.25 mm ATP, 0.25 μg/μl bovine serum albumin. The poly(A)-tailed RNAs were then denatured with CH3HgOH (hydroxymethyl mercuric hydroxide) as described (26Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar), and cDNA was synthesized using oligonucleotide Bam-dT16 (5′-CCGGATCCTTTTTTTTTTTTTTTT-3′) and Superscript reverse transcriptase (Life Technologies, Inc.). PCR amplification of the cDNAs was carried out using oligo Bam-dT16 and one of four oligonucleotides that are complementary to the internal regions of W or T dsRNAs located at around 200 nt from the end of the known sequences. The four internal primers used are PG6 (2191–5′-CGAATCGTCGCCAGTAG-3′-2207) and NR2 (175–5′-GACGGCTCCAACCGTAG-3′-159) for the (+) and (−) strands of W dsRNA and NR23 (2716–5′-TTCATGGGCCTTCGCCCC-3′-2733) and NR22 (208- 5′-GAGTCCACGTCGTAACGC-3′-225) for the (+) and (−) strands of T dsRNA, respectively. Amplification was carried out using TaqDNA polymerase (Promega) for 30 cycles (denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, and extension at 70 °C for 1 min 30 s). The products were digested with BamHI and another restriction enzyme that cut in the known sequence of the amplified fragment and then ligated into pBluescript SK+ or KS+ (Stratagene) predigested with appropriate enzymes. pALI17 contained the complete cDNA sequence of 20 S RNA fused to the T7 RNA polymerase promoter and theSmaI site of pBluescript-KS+ vector. Run-off transcription of SmaI-digested pALI17 by T7 RNA polymerase, therefore, gave transcripts that have the entire sequence of 20 S RNA with the correct 5′ and 3′ ends. Plasmid pALI22 contained 20 S RNA cDNA sequences from nt 2288 to 2514 cloned between the HindIII and SmaI sites of pBluescript-SK+ vector. T7 Run-off transcription of SmaI-digested pALI22 gave the 273-nt RNA transcript WHindIII, which contains 227 nt from the 20 S RNA 3′ end with an upstream 46-nt vector sequence. Plasmid pNR27 contained the entire 23 S RNA cDNA nucleotide sequence (2891 base pairs) cloned into the unique SmaI site of pBluescript-SK+ vector. Plasmid pRE443 contained sequences of 23 S RNA cDNA from nt 2750 to 2891 cloned into the SmaI site of pBluescript SK+ vector. T7 run-off transcription of SmaI-digested pRE443 gave the 215-nt RNA transcript TSpeI, which contained 142 nt from 23 S RNA 3′ end and 73-nt upstream sequence derived from the vector. Plasmids pW3-2 and pT3-8 are reverse transcription-PCR-generated cDNA clones containing the last 227 nt of W or the last 143 nt of T (+) strand 3′ ends followed by a poly(A) tract. To confirm the ends of the clones, we performed primer extension analysis (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). We used oligonucleotides NR2 and NR22 as primers to analyze the 5′ ends of W (+) strands (and 20 S RNA) and T (+) strands (and 23 S RNA), respectively. For W (−) strands and T (−) strands, we used oligonucleotides NR1 (2433–5′-GGGCCGGATGGGCGACT-3′-2449) and LM5 (2812–5′-GGCCGCCCGCCACCTTCA-3′-2829), respectively. Primers were labeled at their 5′ ends with [γ-32P]ATP (Amersham Pharmacia Biotech) and T4 polynucleotide kinase (Boehringer Manheim) as described (26Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982Google Scholar). Phosphorylated primers were mixed with purified template RNAs and ethanol-precipitated. The pellets were dissolved in 30 μl of hybridization buffer containing 40 mm Pipes, pH 6.4, 1 mm EDTA, 0.4 m NaCl, 80% formamide and incubated for 10 min at 85 °C. After annealing at 40 °C overnight, nucleic acids were precipitated with ethanol. cDNA synthesis was carried out at 42 °C for 1 h with Superscript reverse transcriptase (Life Technologies, Inc.). The extended products were separated on a 7 m urea 6% acrylamide gel and detected by autoradiography. For RNase protection experiments, we used the RPAIITM ribonuclease protection assay kit from Ambion. 