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Previous Article | Next Article 
Molecular and Cellular Biology, July 2006, p. 5168-5179, Vol. 26, No. 13
0270-7306/06/$08.00+0 doi:10.1128/MCB.00096-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bioscience Building 501, Kashiwa, Chiba 277-8562, Japan
Received 16 January 2006/ Returned for modification 23 February 2006/ Accepted 10 April 2006
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ABSTRACT |
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INTRODUCTION |
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Many non-LTR retrotransposons that belong to the recently branched type have two open reading frames (ORFs), ORF1 and ORF2, whereas ancient-type non-LTR elements have only one ORF (19). In all non-LTR retrotransposons having two ORFs, ORF2 encodes an endonuclease domain, which cuts and determines the target site of integration, and a reverse transcriptase domain, which is responsible for reverse transcribing the RNA template. However, little is known about the structural and functional features of ORF1, except that they are essential for in vivo retrotransposition and that some CCHC-type zinc finger motifs (also called zinc knuckle motifs) located at the C terminus are conserved in many non-LTR elements. It is assumed by in vitro studies that the ORF1 protein (ORF1p) interacts with RNAs and is involved in protein multimerization (4, 15). In addition, some histological studies demonstrated colocalization of ORF1p and the ORF2 protein (ORF2p) (9) and of ORF1p and RNA (28). Based on these observations, it has been hypothesized that ORF1p, ORF2p, and RNA of recently branched-type non-LTR elements interact with each other to form ribonucleoprotein (RNP) complexes. A recent report shows that the human long interspersed nuclear element 1 (L1) RNA and ORF1p form RNPs in vivo and that some ORF1 mutants have decreased RNP formation (15). However, we have no direct evidence in any non-LTR elements for ORF1p-ORF2p interaction, nor do we know what specific domains of ORF1 are responsible for RNP complex formation by ORF1p, ORF2p, and RNA.
Chromosomal ends of eukaryotes generally consist of telomeric repeats, which are synthesized by the reverse transcriptase activity of telomerase (2). Bombyx mori has a TTAGG telomeric repeat at the chromosomal ends, but its telomerase activity is very low. Since many copies of non-LTR retrotransposons such as TRAS1 and SART1 accumulate in the telomere regions of B. mori, it is hypothesized that they compensate the loss of telomere due to the low telomerase activity (6, 22, 27, 30). The complete unit of SART1 is 6.7 kb in length and terminates with a poly(A) stretch (Fig. 1A). There are 600 SART1 copies in the silkworm haploid genome, and most of them are completely conserved in structure without 5' truncation (30), although many non-LTR retrotransposons are 5' truncated due to incomplete reverse transcription (8). And while expression of retroelements is usually suppressed in general, SART1 mRNA is transcribed abundantly and is detected easily by Northern hybridization (31). These data suggest that the telomere-specific SART1 has a high retrotransposable activity and is a good model to study the retrotransposition mechanisms of non-LTR retrotransposons. Recently, we established a baculovirus-mediated retrotransposition system in which the specific integration of SART1 into the telomeric repeats of the host cells could be easily detected (32). Using this system, we have shown that ORF1p of SART1 encodes a novel nuclear localization signal (NLS) and a putative telomere targeting domain, both of which are essential for in vivo retrotransposition (20). We have also recently found that SART1 can retrotranspose by trans complementation when the ORF1 construct and the ORF2 plus 3' untranslated region (ORF2/3' UTR) construct are coexpressed in vivo (13), indicating that ORF1p can recognize and interact in trans with ORF2p and SART1 mRNA in the cells.
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The aim of this study is to clarify the functional roles of the ORF1 protein of SART1, especially in RNP complex formation, and to identify the essential ORF1 domains involved in this process. By in vivo reconstitution of SART1 RNP complex in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus expression system, we demonstrate that the SART1 RNP complex contains the multimerized ORF1p, ORF2p, and SART1 mRNA. We found that several regions of ORF1 protein near the C terminus are essential for interactions between ORF1p and ORF1p and between ORF1p and ORF2p. In particular, 13 amino acids, VARIGECPPDIVK, which are residues 555 to 567, are crucial to both interactions. In addition, we found that all three CCHC motifs are involved in the specific packaging of SART1 mRNA into the RNP complex. We propose that the C-terminal area of the ORF1 protein in SART1 is responsible for RNP complex formation through ORF1p-ORF1p, ORF1p-ORF2p, and ORF1p-mRNA interactions.
