A 5'-3' long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition
2002; Springer Nature; Volume: 21; Issue: 16 Linguagem: Inglês
10.1093/emboj/cdf436
ISSN1460-2075
AutoresGaël Cristofari, Carole Bampi, M.L. Wilhelm, François‐Xavier Wilhelm, Jean‐Luc Darlix,
Tópico(s)Plant Virus Research Studies
ResumoArticle15 August 2002free access A 5′–3′ long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition Gaël Cristofari Gaël Cristofari LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, Cedex 07, France Search for more papers by this author Carole Bampi Carole Bampi LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, Cedex 07, France Search for more papers by this author Marcelle Wilhelm Marcelle Wilhelm Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France Search for more papers by this author François-Xavier Wilhelm François-Xavier Wilhelm Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France Search for more papers by this author Jean-Luc Darlix Corresponding Author Jean-Luc Darlix LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, Cedex 07, France Search for more papers by this author Gaël Cristofari Gaël Cristofari LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, Cedex 07, France Search for more papers by this author Carole Bampi Carole Bampi LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, Cedex 07, France Search for more papers by this author Marcelle Wilhelm Marcelle Wilhelm Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France Search for more papers by this author François-Xavier Wilhelm François-Xavier Wilhelm Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France Search for more papers by this author Jean-Luc Darlix Corresponding Author Jean-Luc Darlix LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, Cedex 07, France Search for more papers by this author Author Information Gaël Cristofari1, Carole Bampi1, Marcelle Wilhelm2, François-Xavier Wilhelm2 and Jean-Luc Darlix 1 1LaboRetro, INSERM U412, Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, 69364 Lyon, Cedex 07, France 2Institut de Biologie Moléculaire et Cellulaire, 15, rue R. Descartes, 67084 Strasbourg, France *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4368-4379https://doi.org/10.1093/emboj/cdf436 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info LTR-retrotransposons are abundant components of all eukaryotic genomes and appear to be key players in their evolution. They share with retroviruses a reverse transcription step during their replication cycle. To better understand the replication of retrotransposons as well as their similarities to and differences from retroviruses, we set up an in vitro model system to examine minus-strand cDNA synthesis of the yeast Ty1 LTR-retrotransposon. Results show that the 5′ and 3′ ends of Ty1 genomic RNA interact through 14 nucleotide 5′–3′ complementary sequences (CYC sequences). This 5′–3′ base pairing results in an efficient initiation of reverse transcription in vitro. Transposition of a marked Ty1 element and Ty1 cDNA synthesis in yeast rely on the ability of the CYC sequences to base pair. This 5′–3′ interaction is also supported by phylogenic analysis of all full-length Ty1 and Ty2 elements present in the Saccharomyces cerevisiae genome. These novel findings lead us to propose that circularization of the Ty1 genomic RNA controls initiation of reverse transcription and may limit reverse transcription of defective retroelements. Introduction Retrotransposons are mobile genetic elements, closely related to retroviruses, which replicate through a genomic single-stranded RNA intermediate that is converted into a double-stranded DNA by reverse transcriptase (RT). This process, called reverse transcription, takes place in a nucleoprotein particle containing the retrotransposon RT (Boeke and Stoye, 1997). Subsequently the newly formed DNA copy is integrated into the host genome. By means of this copy-and-paste mechanism, retrotransposons have efficiently invaded eukaryotic genomes. As a major portion of eukaryotic genomes they are thought to play a central role in their evolution. Retrotransposons have been involved in double-strand break repair (Teng et