32P-Labeled RNA probes with sequences complementary to the 3′ ends of W and T (+) strands were generated by run-off transcription with T3 RNA polymerase fromPvuII-digested pW3-2 and EagI-digested pT3-8, respectively. The 32P-labeled probes were separated in a denaturing acrylamide gel and purified from the gel as mentioned above. The probe was then annealed with 20 S RNA or W (+) strands or with 23 S RNA or T (+) strands, depending on the probe used. The RNA hybrids were digested with a mixture of RNase A and RNase T1 under high salt, and the protected RNA fragments were separated on a 7 m urea 6% polyacryalamide gel and detected by autoradiography. W dsRNA or small RNA transcripts corresponding to the 3′ ends of W (+) strand (20 S RNA) or T (+) strand (23 S RNA) were 3′ end-labeled with [32P]pCp (3000 Ci/mmol, Amersham) and T4 RNA ligase (Life Technologies, Inc.) in a 30-μl reaction mixture. The conditions were as suggested by the enzyme supplier. The labeled RNA was denatured and separated in 5% polyacrylamide strand separation gels (24Rodrı́guez-Cousiño N. Esteban R. Nucleic Acids Res. 1992; 20: 2761-2766Crossref PubMed Scopus (16) Google Scholar). The 3′-end-labeled transcripts or W (+) strands were excised from the gel and extracted with 0.5 ml of 0.5 m ammonium acetate, 1 mmEDTA overnight at room temperature. The samples were then filtered through glass wool to remove polyacrylamide and precipitated with ethanol. The labeled RNA (5000–20000 cpm) was first preincubated in the reaction buffer for 10 min at 37 °C and then digested with Nuclease S1 (Life Technologies, Inc.) or RNase V1 (Amersham). Nuclease S1 digestion was performed in a reaction mixture (6 μl) containing 30 mm sodium acetate, pH 4.6, 1 mm zinc acetate, 5% glycerol, 80 mm NaCl, 0.5 μg of tRNA, and 0.2, 2, or 10 units of S1 nuclease (28Hannig E.M. Leibowitz M.J. Nucleic Acids Res. 1985; 13: 4379-4400Crossref PubMed Scopus (26) Google Scholar). RNase V1 digestion was done in a buffer (6 μl) that contained 25 mm Tris-HCl, pH 7.2, 10 mm MgCl2, 200 mm NaCl, 0.5 μg of tRNA, and 0.009 or 0.018 units of RNase V1. Nuclease treatments were done at 37 °C for 10 min, and the reactions were stopped by the addition of 1 μl of 100 mm EDTA and the same volume of loading buffer (10 m urea, 1.5 mm EDTA, 0.05% xylene cyanol, and 0.05% bromphenol blue). To generate a sequence ladder, alkaline hydrolysis was carried out at 90 °C for 7 min in a 6-μl reaction volume that contained 50 mm sodium bicarbonate/carbonate, pH 9.2, 3 μg of tRNA, and twice the amount of labeled RNA used for the enzymatic digestions. The cleaved products were analyzed on 7 m urea 20% or 10% polyacrylamide gels. Plasmid DNA was sequenced by the dideoxy chain termination method (29Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52865) Google Scholar) using T7 DNA polymerase (Amersham). In vitro run-off RNA transcription by T7 or T3 RNA polymerases (Promega) was carried out using plasmids linearized with appropriate restriction enzymes. Then the DNA template was digested with 1 μg of DNase I (Promega) for 15 min at 37 °C. RNA secondary structure prediction was done using the RNAFOLD program (30Zuker M. Stiegler P. Nucleic Acids Res. 1981; 9: 133-148Crossref PubMed Scopus (2643) Google Scholar). RNA coliphages nucleotide sequences were retrieved from the EMBL data bank. Previously we cloned and sequenced random primer-generated cDNAs from W and T dsRNAs. In these works we obtained 2505- and 2871-base pair nucleotide sequences for W and T dsRNAs, respectively. As judged from the mobilities in denaturing acrylamide gels of in vitro made transcripts with these sequences, we estimated that our cDNA sequences lacked only a few nucleotides at the ends of these RNAs. To understand the replication mechanism of these RNAs, however, it is essential to know the exact nucleotide sequences of these molecules, especially at both ends. To clone the ends of both RNAs, we used the method called 3′-RACE (rapid amplification of cDNAends) (25Frohman M.A. Innis M.A. Gelfand D.H. Sninsky J.J. White T.J. PCR Protocols. A Guide to Methods and Applications. Academic Press, Inc., New York1990: 28-38Google Scholar). W or T dsRNAs were first poly(A)-tailed at the 3′ ends using poly(A) polymerase. The poly(A)-tailed RNA was denatured and annealed with the deoxyoligonucleotide primer Bam-dT16, which could hybridize to the poly(A) tail with its 3′ end oligo dT sequence. The cDNA was then synthesized with reverse transcriptase. The cDNA was amplified by PCR using Bam-dT16 and a second primer. We used four sets of second primers. Each of them had a nucleotide sequence identical to an internal region close to the 3′ end of either strand of W or T dsRNA, thus providing the specificity in cDNA amplification. Finally the amplified cDNAs were cloned into Bluescript vectors. We sequenced 5–10 clones derived from each amplification. TableI summarizes the results of sequencing, which are shown as the (+) strand sequences. The majority of sequences derived from each amplified end had a cluster of four C residues accentuated by a downstream poly(A) tract. If we assume that the fourth C adjacent to the poly(A) tract represents the end of each dsRNA, the total numbers of the nucleotide sequences of W and T dsRNAs are 2514 and 2891 base pairs, respectively.Table I5′ and 3′ end sequences of W and T (+) strands5′ ends3′ endsW (+) strands/20 S RNAGGGGCUGAUCCCAUG...