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MATERIALS AND METHODS |
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Plasmid expressing HA-tagged protein. The hemagglutinin (HA)-SART1ORF2/3' UTR plasmid is the same as construct IV in Kojima et al., and the construction of the vector lacking a glutathione transferase (GST)-His tag, named pAcLTB, was also described previously (13). HA-SART1ORF1pWT was constructed as follows. The portion of SART1 ORF1 was amplified by PCR from plasmid S1ORF1pWT with the primers S1S880NdeI and pAcBA3106. The PCR products were subcloned between the NdeI and BamHI sites of pGADT7 (Clontech). The region including SARTORF1 and the HA tag was amplified by PCR using the primers T7 and S1A3029NotI. The PCR products were subcloned between the NcoI and NotI sites of the vector lacking GST-His tag (pAcLTB).
Plasmid expressing partial SART1 ORF1. We constructed the vector lacking a GST tag from the plasmid pAcGHLTB (Pharmingen) because the GST tag sometimes bound to nonspecific nucleic acids and proteins. The whole pAcGHLTB plasmid except the GST tag sequence was amplified by inverse PCR using the 5' phosphorylated primers pAcBA2214 and pAcBS2863. Phosphorylated primers were made by T4 polynucleotide kinase (TaKaRa) and ATP (Roche). The PCR products were self-ligated, and the resulting plasmid was named pAcHisB. Partial sequences of SART1 were subcloned between the NcoI and NotI sites of pAcHisB.
Mutation introduction. Point mutations were introduced with pairs of primers using a QuikChange mutagenesis kit (Stratagene). The deletion mutants were constructed by inverse PCR and self-ligation using the 5' phosphorylated primers as described above. SART1-pAcGHLTB containing the TRAS1 ORF1 C terminus was constructed as follows. First, the NotI-BglII region of SART1WT-pAcGHLTB was removed by NotI and BglII digestion, T4 DNA polymerase treatment, and self-ligation. Second, the whole region of SART1-pAcGHLTB except the ORF1 C terminus was amplified by inverse PCR with the 5' phosphorylated primers S1A2505NotI and S1S2917BglII. The amplified product was self-ligated. Third, the TRAS1 ORF1 C terminus was amplified with T1S3359NotI and T1A3761BglII and cloned between the NotI and BglII sites of that plasmid.
Target plasmid for in vitro TPRT assay. To obtain Bombyx telomeric repeats (TTAGG/CCTAA)n, PCR amplification was carried out using the primers (CCTAA)6 and (TTAGG)6 without a template (12). The PCR products were cloned by the pGEM-T Easy Vector System (Promega), and a plasmid including (TTAGG)25 was isolated. The (TTAGG)25 sequence was subcloned to pBluescript II SK(+) by EcoRI.
Recombinant AcNPV generation. Recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) generation was carried out according to the BaculoGold (Pharmingen) instructions and a previous study (32).
In vivo retrotransposition assay. The in vivo retrotransposition assay was performed as described previously by Takahashi and Fujiwara (32). Primers S1S6131 and CCTAA+T (see Table S1 in the supplemental material) were used for PCR amplification of the 3' junction of retrotransposed SART1. The PCR signals were quantified using the NIH program Image J (see Fig. 2D).
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Purification of SART1 complex. Sf9 cells were infected with His6-SART1 containing AcNPV at a multiplicity of infection of 1.0 for 72 h, pelleted by centrifugation, and washed with phosphate-buffered saline. Cells were resuspended in ice-cold HG500 (20 mM HEPES-KOH, pH 7.8, 500 mM KCl, 10% glycerol, 50 mM imidazole, 1 mM mercaptoethanol, and 0.1% Triton X-100) buffer and sonicated slightly. The debris was pelleted at 10,000 x g for 30 min, and the lysate was bound to Ni-nitrilotriacetic acid (NTA) agarose (QIAGEN), preequilibrated with the HG500 buffer, for 2 h at 4°C. The resin was washed with 500 volumes of HG500 buffer at 4°C and eluted with 5 volumes of HG500 buffer plus 250 mM imidazole for 30 min at 4°C. For in vitro rescue experiments, wild-type (WT) SART1 ORF1p was prepared in parallel according to the same procedure (see Fig. 3C). Every eluate except the mock, in which only His tag was expressed, was adjusted to 10 ng/µl with HG500 buffer containing 250 mM imidazole. The eluate was stored at –80°C in 10-µl aliquots.