al., 1996; Yu and Gabriel, 1999) and are sources of insertional mutagenesis, homologous recombination and RT activity (Boeke and Stoye, 1997). The latter has been involved in pseudogene formation, gene transduction and exon shuffling, as well as intron loss when RT acts on cellular RNAs rather than on retrotransposon RNA (Fink, 1987; Derr and Strathern, 1993; Flavell et al., 1994; Moran et al., 1999; Esnault et al., 2000; Elrouby and Bureau, 2001). Therefore, understanding the mechanism of endogenous reverse transcription and the way it is controlled are major challenges. Long terminal repeat-containing retrotransposons (LTR-retrotransposons) share with oncoretroviruses a common organization of their genome. The close relationships between LTR-retrotransposons and retroviruses have led to the assumption that reverse transcription is identical in both retroelements. However, few studies have investigated reverse transcription at the molecular level in LTR-retrotransposons. The best-studied LTR-retrotransposons are Saccharomyces cerevisiae Ty1 and Ty3, and Schizosaccharomyces pombe Tf1. Initiation of reverse transcription, thought to be a strictly controlled step, has been studied in these three retrotransposons and striking differences from the retroviral modus operandi have been found. In retroviruses a specific cellular tRNA is annealed to an 18 nucleotide (nt) region called the primer binding site (PBS) near the 5′ end of genomic RNA. In Ty1 and Ty3 the primer for reverse transcription, initiator methionine tRNA [tRNA(iMet)], binds to canonical PBSs, which are much shorter (10 and 8 nt, respectively) than the 18 nt retroviral PBS. This has led to a search for additional contacts between tRNA(iMet) and genomic RNA that could account for the stability and specificity of the RT priming process. In these two elements a functional PBS was found split into at least three separate regions. In Ty1, tRNA(iMet) is annealed to boxes 0, 1 and 2.1 in the 5′ coding region of Ty1 RNA in addition to the canonical PBS (Figure 1) (Chapman et al., 1992; Keeney et al., 1995; Friant et al., 1996, 1998). In Ty3, tRNA(iMet) is annealed to sequences located at opposite ends of the genomic RNA, causing circularization of Ty3 RNA through a tRNA bridge (Gabus et al., 1998). Tf1 priming differs drastically from that of retroviruses, since the 5′ end of the genomic RNA folds back on itself, is cleaved and used as primer (Levin, 1995, 1996). Figure 1.Scheme of Ty1 reverse transcription. (A) Current model of Ty1 (−) strand cDNA synthesis. (1) Primer tRNA(iMet) is annealed to the primer binding site (PBS) and to boxes 0, 1 and 2.1 by the RNA-chaperone properties of the Ty1 Gag peptide, called TYA1-D. (2) The 3′ OH of tRNA(iMet) is elongated by Ty1 RT using Ty1 RNA as template (thin line) to generate minus-strand strong-stop cDNA (ss-cDNA, thick line). During the elongation process, RT RNase H degrades the RNA template (dotted line). (3) ss-cDNA is transferred to the 3′ end of Ty1 RNA genome (either intra- or intermolecular) by R sequences pairing and conducted by TYA1-D. (4) ss-cDNA is elongated by RT generating minus-strand cDNA product (st-cDNA). (B) WT RNAs used in the present study. Ty1 5′ RNA contains the 5′ repeat sequence R, U5 of the LTR, PBS and boxes 0, 1 and 2.1. Ty1 3′ RNA contains the polypurine tract (PPT1), U3 and 3′ R sequences of the LTR. Numbers indicate nucleotide positions with respect to the Ty1-H3 molecular clone. Download figure Download PowerPoint Since the PBSs of Ty1 and Ty3 are multipartite, we wondered whether reverse transcription was strictly identical to the retroviral mode or might substantially diverge. To answer this question, we have set up an in vitro Ty1 model system to examine minus-strand cDNA synthesis. Surprisingly, this reconstituted system revealed that the 3′ region of Ty1 RNA greatly enhances the initiation efficiency, independently of minus-strand DNA transfer. This functional interaction requires base pairing between a 5′ Gag coding sequence of 14 nt and a complementary sequence located in the 3′ UTR. Furthermore, this 5′–3′ interaction is required in vivo for efficient transposition of a marked Ty1 element. The