Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama, Kanagawa 226-8501, Japan
Correspondence: E-mail: nokada{at}bio.titech.ac.jp.
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Abstract |
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Key Words: transposable element retrotransposon retrotransposition reverse transcriptase endonuclease
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Introduction |
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LINEs are 4-7 kb and comprise a 5' untranslated region (UTR), open reading frames (ORFs), and a 3' UTR. LINEs usually encode two ORFs. ORF1 encodes a nucleic acidbinding protein that can bind its own RNA (Hohjoh and Singer 1997; Kolosha and Martin 1997) and is thought to form a retrotransposition intermediate. In addition, it has been reported that this protein also possesses a nucleic acid chaperone activity (Martin and Bushman 2001), although its role in retrotransposition processes is unclear. ORF2 encodes an endonuclease (EN; Feng et al. 1996) and a reverse transcriptase (RT; Xiong and Eickbush 1990). Through phylogenetic analysis of LINE RTs, LINEs can be divided into 12 families or clades (Malik, Burke, and Eickbush 1999; Lovsin, Gubensek, and Kordis 2001; Ogiwara et al. 2002).
A biochemical analysis of the amplification mechanism of LINEs was first performed using the silkworm LINE R2Bm (Luan et al. 1993; Luan and Eickbush 1995; Yang and Eickbush 1998). By the analysis it is revealed that the LINE EN nicks a target site in the host genome, and the LINE RT initiates reverse transcription of its own RNA from the 3' hydroxyl group that is generated by the nick (Luan et al. 1993). This reaction, in which the cleavage and the reverse transcription occur simultaneously, is called target-primed reverse transcription (TPRT). Besides R2Bm, the amplification mechanism of the human L1 has also been well studied via a retrotransposition assay in mammalian cells (mainly in HeLa cells; e.g., Moran et al. 1996). L1 elements retrotranspose at high frequency in HeLa cells (Moran et al. 1996), and L1 ORF1 and ORF2 (EN and RT) are required for L1 retrotransposition (Feng et al. 1996; Moran et al. 1996). Perhaps 80100 copies of retrotransposition-competent L1 are present in the human genome (Brouha et al. 2003), and L1 can mobilize in trans and generate processed pseudogenes (Esnault, Maestre, and Heidmann 2000; Wei et al. 2001). L1 can also mobilize human SINE; Alu in trans (Dewannieux, Esnault, and Heidmann 2003). L1s, along with sequences derived from their 3' flanking regions, retrotranspose to new locations (3' transduction; Moran, DeBerardinis, and Kazazian 1999). Furthermore, a large-scale deletion or inversion can be generated during L1 retrotransposition (Gilbert, Lutz-Prigge, and Moran 2002; Symer et al. 2002). These data provide convincing evidence for detailed mechanisms of mammalian genome diversification. One intriguing discovery of L1 elements is that, except for the poly(A) tail, there is apparently no strict sequence requirement in the 3' tail for recognition by RT during retrotransposition (Moran et al. 1996).
SINEs are 100-500 bp long. They are composed of a tRNA-related region and a tRNA-unrelated region, except for the human SINE Alu, which has a 7SL RNA-related region instead of the tRNA-related region. SINEs do not encode any protein required for retrotransposition, so SINEs must recruit the enzymatic machinery for their own retrotransposition and are thus non-autonomous transposable elements. We previously reported that there are several LINE/SINE pairs that share a similar 3' tail in eukaryotic genomes (Okada et al. 1997). These pairs are distributed in several clades, such as the LINE2 (L2) clade (Smit 1996; Terai, Takahashi, and Okada 1998; Ogiwara et al. 2002), the CR1 clade (Ohshima et al. 1996; Kajikawa, Ohshima, and Okada, 1997; Ogiwara et al. 1999; Ogiwara et al. 2002), the RTE clade (Okada and Hamada 1997), and the Tad1 clade (Okada et al. 1997). Based on the TPRT reaction, the initiation of reverse transcription occurs at the 3' end of LINEs. Hence we proposed that these SINEs are mobilized by their partner LINEs (Ohshima et al. 1996; Okada et al. 1997). The RT of silkworm LINE R2Bm specifically recognizes the 3' UTR of its own RNA for the initiation of reverse transcription (Luan and Eickbush 1995; Mathews et al. 1997), although a partner SINE that shares the same 3' tail with R2Bm has not been discovered. We therefore proposed that LINEs can be divided in two groups, the stringent type and the relaxed type, based on 3' tail recognition (Okada et al. 1997). The stringent type strictly recognizes its own 3' tail for retrotransposition, whereas the relaxed type does not require a specific sequence at the 3' tail, except for the poly(A), for retrotransposition. At present, human L1 is the only example of the relaxed type, so it is thought that the majority of LINEs are of the stringent type.