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Glycerol gradient sedimentation. Purified SART1 complex was overlaid onto 3.3 ml of 10 to 40% glycerol gradient in the HG500 buffer. Centrifugation was carried out using a swing rotor P55ST2 (Hitachi) for 16 h at 40,000 rpm at 4°C. Thyroglobulin (670 kDa; SIGMA) and BSA (67 kDa; Biomedical Technologies, Inc.) were used as protein molecular weight standards. A total of 300 µl of each fraction was collected from the bottom and stored at –80°C. The fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on an 8% gel, followed by electrophoretic transfer to polyvinylidene difluoride membrane (Pall Corp.) and immunoblot detection using anti-His antibody (Amersham). The absence and presence of SART1 ORF2/3' UTR mRNA (SART1 residues 6131 to 6702) in each fraction were analyzed by subsequent reverse transcription-PCR (RT-PCR).
RT-PCR. RNA was isolated from the purified SART1 RNP and the glycerol gradient fraction by a TRI Reagent LS kit (SIGMA). The RNA mixture was treated by DNase I (TaKaRa) to exclude DNA contamination. Components of the reverse transcriptase reaction mixture included the following: 0.1 µM pd(N)6 primer, 0.5 mM concentrations of each dNTP, 50 mM Tris-HCl, pH 8.3, 75 mM KCl, and 3 mM MgCl2. One microliter of RNA solution was added to the buffer and denatured at 65°C for 5 min and then incubated in the presence of 100 units of Moloney murine leukemia virus (Mo-MuLV) reverse transcriptase (Invitrogen) at 37°C for 50 min. After heat inactivation at 70°C for 15 min, 1 µl of the resulting mixture was amplified by PCR. The PCR assays were conducted using the Ex-Taq DNA polymerase (TaKaRa) with the respective primer sets (see Table S1 in the supplemental material). The PCR mixture was denatured at 96°C for 2 min, followed by 35 cycles of 98°C for 20 s, 60°C for 30 s, and 72°C for 1 min.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We tried to determine whether the three CCHC motifs between SART1 and TRAS1, the telomere-specific non-LTR retrotransposons in B. mori, can be exchanged functionally. We constructed a chimeric SART1-TRAS1 CCHC motif element by replacing the SART1 C terminus, which includes three CCHC motifs, with TRAS1 (Fig. 1C). In all constructs, the N terminus of ORF1 was fused with the GST-His6 tag. From previous studies, it is shown that SART1 expressed under the polyhedrin promoter of AcNPV in Sf9 cells transposes into the specific site at telomeric repeats (TTAGG)n, which can be detected by PCR using a specific primer set, S1S6131 and CCTAA+T (32). The WT SART1 actively retrotransposed 72 h postinfection (Fig. 1D, lane 1), but the chimeric SART1 element did not (lane 2). Coexpression of the SART1 ORF1p rescued the retrotransposition of the chimeric construct by trans complementation (lane 4), but the TRAS1 ORF1p did not (lane 3). We confirmed the expression of proteins in all constructs by immunoblot detection (Fig. 1E). These results indicate that, for retrotransposition, SART1 requires its own CCHC motifs in ORF1 and that they cannot be replaced by motifs of other non-LTR retrotransposons.
Any CCHC motifs of ORF1 are essential for SART1 retrotransposition in vivo.
To further examine whether three CCHC motifs are crucial for SART1 retrotransposition, we generated a series of SART1 constructs containing point mutations in every motif (Fig. 2A). H626P (626-His
Pro at motif 1), H649A (649-HisAla at motif 2), and H672A (672-HisAla at motif 3) were assumed as severe mutants for the zinc finger function. We changed the CCHC motif at motif 1 to the CCCC type in H626C (626-HisCys) and to the CCHH type in C631H (631-CysHis). Also, to understand the role of spacing in the CX2CX4HX4C (X indicates spacing) sequence, we changed the spacing in motif 1. The construct 623+A had one additional alanine residue between 623-Ala and 624-Leu, and the construct 623-AL lost the residues 623-Ala and 624-Leu.