importance of the 5′–3′ RNA interaction is also strengthened by conservation or covariation in all full-length Ty1 and Ty2 elements present in the yeast genome. Based on these findings we propose that genomic RNA circularization, either directly, as in Ty1, or indirectly, as in Ty3, may be a common feature of reverse transcription in LTR-retrotransposons. This novel mechanism may limit reverse transcription of defective retroelements. Results A reconstituted in vitro Ty1 reverse transcription system Ty1 cDNA synthesis occurs in a cytoplasmic nucleoprotein shell, called the virus-like particle (VLP), composed of Ty1 Gag (Garfinkel et al., 1985; Mellor et al., 1985), the dimeric RNA genome, primer tRNA(iMet), protease (PR), integrase and RT (Garfinkel et al., 1985; Mellor et al., 1985; Adams et al., 1987; Eichinger and Boeke, 1988; Chapman et al., 1992; Feng et al., 2000). Ty1 Gag is not processed further into matrix, capsid and nucleocapsid proteins (NCp) by PR, but rather the C-terminal 40 amino acids are cleaved by PR and then probably degraded (Merkulov et al., 1996). In a previous report, we showed that the C-terminal region of mature Ty1 Gag has in vitro nucleic acid chaperone properties similar to retroviral NCp (Cristofari et al., 2000). As a synthetic peptide called TYA1-D, this domain allows specific initiation of reverse transcription in vitro by annealing primer tRNA(iMet) to the PBS in physiological conditions and by inhibiting non-specific priming events (Cristofari et al., 2000). Retroviral NCps are nucleic acid chaperones that direct specific reverse transcription initiation and the two obligatory strand transfers required to generate the LTRs and complete proviral DNA by RT (Darlix et al., 2000). By taking advantage of the recent characterization of both Ty1 RT and the Gag peptide with nucleic acid chaperone properties (Cristofari et al., 2000; Wilhelm et al., 2000), we reconstituted functional Ty1 nucleoprotein complexes to study minus-strand cDNA synthesis in vitro. The reconstituted Ty1 complexes comprise two in vitro generated RNAs (5′ and 3′ Ty1 RNA) mimicking the 5′ and 3′ regions of Ty1 genomic RNA, containing all sequences in 5′ (R, PBS and boxes 0, 1 and 2.1) or in 3′ (PPT1, R), respectively, thought to be required for transposition (Figure 1B) (Xu and Boeke, 1990). The Ty1 complexes also contain primer [32P]tRNA(iMet), TYA1-D peptide and recombinant Ty1 RT (Wilhelm et al., 2000). Minus-strand strong-stop cDNA synthesis and transfer 5′ and 3′ Ty1 RNAs and 5′ 32P-labelled tRNA were first incubated with TYA1-D to form nucleoprotein complexes and to direct the annealing of tRNA to the multipartite PBS (PBS, box 0, box 1 and box 2.1; Figure 1A, step 1). Then Ty1 RT and dNTPs were added to start cDNA synthesis. All steps were carried out at 25°C, the optimal temperature for Ty1 transposition in yeast and for activity of the recombinant Ty1 RT. Two cDNA were synthesized (Figure 2A, lanes 6–10). The shorter one corresponds to the minus-strand strong-stop cDNA (ss-cDNA), i.e. the cDNA starting at the 3′ end of the tRNA and ending at the 5′ end of Ty1 5′ RNA (Figure 1A, step 2). The longer cDNA was of the expected size for the minus-strand transfer product (st-cDNA) (i.e. elongation of ss-cDNA after transfer to Ty1 3′ RNA; Figure 1A, steps 3 and 4). The maximal strand transfer efficiency was 40% st-cDNA of total cDNA synthesis (Figure 2A, lanes 7 and 8). In the absence of Ty1 3′ RNA, only ss-cDNA was detected (Figure 2A, lanes 1–5). A surprising observation was that Ty1 3′ RNA increased 10- to 20-fold the total level of ss-cDNA synthesis (Figure 2A, compare lanes 1–5 with 6–10, and B for quantification; see also Figure 6). In a control experiment with no Ty1 5′ RNA, no cDNA was detected (Figure 2A, lanes 11–15). DNA synthesis was strictly dependent on TYA1-D (Figure 2A, compare lanes 1 or 6 with 2–4 or 7–9, respectively). However, an excess of TYA1-D resulted in the inhibition of reverse transcription, as already observed with retroviral NCp (Guo et al., 1997; Lapadat-Tapolsky et al., 1997). Together, these two phenomena led to a sharp peak of cDNA synthesis. Therefore, several TYA1-D concentrations were always used