Here we use the retrotransposition assay in a cultured cell line to analyze the amplification mechanism of LINEs (and SINEs) of the stringent type. We previously demonstrated that UnaL2, a stringent LINE from the eel, can retrotranspose in HeLa cells and that UnaSINE1, which has a similar 3' tail, can retrotranspose using the enzymatic machinery of UnaL2 (Kajikawa and Okada 2002). We have now isolated a second SINE from the eel genome, UnaSINE2, and report here the further characterization of the eel LINE and SINEs.
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Materials and Methods |
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Estimation of the Copy Number of UnaL2 and UnaSINEs
For dot blot analyses, progressively decreasing amounts of genomic DNA from A. japonica and cloned DNA were dotted on a membrane. Polymerase chain reaction (PCR) products of 150 bp that corresponded to sequences from UnaL2, UnaSINE1, or UnaSINE2 were labeled by BcaBEST DNA polymerase (TaKaRa) using [
-32P]dCTP. Hybridization was performed at 42°C in 50% formamide. Washing was performed in 2x SSC and 1% sodium dodecyl sulfate (SDS) at 55°C for 30 min. From comparisons of the intensities of spots obtained with the genomic DNA and the cloned DNA, we estimated the element copy number. The haploid genome of the eel was assumed to contain 1 x 109 bp.
Retrotransposition Assay
Plasmids used for the cis and trans retrotransposition assay (Kajikawa and Okada 2002) were purified by the QIAfilter Plasmid Midi kit (Qiagen) according to the manufacturer's instructions. Complete experimental procedures are available on request.
HeLa cells were grown in high-glucose Dulbecco's modified Eagle's medium (Invitrogen) in the absence of pyruvate and supplemented with 2 mM L-glutamine and 10% fetal bovine serum. All cell cultures were maintained at 37°C in a humidified atmosphere of 5% CO2. For the assay, HeLa cells (2 x 105 cells/well) were seeded in six-well plates and transfected with 1 µg plasmid DNA with 3 µl of FuGENE6 reagent (Roche) according to the manufacturer's instructions. For co-transfection, we used 6 µl of FuGENE6 reagent with 1 µg of each plasmid DNA (2 µg DNA total). After transfection, HygR cells were selected with 200 µg/ml hygromycin. In co-transfections, the selection was performed with 200 µg/ml hygromycin and 15 mM L-histidinol. By comparing the data with cell survival results from negative controls, we estimated that 95% of transfected cells became antibiotic resistant (HygR cells or HygR and HisR cells). The antibiotic-resistant cells were then trypsinized and seeded to a density of
2 x 105 to 4 x 106 cells per 100-mm plate and grown in medium containing 400 µg/ml G-418. After G-418 selection, plates were fixed with 100% ethanol and stained with 2% Giemsa's solution. G-418R colonies were counted, and the retrotransposition frequencies were calculated as the number of G-418R colonies per one plated cell with HygR, or one plated cell with HygR and HisR.
Accession Numbers
The UnaL2 nucleotide sequence of the clone Aja 6-15 has been submitted to the DNA Databank of Japan (DDBJ) and assigned the accession number AB179624. Nucleotide sequences of seven UnaSINE1s and four UnaSINE2s have been submitted to DDBJ and assigned accession numbers AB179625, AB179626, AB179627, AB179628, AB179629, AB179630, and AB179631 for the UnaSINE1s; accession numbers AB179632, AB179633, AB179634, and AB179635 for the UnaSINE2s.
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Results and Discussion |
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Characterization of the 5' UTR of UnaL2
The 5' UTR of UnaL2 is 400 bp and includes a series of 39-bp repeats (fig. 1B, underlined sequence) that are separated by an internal sequence of 24 bp. In some UnaL2s, this 63-bp unit (dashed underline) is also repeated in tandem, with the number of repeats varying in different UnaL2s (one to several units). In comparison, the 5' UTR of ZfL2 is
1.1 kb, most of which is composed of a tandem repeat of an
300-bp unit (Kapitonov 2002). Repeat sequences occur in 5' UTRs of other LINEs, but the length and sequence of the repeats vary (Kajikawa, Ohshima, and Okada 1997; for a review on mouse L1s, see Moran and Gilbert 2002). It is thought that LINEs have an internal promoter in the 5' UTR, so the repeat sequences may be important for promoter activity. In the case of the mouse L1 LINE subfamily, TF, the 5' UTR contains a tandem repeat of a monomer unit (
200 bp), and the promoter activity of mouse L1 is proportional to the number of monomers (DeBerardinis and Kazazian 1999). Hence, the repeat sequences of UnaL2 might also have promoter activity.