Although the retrotransposition of WT SART1 occurred actively, H626P, H649A, and H672A did not show a significant PCR band (Fig. 2B, lanes 1 to 4). The expression of proteins in the three constructs at expected sizes was confirmed by immunoblot detection (Fig. 2C). The retrotransposition activities in the three mutants were rescued by coinfection of the WT SART1 ORF1p construct by trans complementation (Fig. 2B, lanes 9 to 11). These results indicate that all three CCHC motifs of ORF1 are necessary for retrotransposition of SART1 in vivo. Interestingly, the CCCC mutant H626C retrotransposed into telomeric repeats, whereas C631H (CCHH mutant), 623+A, and 623-AL could not (Fig. 2B, lanes 5 to 8). trans Complementation with normal ORF1p expression also rescued the retrotransposition activity (Fig. 2B, lanes 13 to 15). These results indicate that both the CCHC sequence itself and the spacing of amino acids are essential and that the CCCC type, but not the CCHH type, exhibits equivalent function of with the CCHC type, at least for motif 1 in the SART1 retrotransposition.
We wondered if the CCHC motif mutants had completely lost the retrotransposition activity by just a single amino acid mutation. To quantitatively evaluate retrotransposition activity, we changed the amount of genomic DNA used for the PCR assays. When we increased the amount of template genomic DNA by 10-fold (to calculate roughly, genomic DNAs extracted from 104 cells), we detected very weak signals for retrotransposition from every mutant (Fig. 2D, 104). Next, we analyzed the retrotransposition activity of WT using a 10-fold dilution series of template DNA (Fig. 2E) (roughly calculating, DNA was extracted from 104 to 1/1,000 cells). The retrotransposition of WT was still detected when the template was diluted to 1/1,000 of the original PCR condition (DNAs roughly equivalent to 1 cell). These results indicate that the CCHC mutants do not completely lose retrotransposition activities, but they are extremely suppressed compared to WT (roughly calculating, the CCHC mutants have only 1/1,000 the activity of the WT) (Fig. 2D). We previously reported that the deletion of the N-terminal NLS (residues 1 to 142 [
1-142]) or the central basic domain (440-447) of ORF1, which controls cellular localization, strongly decreases the retrotransposition activity of SART1. However, these mutants retain very low activity under the same experimental condition of the WT, unlike CCHC motif mutants (20). These data suggest that the CCHC motifs of ORF1 are indispensable to SART1 retrotransposition and that mutations to them are critical.
CCHC-mutated SART1 complexes reconstituted in vivo cannot be rescued in vitro. We next questioned how the CCHC motif works in the processes of SART1 retrotransposition. Because the CCHC motifs in retroviruses are thought to be involved in the complex formation of viral proteins and genomic RNAs (5, 33), we next studied the complex formation through the SART1 CCHC motifs by a new in vitro retrotransposition analysis. We infected Sf9 cells with full-length WT SART1-AcNPV and purified the ORF1 protein using a His tag. When we incubated this purified "ORF1" protein with plasmid including telomeric repeat sequences, it showed TPRT activity without the addition of template RNA or ORF2 protein. Therefore, we assumed that this "purified" ORF1p fraction involves the ORF2p and SART1 mRNA as a putative RNP complex, which is retained even during the elution steps in vitro.