to avoid missing the optimum (usually around 1:12 or 1:10 peptide to nucleotide ratio). Figure 2.In vitro synthesis of Ty1 minus-strand cDNA. (A) Ty1 nucleoprotein complexes containing 5′ 32P-labelled tRNA and Ty1 RNAs were incubated with Ty1 RT and dNTP. Ty1 5′ RNA alone (lanes 1–5), 5′ and 3′ RNAs (lanes 6–10) or 3′ RNA only (lanes 11–15) were used. After reverse transcription, nucleic acids were purified, analysed by 6% PAGE in denaturing conditions and the gel was autoradiographed. TYA1-D to nucleotide molar ratios were 0, 1:15, 1:12, 1:10 and 1:8. Arrowheads indicate minus-strand strong-stop cDNA (ss-cDNA, 167 nt) and strand transfer product (st-cDNA, 439 nt), covalently linked to [32P]tRNA(iMet). (B) Quantification of the gel shown in (A). Quantifications of lanes 1–5 are shown as black bars and those of lanes 6–10 as grey bars. Download figure Download PowerPoint Figure 3.Molecular requirements for minus-strand DNA transfer. Experiments were performed as in Figure 2. Ty1 5′ RNA was WT. Ty1 3′ RNA was either WT (lanes 1–4 and 9–12) or deleted of the repeated 3′ region (ΔR, lanes 5–8 and 13–16). Ty1 RT was either WT (lanes 1–8) or RNase H(−) (lanes 9–16). TYA1-D to nucleotide molar ratios were 0, 1:15, 1:10 and 1:8. Black arrowheads indicate strong-stop cDNA (ss-cDNA, 167 nt) and strand transfer product (st-cDNA, 439 nt), covalently linked to [32P]tRNA(iMet). Note that several premature stops before ss-cDNA completion were observed with the RT RNase H(−) mutant (white arrowheads). Download figure Download PowerPoint Since this model system was able to recapitulate the main steps of Ty1 minus-strand DNA synthesis in vitro, we examined the requirement for cis elements and trans-acting factors. For example, the 3′ R sequence and RT-associated RNase H are both necessary in retroviral strand transfers (Peliska and Benkovic, 1992; Allain et al., 1994). First, deletion of the 3′ R caused a drastic reduction of st-cDNA synthesis from 40% to <5% of strand transfer efficiency, whereas ss-cDNA level was intact (Figure 3, compare lanes 1–4 with 5–8). Next, Ty1 RT with the D468S mutation in the RNase H active site was used since it has a wild-type level of polymerase activity on homopolymeric template but no RNase H activity (Wilhelm et al., 2001). In vitro Ty1 RT D468S was able to synthesize ss-cDNA at similar levels to that of wild-type (WT) RT, whereas no st-cDNA was detected (Figure 3, compare lanes 1–4 with 9–12). Premature pauses before completion of ss-cDNA synthesis were observed with this mutant (Figure 3, lanes 9–16, white arrowheads), as already described for retroviral RNase H(−) RT (Dudding and Mizrahi, 1993). These results are in agreement with the effect of the D468S mutation on Ty1 cDNA synthesis in vivo (Uzun and Gabriel, 2001). Figure 4.The 3′ end of Ty1 RNA specifically enhances ss-cDNA synthesis. (A) Reverse transcription was performed in the presence of Ty1 RT RNase H(−) with WT (lanes 1–10) or pbs− (lanes 11–20) 5′ Ty1 RNA and with or without WT 3′ Ty1 RNA (lanes 1–5 and 11–15, or lanes 6–10 and 16–20, respectively). TYA1-D to nucleotide molar ratios were 0, 1:15, 1:12, 1:10 and 1:8. (B) Specificity of the Ty1 3′ RNA on ss-cDNA synthesis. Assays were performed with a constant amount of Ty1 5′ RNA (0.25 pmol) and a constant 1:10 TYA1-D protein to nucleotide molar ratio. An increasing 3′ to 5′ RNA molar ratio was used (0; 1; 2; 5). 3′ RNA was either Ty1 3′ RNA (lanes 1–4), HIV-1-derived 5′ RNA (nt 1–415) (lanes 5–8) or yeast poly(A)+ RNAs (lanes 9–12). Ty1 RT RNase H(−) was used in order to block strand transfer. The arrowhead indicates strong-stop cDNA covalently linked to [32P]tRNA(iMet) (ss-cDNA, 167 nt). Download figure Download PowerPoint The 3′ region of Ty1 RNA is required for efficient minus-strand ss-cDNA synthesis As underlined above, Ty1 3′ RNA seemed to increase the total level of ss-cDNA synthesis (Figure 2). To determine whether strand transfer could be indirectly required for efficient ss-cDNA synthesis, we compared the level of ss-cDNA with the RNase H defective RT, in the presence or absence of Ty1 3′ RNA. ss-cDNA synthesis was increased by Ty1 3′ RNA, even when strand transfer was impaired due to the