A 5'-3' long-range interaction in Ty1 RNA controls its reverse transcription and retrotransposition

Abstract

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.

Fig. 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.

Fig. 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.

Fig. 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).

Fig. 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).

Fig. 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).

Fig. 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).

Fig. 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).

Fig. 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).

Fig. 9. Pairing of CYC5 and CYC3 sequences is required for Ty1 cDNA synthesis. (A and B) Transposition of WT or mutants of HIS3AI-marked Ty1 element in AGY49 cells at 22°C. Descending spots correspond to 5-fold dilutions. Transposition was assayed under repressing (lanes 1–6, glucose medium) and inducing (lanes 7–12, galactose medium) conditions by comparing growth on non-selective medium (A, SC) and on selective medium lacking histidine (B, SC–His). (C) Steady-state levels of Ty1 Gag protein as determined by immunoblotting of total protein extracts using anti-Gag antibodies. The same amount of total proteins was loaded in each lane. Molecular weights (left) are in kDa. (D) Steady-state levels of Ty1 cDNA as determined by Southern blot analysis of total DNA. The HIS3 probe hybridized to Ty1 cDNA (1.2 kbp) as well as to the pGTy1-H3mHIS3AI donor plasmid (14.5 kbp; all lanes except 2 and 7; lane 13 is a control with pGTy1-H3mHIS3AI alone). Molecular weights (left) are in kbp. Plasmid bands in lanes 2 and 7 are lower since they correspond to plasmid pGmHIS3AI (Ct) and the plasmid copy number increased when cells were grown in glucose rather than in galactose. Samples from the same cultures were used in experiments (A)–(D).

Fig. 10. Covariation of CYC sequences in all complete copies of Ty1 and Ty2 present in the S.cerevisiae genome. (A) Conservation and covariation of CYC sequences. Numbers indicate how many Ty1 elements contain the depicted changes. (B) Placement of CYC variants on an unrooted phylogenic tree based on the alignment of full Ty1–Ty2 sequences. Asterisks indicate mutations found in only one LTR, suggesting post-integration divergence. Note that CYC sequences are strictly conserved in all Ty2 elements, which are thought to form the most recent and active retrotransposon family in yeast (Kim et al., 1998).