To verify this assumption, we coinfected two AcNPV constructs, SART1 ORF1 (His tag fused) and SART1 ORF2/3' UTR (HA tag fused), into the Sf9 cells, purified the ORF1p fraction by Ni-NTA agarose, and tested whether it included ORF2p and ORF2/3' UTR mRNA using an in vitro retrotransposition assay (summarized in Fig. 3A). The eluted fraction showed only one strong band corresponding to the SART1 ORF1p by Coomassie brilliant blue staining after SDS-PAGE (Fig. 3B). After the eluted fraction was incubated with the plasmid containing Bombyx telomeric repeats and amplified by PCR with the primer set S1S6449 and CCTAA+T, we detected a band representing retrotransposition at the expected size in the WT ORF1 and H626C (CCCC mutant) ORF1 constructs (Fig. 3C, lanes 1 and 5, arrowhead) but not in any other CCHC motif mutants (lanes 2 to 4 and 6 to 8). These results are consistent with those of the in vivo retrotransposition assay (Fig. 2). We further cloned and sequenced the PCR products (Fig. 3C, lane 1, WT, and lane 4, CCCC-type mutant) (see Fig. S2A in the supplemental material) and found that only ORF2/3' UTR mRNA (not ORF1 mRNA) was reverse transcribed and inserted specifically into various sites in telomeric repeats of the target plasmid (see Fig. S2B in the supplemental material). This result indicates that ORF1p of SART1 interacts with the ORF2p/3' UTR mRNA to form the retrotransposable machinery and that the CCHC motifs are involved in some processes in these interactions.
Interestingly, the in vitro addition of purified WT ORF1p into the reaction buffer did not rescue the TPRT activity of the CCHC motif mutants (Fig. 3C, lanes 9 to 15), which is not consistent with the above results in coexpression in vivo (Fig. 2C, lanes 9 to 15). This result implies that the involvement of host factors in the cell or de novo complex formation immediately after translation is essential to generate the intact RNP unit of SART1.
The retrotransposition activity of H626C (lane 5) somewhat decreased when His-tagged ORF1p WT was added (Fig. 3C, lane 12). We have data indicating that the SART1 ORF1p binds to telomeric repeats (T. Matsumoto and H. Fujiwara, unpublished data). Thus, in the case of in vitro assays, excess WT ORF1p may bind to and cover telomeric repeats of the target plasmid and inhibit retrotransposition.
The specific domain but not the CCHC motifs in SART1 ORF1 directs the ORF1p-ORF1p interaction. To directly analyze the protein-protein interaction with SART1 ORF1p, we used the baculovirus expression system to coexpress various ORF1p constructs fused with His tag and ORF1p (Fig. 4) or ORF2p (see Fig. 5) fused with HA tag and examined their interactions in the Sf9 cells. WT or mutated ORF1 proteins with a His tag at their N termini and a WT ORF1p with an HA tag at its N terminus were coexpressed (extract fraction) and purified by Ni-NTA agarose (eluate fraction) (Fig. 4A). HA-tagged ORF1p was detected by immunoblot analysis in the His-tag-based eluate fraction of WT ORF1p, which we had expected it to form protein complexes with (Fig. 4B, eluate-anti-HA, WT). This result indicates that the His-tagged WT SART1 ORF1p coprecipitated HA-tagged SART1 ORF1p through some form of interaction. In contrast, the mock construct expressing only the His tag did not coprecipitate HA-ORF1p (Fig. 4B, eluate-anti-HA, Mock).
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354-554 (354 to 554 deleted) showed strong signals similar to those of the WT. The HA-tagged ORF1p constructs were almost equally expressed (Fig. 4B, extract-anti-HA) and various His-tagged ORF1p constructs were recovered to a similar extent (Fig. 4B, eluate-anti-His) (SDS-PAGE results not shown).
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SART1 ORF1p binds directly to ORF2p.
Using a similar assay, we next examined whether the His-tagged ORF1p also interacts with the HA-tagged ORF2p of SART1 (Fig. 5). His-tagged ORF1p and HA-tagged ORF2p constructs were coexpressed in Sf9 cells in the baculovirus expression system (extract fraction), and the extract was purified by Ni-NTA agarose (eluate fraction). In the eluate fraction, the anti-HA antibody detected a strong band representing the HA-tagged ORF2p, which was trapped by the WT His-tagged ORF1p. In contrast, no HA-tagged ORF2p coprecipitated with the His-tagged mock protein (Fig. 5A, eluate-anti-HA, WT and Mock). We also observed a clear band of HA-tagged ORF2p showing interaction with three His-tagged ORF1p constructs, 1-567 (residues 568 to 712 deleted), 1-580 (581 to 712 deleted), and 285-712 (1 to 284 deleted) but not in constructs 1-483, 1-519, and 1-554, which lacked a large C-terminal region of ORF1p, nor in 354-554, the deleted central region (Fig. 5A, eluate-anti-HA). Interestingly, the CCHC motif mutants of ORF1p interacted with ORF2p in the same way as WT-ORF1p (Fig. 5B, eluate-anti-HA). In all cases, HA-tagged ORF2p was normally expressed (extract-anti-HA), and His-tagged ORF1p was purified to a similar extent (Fig. 5, eluate-anti-His) (SDS-PAGE results not shown). Based on these results, we conclude that (i) residues 555 to 567 of ORF1p are involved in the ORF1p-ORF1p interaction and are essential for the ORF1p-ORF2p interaction, (ii) that the ORF1p-ORF2p interaction requires another domain within residues 285 to 554 of ORF1p, and (iii) that CCHC motifs are not involved in the ORF1p-ORF1p or ORF1p-ORF2p interactions.