use of RT RNase H(−) (Figure 4A, compare lanes 1–5 with 6–10). The 5′ RNA PBS was also mutated to verify that no other initiation pathway was involved in the process. We used 5 nt point mutations preventing tRNA(iMet) pairing to PBS (Cristofari et al., 2000). Indeed, this mutation completely abolished ss-cDNA synthesis independently of added Ty1 3′ RNA (Figure 4A, compare lanes 1–10 with 11–20). Figure 5.Direct interaction between the 5′ and 3′ ends of Ty1 RNA. (A) The ends of Ty1 RNA can interact in vitro. Equimolar amounts of Ty1 5′ and 3′ RNAs were incubated with increasing amounts of TYA1-D, at nt molar ratios of 0, 1:20, 1:10 and 1:5. 5′ RNA alone (lanes 1–4), 5′ and 3′ RNAs (lanes 5–8) or 3′ RNA alone (lanes 9–12) were used. After incubation, nucleic acids were purified and analysed in native conditions by agarose gel electrophoresis, followed by EtBr staining. (B) Putative 5′–3′ interacting sequences. 5′ and 3′ sequences have been called CYC5 and CYC3, respectively. (C) Mutations introduced to destabilize putative CYC pairing. RNA mutants are cyc5− and cyc3−, respectively. (D) 5′–3′ interaction is mediated by CYC sequence pairing. Interaction between WT or mutant RNAs (cyc) was analysed as in (A) with a TYA1-D to nucleotide molar ratio of 1:10 (even lanes) or without TYA1-D (odd lanes). Note that mutant 3′ RNA is smaller than WT 3′ RNA because unlike WT it does not contain a poly(A) tail. Similar results were obtained when poly(A)− WT RNA was used (data not shown). Download figure Download PowerPoint These findings suggest that the Ty1 3′ RNA contains a sequence required for efficient ss-cDNA synthesis independently of the other reverse transcription steps. To assess the specificity of the Ty1 3′ RNA effect on ss-cDNA synthesis, Ty1 3′ RNA was replaced by heterologous RNA and reverse transcription was performed with RT RNase H(−) mutant to block strand transfer. Neither an in vitro-generated RNA encompassing nt 1–415 of HIV-1 RNA (Lapadat-Tapolsky et al., 1997), nor YH50 yeast poly(A)+ RNAs were able to increase the level of ss-cDNA synthesis as Ty1 3′ RNA did (Figure 4B). This indicated that Ty1 3′ RNA can specifically enhance ss-cDNA synthesis. Direct interaction between the 5′ and 3′ ends of Ty1 genomic RNA Since functional relationships appear to take place between the two ends of Ty1 genomic RNA, we wondered whether this could rely on direct physical interactions. To test this hypothesis, 5′ and 3′ Ty1 RNAs were incubated with TYA1-D. Following protein extraction, RNAs were analysed by electrophoresis in native conditions. With both Ty1 RNAs, a new RNA species with an apparent higher molecular weight of 930 nt was formed (Figure 5A, lanes 5–8). Conversely, incubation of 5′ RNA or 3′ RNA alone with TYA1-D did not generate this new RNA species (lanes 1–4 and 9–12). Interaction between 5′ and 3′ RNAs was strongly favoured by the TYA1-D RNA chaperone peptide (Figure 5A, compare lanes 5 and 8). When primer tRNA(iMet) was included in the assay, identical results were obtained (data not shown). This prompted us to perform a computer local alignment search for sequence complementarities at the 5′ and 3′ ends of Ty1 RNA. A 3′ sequence of 14 nt was discovered that perfectly matches a region downstream of the PBS, encompassing box 2.1 (Figure 5B). The 5′ and 3′ sequences were called CYC5 and CYC3, for cyclization in 5′ or in 3′, respectively. To examine whether these complementary sequences mediate the interaction between the 5′ and 3′ RNAs, we introduced point mutations in CYC5 to disrupt sequence complementarities. Compensatory point mutations were inserted in CYC3 to compensate for the CYC5 mutations (Figure 5C). Mutations in CYC5 or in CYC3 completely abolished RNA–RNA interactions (Figure 5D, compare lanes 9–10 with 11–14). In contrast, the use of both cyc5− and cyc3− mutations restored the 5′–3′ interaction (lanes 15–16). These results indicate that CYC5–CYC3 pairing mediates a direct molecular interaction between the ends of Ty1 RNA. Figure 6.Interaction between the ends of Ty1 RNA is required for efficient reverse transcription initiation. (A) ss-cDNA synthesis was performed with WT (lanes 1–9) or cyc5− (lanes 10–18) 5′ RNA, in the absence (lanes 1–3) or presence of WT (lanes 4–6 and 14–16) or cyc3− (lanes 7–9 and 16–18) 3′ RNA. TYA1-D to nucleotide molar ratios were 1:15, 1:12, 1:10 and 1:8. Ty1 RT RNase H(−) was used as before. (B) Quantification of the peak of ss-cDNA synthesis of the gel in (A) (lanes 2, 6, 10, 14 and 18, respectively). Download figure Download PowerPoint Base pairing between the 5′ and 3′ ends of Ty1 genomic RNA is required for efficient ss-cDNA synthesis in vitro To investigate the requirement of CYC pairing for optimal ss-cDNA synthesis, the mutated 5′ and 3′ RNAs were used in the reconstituted reverse transcription system with RT RNase H(−) mutant to block strand transfer. Mutant cyc5− RNA with WT 3′ RNA or WT 5′ RNA with mutant cyc3− RNA were much less efficient than both 5′ and 3′ WT RNAs in promoting ss-cDNA synthesis (12 and 2% of the WT level, respectively; Figure 6A, compare lanes 4–6 with 7–9 and 13–15; Figure 6B for quantification). On the other hand, when both mutant RNAs were used, cDNA synthesis was restored to a level close to that of the wild type (Figure 6A, compare lanes 7–9, 13–15 and 16–18; Figure 6B for quantification). Thus CYC5–CYC3 base pairings are required for efficient ss-cDNA synthesis in vitro. Ty1 3′ RNA enhances ss-cDNA synthesis by acting at the level of reverse transcription initiation The Ty1 3′ RNA-mediated enhancement of ss-cDNA synthesis can take place at one (or more) of the following steps: tRNA(iMet) annealing, initiation, initiation-to-elongation transition or elongation. We have compared the annealing of tRNA(iMet) to the 5′ PBS with or without 3′ RNA. Results show that the level of tRNA(iMet) annealed to PBS was not increased by the 3′ RNA but was rather slightly decreased (Figure 7B, compare free tRNA(iMet) in lanes 5 and 10). Furthermore disrupting CYC pairing does not prevent tRNA(iMet) annealing to the PBS (see Supplementary figure 1 available at The EMBO Journal Online). This indicates that the 3′ RNA acts during a later step of ss-cDNA synthesis. Figure 7.The 3′ RNA acts at the level of reverse transcription initiation. (A and B) Annealing of tRNA(iMet) to 5′ RNA without (lanes 1–5) or with 3′ RNA (lanes 6–10). Experiments were as described in Figure 5, with an excess of [32P]tRNA(iMet). (A) and (B) are EtBr staining and autoradiography of the same gel, respectively. (C) Sensu stricto initiation of reverse transcription. Reverse transcription was performed as described in Figure 2, but an unlabelled tRNA(iMet) and solely [α-32P]dTTP were used instead of all four dNTPs. TYA1-D to nucleotide molar ratios were 1:30, 1:25, 1:20, 1:15, 1:12, 1:10 and 1:8. The arrowhead indicates [32P]dTMP covalently linked to tRNA(iMet) (76 nt). Labelled tRNA(iMet) and φX174 DNA HinfI markers (Promega) were used for size determination (not shown). Download figure Download PowerPoint Next, we examined the influence of the 3′ RNA at the initiation step. To this end, we used an unlabelled tRNA(iMet) and solely [α-32P]dTTP instead of all four dNTPs to initiate reverse transcription. Since dTMP is the first nucleotide added by RT to tRNA(iMet), a 76 nt tRNA–[32P]dTMP product must arise upon addition of RT if initiation of reverse transcription occurs (scheme in Figure 7B). Indeed, we observed an initiation product with both 5′ and 3′ RNAs, whereas virtually no initiation product was detected with 5′ or 3′ RNA alone. Increasing the incubation time from 30 to 60 min did not increase initiation level. This suggests that tRNA(iMet)–5′ RNA–3′ RNA complexes are more competent for reverse transcription initiation than are tRNA(iMet)–Ty1 5′ RNA complexes. CYC pairing is required for efficient cDNA synthesis in vivo and transposition To analyse the role of the CYC sequences in vivo, we introduced a plasmid-borne Ty1 element under the control of the inducible GAL1 promoter (pGTy1-H3mHIS3AI) into a His− yeast strain. This element is genetically marked with a HIS3 reporter gene in the antisense orientation and interrupted by an artificial intron (AI) in the same orientation as Ty1 (Figure 8A). Thus the level of His+ revertants is an indicator of retrotransposition frequency since HIS3 expression only occurs after