(7)...UGAGGCCACGGCCCC(5)T (+) strands/23 S RNAGGGGCCA UG...(6)...CCGGGCCUGAGCCCC(5) GGGCCA UG...(2)...CCGGGCCUGAGCC(1)GGCCA UG...(1)...CCGGGCCU(1)Newly acquired nucleotide sequences in the reverse transcription-PCR-generated clones are underlined. The number of independent clones in each case are indicated in parenthesis. Initiation and termination codons for p91 (20 S RNA) or p104 (23 S RNA) are in boldface. Open table in a new tab Newly acquired nucleotide sequences in the reverse transcription-PCR-generated clones are underlined. The number of independent clones in each case are indicated in parenthesis. Initiation and termination codons for p91 (20 S RNA) or p104 (23 S RNA) are in boldface. We added poly(A) tails at the 3′ ends of W and T dsRNAs during cloning. If W or T dsRNA had extra A residues downstream of the successive 4 C residues at the 3′ ends, these A residues could not be distinguished from the poly(A) tail attached by the poly(A) polymerase. This is the intrinsic problem associated with the 3′ RACE method. Therefore we needed to evaluate whether these successive 4 C residues were the true ends of these RNAs. For this purpose, two approaches were undertaken: primer extension analysis and RNase protection experiments. In the primer extension experiments, purified (+) or (−) strands of W dsRNA or denatured T dsRNA were mixed with a 5′ end-labeled oligo primer that was complementary to the 5′ end region of the RNA. Then the labeled primer was extended toward the 5′ end of the template by reverse transcription. As shown in Fig.2 A, lane 1, the primer complementary to the 5′ end region of W (+) strand was fully extended and terminated as a single band at the position corresponding to the last 3′ end C of the W (−) strand. This result clearly indicates that the 5′ end sequence of W (+) strand shown in Table I is correct and has no preceding extra Ts. When the T (+) strands were examined similarly by primer extension, we obtained again a single band of the extended primer terminating at the position corresponding to the last C of the clustered 4 C residues nested at the 3′ end of T (−) strands (Fig. 2 B, lane 1). This result again indicates that the 5′ end sequence of T (+) strands shown in Table I is correct and has no extra nucleotide sequences at the 5′ end. We also examined the (−) strands of W and T dsRNAs and obtained the same results; that is, the first nucleotides of the 5′ ends of W and T (−) strands are G and there are no extra Ts attached to them (not shown). It should be pointed out that although the cloning of each end of W and T dsRNA by 3′ RACE was manipulated at their 3′ ends, the primer extension experiments shown in Fig. 2 directly analyzed the 5′ end of each RNA strand. The fact that these two independent but complementary experiments gave consistent results strongly suggests that our cloned sequences are correct and represent the real W and T end sequences. Logically, however, the possibility still remains that there exists a nonbase-pairing poly(A) tail at the 3′ end of W or T (+) strands. To rule out this possibility, we undertook a second experimental approach; RNase protection experiments. We made uniformly labeled RNA in vitro that had the nucleotide sequence from base 2514 to 2288 (numbering refers to the (+) strand sequence) of the W (−) strand attached to the 5′ upstream poly(T) sequence (Fig.3 A). This probe therefore can hybridize to the 3′ end region of W (+) strand. If W (+) strands have poly(A) tails at their 3′ ends, a part of the poly(T) sequence of the probe complementary to the poly(A) tail should be protected from RNase digestion. As shown in Fig. 3 A, a part of the probe corresponding to W (−) strand from base 2514 to 2288 was fully protected, but the 5′ end poly(T) sequence was completely digested with the RNases (lanes 4 and 5). When the 3′ end of the T (+) strand was examined using a similar probe, a portion of the probe corresponding to the T (−) strand sequence from base 2891 to 2812 was fully protected, but again, the adjacent upstream poly(T) sequence was