We performed an in vivo retrotransposition assay on SART1 constructs lacking the ORF1 regions implicated in the ORF1p-ORF1p and ORF1p-ORF2p interactions discussed earlier (Fig. 5C). When the WT SART1 ORF1 and ORF2/3' UTR were coexpressed in Sf9 cells, we observed a strong PCR band representing SART1 (precisely mRNA for ORF2/3' UTR) retrotransposed to the telomere of the genome by trans action between ORF1p, ORF2p, and SART1 mRNA (13) (Fig. 5C, WT). When three ORF1 constructs 484-580, 555-567, and 520-580, which lacked the regions involved in the protein interactions, were coexpressed with ORF2/3' UTR, no bands for retrotransposition were observed (Fig. 5C), although the mutated constructs expressed ORF1p to the same extent as the WT construct (Fig. 5D). These results indicate that the ORF1p-ORF1p and ORF1p-ORF2p interactions are conducted via specific domains of ORF1p and are essential for the retrotransposition activity of SART1.
CCHC motifs of ORF1 play a role in the packaging of SART1 RNA into the RNP complex. Our data show that the three CCHC motifs are essential for the SART1 retrotransposition but are not involved in the interactions between ORF proteins. Consequently, we tested the involvement of CCHC motifs in the ORF1p-RNA interaction, because in vitro TPRT (Fig. 3) showed that the SART1 complex included its own mRNA autonomously, without the addition of an extra RNA template. In a system similar to that shown in Fig. 3, His-tagged ORF1p and HA-tagged ORF2/3' UTR were coexpressed in Sf9 cells in the baculovirus expression system (extract fraction), and the protein complex was purified by Ni-NTA agarose (eluate fraction) (Fig. 6). Using RT-PCR with three primer sets, we detected mRNAs for S. frugiperda ribosomal protein L3 gene (rPL3), ORF1 (the region of residues 1785 to 2585), and ORF2/3' UTR (the 3' UTR region of residues 6131 to 6702) in the extract fraction of Sf9 cells. However, in the eluate fraction purified by His-tagged WT ORF1p, we detected the ORF2/3' UTR mRNA (Fig. 6, II, WT) but not the ORF1p and rPL3 mRNAs (Fig. 6, rPL3 and I, WT). This result indicates that mRNA of SART1 ORF2/3' UTR but not of ORF1 is recognized by SART1 ORF1p and packaged into the ORF1p-ORF2p complex, which is consistent with the trans complemented retrotransposition between ORF1p and ORF2/3' UTR (Fig. 5C, WT) (13). It is of interest that three CCHC motif mutants of ORF1p, H626P, H649A, and H672A, lost their binding activity to SART1 ORF2/3' UTR mRNA (Fig. 6), indicating that the three CCHC motifs of ORF1 are essential for the packaging of their own mRNA into the SART1 complex. Unexpectedly, other ORF1p mutants in the first CCHC motif, C631H (CCHH), 623+A, and 623-AL (Fig. 2), packaged the ORF2/3' UTR mRNA into the protein complex (Fig. 6, II) despite the loss of retrotransposition activity (Fig. 2B). These results indicate that the three zinc fingers are involved in the packaging of SART1 RNA into the RNP complex and that H626P, H649A, and H672A mutations cause defects in RNA packaging. Since the C631H, 623+A, and 623-AL mutants can package SART1 mRNA but cannot retrotranspose, the zinc fingers may have other roles in retrotransposition, such as nucleic acid annealing shown in some retroviruses (35).