Characterization of the 3' UTR of UnaL2
The 3' UTR of UnaL2 is 330 bp, and although it is not well conserved with the 230-bp ZfL2 3' UTR, their 3'-terminal regions are well conserved (fig. 1C). This suggests that the 3' tails of UnaL2 and ZfL2 are important for retrotransposition. The conserved tail is composed of two parts, the stem-loop region and the terminal repeat region (figs. 1C and D; Kajikawa and Okada 2002). Both regions of UnaL2 are required for retrotransposition (Kajikawa and Okada 2002). The stem-loop region is thought to function as a recognition site for the UnaL2 protein (UnaL2p) when this region is transcribed in the RNA (Baba et al. 2004). The terminal repeat region of UnaL2 is probably required for the slippage reaction during reverse transcription initiation (Kajikawa and Okada 2002). These data indicate that UnaL2 and ZfL2 belong to the stringent type of LINEs. The 3' tail of UnaL2 contains a putative poly(A) signal (fig. 1C, underline), whereas ZfL2 does not. Thus, the poly(A) signal in these LINEs may be dispensable for retrotransposition, and the poly(A) signal of the host genome located downstream of the LINE might lead to poly(A) addition to the RNA of these LINEs.
Implication from Structural Modeling of UnaL2
The amino acid sequence of the N-terminal region (1250) of UnaL2p shows similarity to members of the apurine/apyrimidine (AP) endonuclease family (figs. 1A and 2A), most of which possess an activity specific to AP sites in duplex DNA. The co-crystal structure of the AP endonuclease of hHAP1 with substrate DNA has revealed that AP site recognition uses two protruding elements (AP-pinch1 and AP-pinch2; figs. 2A and 2B), which together pinch the AP site duplex from both sides of the helix (Mol et al. 2000). Moreover, Phe-266, Trp-280, and Leu-282 (fig. 2A, triangles) comprise a pocket for binding of the "flipped-out" abasic deoxyribose. A sequence alignment based on 3D1D profiles made by the program 3D-PSSM (Fischer et al. 1999) suggests that UnaL2p lacks these elements and residues. Using this alignment, we modeled a 3D structure of the UnaL2p endonuclease domain using the program MODELLER (Sali and Blundell 1993; fig. 2B). Although the structure of the endonuclease core is very similar to that of hHAP1, the modeled structure does not possess the AP-pinch regions. Moreover, UnaL2p lacks the pocket that packs the flipped-out abasic deoxyribose. Weichenrieder, Repanas, and Perrakis (2004) recently resolved the structure of the L1Hs endonuclease and suggested that Phe-193, Ser-202, and Ile-204 (which correspond to Phe-266, Trp-280, and Leu-282 of hHAP1) accommodate an extrahelical adenosine next to the scissile bond. Because its substrate (5'TTTT-AA3', hyphen indicates cleavage) is unusual, as the adenosine next to the scissile bond tends to be flipped out, it is still pending if LINE endonucleases generally recognize an extrahelical nucleotide. The lack of a corresponding pocket in UnaL2p (and Tad endonuclease) argues against the generality of the extrahelical nucleotide recognition. In addition, the sequence alignment suggests the absence of the AP-pinch regions in many LINE endonucleases, which may explain the fact that they are not specific to AP sites.
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The Stem-Loop of the 3' Tail is Conserved Among LINEs and SINEs of the L2 Clade
The 3' tails of UnaL2 and UnaSINEs are conserved and required for retrotransposition (figs. 4A and 5). The RNA transcribed from the conserved tail is proposed to form a stem-loop (Fig. 1D; Kajikawa and Okada 2002). Most LINEs (and SINEs) belonging to the L2 clade also have a 3' tail that purportedly forms a stem-loop similar to that of UnaL2. Alignment of the 3' tails from the L2 clade LINEs and SINEs is shown in figure 6. All of the L2 clade LINEs end with a repeat sequence, although the nucleotide sequence of the repeats differs among the members. The stem region (underlined) is well conserved, whereas the loop region varies in length and sequence. The sequences that are 5' and 3' to the stem are also well conserved (dashed underlines). This conservation suggests that LINE proteins may use a common mechanism for recognizing the 3' tails of LINE RNAs. The retrotransposition machinery of UnaL2 can recognize the 3' tail of UnaSINE1 and UnaSINE2 as well as its own 3' tail, but it cannot recognize those of CiLINE2 and Af1SINE (Kajikawa and Okada 2002). These tails differ substantially in the loop region. Thus, we speculate that the stem is used as a common binding site for LINE proteins, and the loop region is used for LINE-specific recognition (Baba et al. 2004).
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Conclusions |
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Acknowledgements |
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Footnotes |
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References |
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