completely digested (Fig. 3B, lanes 4 and 5). These results, therefore, (i) confirm the correctness of our 3′ end nucleotide sequences of W and T (+) strands and (ii) clearly rule out the possibility that the W and T (+) strands have non-base paring poly(A) tails at their 3′ ends. Altogether, the results from primer extension analysis and RNase protection experiments indicate that the 3′ end sequences of W and T dsRNAs obtained by 3′ RACE are genuine, and that there are no non-base paring poly(A) tails at their 3′ ends. Thus we have now established the complete nucleotide sequences of W and T dsRNAs.Figure 5Nuclease S1 and V1 mapping of W (+) strand 3′ end. A, S1 mapping. 3′ end-labeled W (+) strands (lanes 8 to 10) or WHindIII transcript (lanes 3 to 5) were digested with various amounts of nuclease S1 (0.2, 2, or 10 units (U)) or mocked-treated (lane C) and separated on a 7 m urea 20% acrylamide gel. Cleaved products were detected by autoradiography. Arrows indicate the positions of the two most sensitive regions to S1 digestion, designated as loop I and loop II. The size of the cleaved products was estimated from an RNA ladder obtained by alkaline hydrolysis of the samples (lane -OH). The larger RNA fragments from the W (+) strands were resolved in a 10% acrylamide gel and shown on the upper right panel. Diagrams of the W (+) strand and the WHindIII transcripts are shown at the bottom of the autoradiograms. WHindIII transcripts contain the 3′ end fragment of W (+) strand from nt 2288 to 2514 and the upstream vector-derived sequence (open square). B, V1 mapping. The same samples as in A were digested with 0.009 or 0.018 units of RNase V1 (lanes 3 and 4 for the WHindIII transcript or lanes 9 and 10 for W (+) strands) or mocked-treated (lane C). The cleaved products were separated on a 7 m urea 20% acrylamide gel and detected by autoradiography. For comparison, we analyzed the same samples digested with 2 units of nuclease S1 in the same gel (lanes 6 and12). Arrows indicate positions highly sensitive to V1 digestion (numbered from the 3′ end). The positions of loop I and loop II from the S1 digestion are also indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Previously we proposed that 20 S and 23 S RNAs are identical to the (+) strands of W and T dsRNA, respectively, based on the following evidence. (i) All the strains carrying W and T dsRNA also harbor 20 S and 23 S RNA, respectively, and vice versa. (ii) Specific probes for the (+) strands of W (or T) dsRNA hybridize with 20 S (or 23S) RNA. (iii) 20 S (or 23 S) RNA was indistinguishable from the (+) strands of W (or T) dsRNA in denaturing and strand separation acrylamide gels. (iv) The known nucleotide sequence of 20 S RNA (2479 base pairs) (12Matsumoto Y. Wickner R.B. J. Biol. Chem. 1991; 266: 12779-12783Abstract Full Text PDF PubMed Google Scholar) is identical to the W (+) strand sequence from base 13 to base 2491. Since we have obtained the complete sequences of W and T dsRNAs, we asked whether 20 S and 23 S RNA have the same corresponding sequences at their ends. The 5′ end of 20 S RNA was examined by primer extension with the same oligonucleotide used for W (+) strands. As shown in Fig.2 A, lane 2, the primer was extended and terminated as a single band at the same position where the 5′ end of the W (+) strand terminates. This result indicates that 20 S RNA has the same primer binding site at the same distance from the 5′ end as the W (+) strand. When the 5′ end of 23 S RNA was analyzed similarly with the primer used for the T (+) strands, we obtained the same result; that is, the primer was terminated as a single band at the same position corresponding to the 5′ end terminus of the T (+) strand (Fig.2 B, lane 2). Therefore, the primer extension analysis indicates that the 5′ ends of 20 S RNA and 23 S RNA are indistinguishable from those of W and T (+) strands, respectively. The 3′ end regions of 20 S and 23 S RNAs were analyzed by RNase protection experiments with t" @default.
• W2021983301 created "2016-06-24" @default.
• W2021983301 creator A5021234627 @default.
• W2021983301 creator A5051003882 @default.
• W2021983301 creator A5068143450 @default.
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• W2021983301 date "1998-08-01" @default.
• W2021983301 modified "2023-10-02" @default.
• W2021983301 title "Yeast Positive-stranded Virus-like RNA Replicons" @default.
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