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In vivo reconstitution of the functional SART1 RNP complex. We succeeded in purifying functional SART1 RNP by coexpressing ORF1 and ORF2/3' UTR in Sf9 cells using the baculovirus system, which expresses these products effectively. The SART1 RNP purified by this system retains the TPRT activity (Fig. 3). These novel reconstitution and retrotransposition assays using the baculovirus expression system will lead to better understanding of RNP formation in non-LTR elements.
The results shown in Fig. 4 and 5 clearly demonstrate that ORF1p and ORF2p of SART1 can be reconstituted when two differently tagged constructs are expressed in the same Sf9 cells. However, when the two constructs were expressed and purified separately in the Sf9 cells and the products were mixed in vitro, we could not detect the ORF1p-ORF1p or the ORF1p-ORF2p interactions (data not shown). In addition, three CCHC motif mutants were rescued in vivo by coexpression of WT ORF1p (Fig. 2B), but the purified ORF1p could not rescue the CCHC motif mutants in vitro (Fig. 3C). Because purified H626P, H649A, and H672A complexes lack template RNAs (Fig. 6), it is not strange that they do not have TPRT activity even when the purified WT ORF1p is added (Fig. 3C). However, the purified C631H, 623+A, and 623-AL mutants, which retain the SART1 RNAs (Fig. 6), lacked the TPRT activity even when WT ORF1p was added (Fig. 3C). These data suggest that some host factors in the insect cells may be involved in the SART1 complex reconstitution. Luschnig et al. reported that when Ty1, a yeast LTR-retrotransposon was expressed in Escherichia coli, the VLP was not identical to that constituted in Saccharomyces cerevisiae, supporting the notion mentioned above (18). Cellular chaperone activities or immediate reconstitution after de novo translation in the Sf9 cells may be necessary for formation of the functional SART1 RNP. A full understanding of the involvement between non-LTR retrotransposons and host factors in RNP formation awaits future studies using cell-free biosynthesis and other experiments.
The domains involved in the protein interaction in SART1 and other retroelements. The amino acid residues 555 to 567 of ORF1p are essential for both the ORF1p-ORF1p and ORF1p-ORF2p interactions (Fig. 4 and 5) and for retrotransposition in vivo (Fig. 5C and 8A). In retroviruses, Gag proteins have a highly conserved stretch of 20 amino acids, termed the major homology region (MHR) (7), which is also observed in the yeast LTR retrotransposon, Ty3 (23). Several mutation studies have shown that MHR plays an important role in the Gag protein interaction, assembly, and maturation steps (3, 29). Recently, Rashkova et al. reported that non-LTR retrotransposons of Drosophila, HeT-A and TART, also have the MHR-like domain just before the CCHC motifs in ORF1, and colocalization analysis has shown that it is important for the ORF1p-ORF1p interactions (25). In contrast, even though we searched the whole ORF1 region, we did not find MHR-conserved amino acids in SART1. We found no sequence similarity between the protein-binding regions of SART1 ORF1 (the region of residues 555 to 567 and its surrounding area) and MHR, although their locations just before the CCHC motifs are analogous (see Fig. S4A in the supplemental material). In addition, computer simulation (http://www.cbrc.jp/papia-cgi/ssp_menuJ.pl) suggests that this SART1 protein-binding region includes two helices (residues 542 to 558 and 565 to 573) (see Fig. S4B in the supplemental material). The crystal structure of HIV-1 shows that two helices typically found in the MHR of nucleocapsid protein are critical for protein binding (7). These observations imply that, although the protein-binding domain in SART1 ORF1p has a different primary structure from MHR, it functions in a similar way to retrovirus MHR.
It is interesting that CCHC-motif mutants of SART1 did not affect the ORF1p-ORF1p and ORF1p-ORF2p interactions (Fig. 4, 5, and 8A). In retroviruses, the CCHC motifs in Gag protein are thought to contribute the interactions between the Gag protein monomers (33). The histochemical studies indicated that HeT-A Gag protein lacking CCHC motifs could not interact with proteins (25). In contrast, ORF1p of human L1, which has no CCHC motif, multimerizes to form protein complexes (15). These data suggest that not all retroelements use CCHC motifs to multimerize and also that some non-LTR elements such as SART1 and L1 can form the protein complex without the help of CCHC motifs, unlike retroviruses.