splicing, reverse transcription and integration (Curcio and Garfinkel, 1991). The YH50 strain and its isogenic rad52− counterpart AGY49 used here are spt3− to limit expression of endogenous Ty1s, which might complement in trans the HIS3-marked element (Boeke et al., 1986). Retrotrans position efficiencies in RAD52 and rad52− yeast were compared since cDNA incorporation into the yeast genome is known to occur by at least two pathways: one requires Ty1 integrase and a complete, double-stranded Ty1 DNA, and the second is dependent on homologous recombination (therefore on Rad52p) and could mediate integration of aberrant reverse transcripts (Sharon et al., 1994). Figure 8.Pairing of CYC5 and CYC3 sequences is required for Ty1 transposition. (A) Scheme of the Ty1 transposition assay. pGTy1-H3mHIS3AI plasmid contains the Ty1-H3 molecular clone, the expression of which is under the control of the inducible GAL1 promoter and marked with a HIS3 reporter gene in the antisense orientation. An artificial intron (AI) in the sense orientation has been inserted in the HIS3 gene. The plasmid also contains a URA3 gene to permit transformant selection and a high copy number origin of replication (μ ORI). (B) Effect of CYC mutations on Ty1 transposition. The WT plasmid or one of its mutant counterparts was transformed in a ura3− and his3− strain. Transformants were selected in synthetic medium containing glucose and lacking uracil (SC−Ura+Glc) and patched onto SC−Ura+ Glc plates. The position of each transformant is indicated in the grey squares. Transposition induction was performed by replica plating patches onto medium containing either glucose or galactose and lacking uracil (SC−Ura+Gal) at 30°C. Finally, transposition events were selected by replica plating colonies on medium containing glucose and lacking histidine (SC−His+Glc, plate shown). Strains were YH50 or its rad52 counterpart AGY49. Ct stands for plasmid pGmHIS3AI (see text). Download figure Download PowerPoint Yeasts were transformed with pGTy1-H3mHIS3AI plasmid (WT Ty1) or with its mutated counterparts (cyc5−, cyc3− and double mutant cyc5−/cyc3−). Two negative controls included a pbs− mutant defective in the initiation of reverse transcription (Chapman et al., 1992) and the HIS3AI cassette alone under the GAL1 promoter as a control (pGmHIS3AI plasmid, Ct) of HIS3AI reverse transcription (Curcio and Garfinkel, 1991). The mutations chosen in vivo were those used in vitro. Since cyc5− and pbs− mutations were localized in the Gag coding sequence, silent mutations were chosen. WT or mutated Ty1 elements were induced by growth on galactose and then replica-plated onto media lacking histidine. The level of His+ prototrophy was greatly decreased with single cyc5− and cyc3− mutations compared with WT (Figure 8B), but the number of His+ colonies was restored to a WT level when cyc5−/cyc3− compensatory mutations were inserted into the marked Ty1 element. The effect of the CYC mutations on transposition was qualitatively similar in RAD52 and rad52− strains. However, a number of cDNA insertions in the RAD52 strain did not depend on an intact PBS and therefore did not rely on accurate reverse transcription. In the RAD52 genetic background, the transposition rate of cyc5− or cyc3− mutants was similar to that of the pbs− mutant. Transposition frequencies of the Ty1 mutants were quantified and compared with that of the WT element in the rad52− strain in which only integrase-dependent events are observed. Levels of cyc5− mutant and cyc3− mutant transposition were 5 and <1% that of WT Ty1, respectively (Table I). In contrast, the transposition level of the double cyc5−/cyc3− mutant was 33% higher than that of WT Ty1. Table 1. Transposition frequency of cyc and pbs mutants Ty1 plasmid Transposition frequency Relative transposition frequency WT 3.2 (± 0.8) × 10−3 1.00 Ct <1.8 (± 0.4) × 10−5 <0.01 cyc5− 1.5 (± 0.6) × 10−4 0.05 cyc3− <2.0 (± 0.2) × 10−5 <0.01 pbs− 2.0 (± 2.2) × 10−5 0.01 cyc5−/cyc3− 4.3 (± 0.6) × 10−3 1.33 The transposition frequency was determined as the number of His+ cells/total number of cells. Transposition frequencies shown are the mea
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