Do CCHC motifs of SART1 ORF1 work at various steps of the life cycle? We found that three CCHC mutants in which the third H was replaced lost both the ability to package RNA into RNP and the retrotransposition activity (Fig. 8A, H626P, H649A, and H672A). Based on the structural features, we speculate that these mutants lack the ability to coordinate Zn2+ appropriately. Interestingly, a CCCC mutant at motif 1 (Fig. 8A, H626C) can bind RNA and retrotranspose normally, but a CCHH mutant at motif 1 (Fig. 8A, C631H), which retains an RNA-packaging ability, lacks the retrotransposition ability. In Mo-MuLV, CCCC and CCHH mutants retaining the ability to coordinate Zn2+ can package their genomic RNA normally but are replication defective (11), which seems to parallel the RNA-binding abilities of SART1 CCCC and CCHH mutants. In HIV-1, which is more similar to SART1, the CCCC mutant packages viral RNA and retains a reduced level of infectivity, whereas the CCHH mutant packages 32% of RNA but is replication defective (10). We also found that changing the spacing of CCHC motif 1 retained the RNA-packaging ability but lost the retrotransposition activity (Fig. 8A, 623+A and 623-AL). By analogy to the retroviruses described above, mutations at CCHH, 623+A, or 623-AL of SART1 may affect some steps such as nucleic chaperone activity rather than RNA packaging activity. L1 ORF1 also has a region involved in both the RNP formation and downstream steps in the retrotransposition pathway (15). Therefore, retroelements including non-LTR retrotransposons have a domain that works in multiple steps in the life cycle to pack various functional domains in the limited space of the ORF.
RNA-binding specificity of retroelements. As shown in Fig. 6, RNA packaging into the RNP complex requires three CCHC motifs of ORF1. We detected mRNA for the ORF2/3' UTR portion only but not for ORF1 in the purified SART1 RNP. This suggests that the SART1 ORF protein specifically recognizes and interacts with mRNA of a portion of ORF2/3' UTR through the function of the CCHC motifs of ORF1p.
When we expressed SART1 without the 3' UTR and green fluorescent protein mRNA fused with the SART1 3' UTR in Sf9 cells, the green fluorescent protein mRNA retrotransposed into telomeric repeats by trans complementation between SART1 ORF proteins and SART1 3' UTR (24). Therefore, some unknown structure in SART1 ORF proteins should strictly recognize the 3' UTR of SART1 mRNA. This implies that the CCHC motifs in ORF1p may recognize the 3' UTR of SART mRNA, although we have no direct evidence at present. In contrast, several retroelements occasionally generate processed pseudogenes by reverse transcribing cellular mRNAs. For example, human L1 recognizes only the poly(A) tract at the 3' end of its mRNA and possibly misidentifies cellular poly(A) mRNAs as a target. To repress this false recognition, which is harmful for the host, human L1 is thought to employ the cis preference rather than trans complementation (34). Some reports indicate that the L1 ORF1 protein binds non-sequence-specific RNAs in vitro (14) and that the Ty1 VLP contains other cellular RNAs as well as Ty1 RNA, which results in the pseudogene production (21). It is notable that L1 and Ty1 lack the CCHC motif in their ORF1p and Gag protein. Thus, the absence of the CCHC motif in these retroelements may lead to weak recognition of the template RNA.
We do not yet know which region of SART1 mRNA in ORF2 or the 3' UTR is recognized by the CCHC motifs of SART1 ORF1. In addition, the detailed features of the ORF1p-ORF1p and ORF1p-ORF2p interactions are still obscure. Further biochemical and structural biological studies will help clarify the ORF1p-ORF1p, ORF1p-ORF2p, and ORF1p-mRNA interactions and the entire image of the RNP complex of SART1, which should lead to better understanding of the retrotransposition mechanism of non-LTR retrotransposons.
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ACKNOWLEDGMENTS |
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Supplemental material for this article may be found at http://mcb.asm.org.
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