Paul-Ehrlich-Institut, Langen, Germany
Correspondence: E-mail: loero{at}pei.de.
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Abstract |
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Key Words: SVA SINE-R leptin receptor retrotransposition HERV-K L1
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Introduction |
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Ono, Kawakami, and Takezawa (1987) estimated the copy number of the SINE-R/SVAs to be about 4,500 per haploid human genome. In the following years, Kim and coworkers performed detailed phylogenetic analyses of the SINE-R/SVA family of retroposons in African and Asian apes, as well as on different human chromosomes (Kim and Takenaka [2001] and references therein). They showed that SINE-R/SVAtype retroposons are present only in hominoid primates (Kim, Takenaka, and Crow 1999). SVAs show characteristics such as a poly A tail in close proximity to a polyadenylation signal and target site duplications and can thus be considered to represent processed pseudogenes. Esnault and coworkers directly showed that processed pseudogenes are retrotransposed by the L1 machinery (Esnault, Maestre, and Heidmann 2000).
To date, insertions of SVA/ SINE-R elements into genes have been described only in few cases. They occur in the human complement C2 gene, where the repetitive element is part of an intronic polymorphic region (Zhu et al. 1992), in the RP1 (STK19) gene, which is in close proximity to the complement C4 gene (Shen et al. 1994), and in the human leptin receptor (LEPR) locus (Bennett et al. 1996; Cioffi et al. 1996).
In humans, there are four splice isoforms of the leptin receptor. They differ by the last exon (exon 20 in humans), which codes for the cytoplasmic tail of the receptor. One of these alternatively spliced C-terminal exons (219.1) was found to be homologous to the HERV-K derived part of the SINE-R/SVA family of primate retroposons (Cioffi et al. 1996). To date, the 219.1 isoform is the only known case in which an SVA element has coding capability. Expression of the isoform on RNA level has been demonstrated in fetal liver and CD34+ hematopoietic stem cells (Bennett et al. 1996; Cioffi et al. 1996), whereas data on protein expression and function are still lacking. As a first step in investigating SVA elements as protein-coding domains, we set out to trace the evolutionary origin of this specific SVA sequence. Towards this aim, we analyzed the leptin receptor locus in humans and nonhuman primates and found the insertion to be human specific.
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Materials and Methods |
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Amplification, Cloning, and Analysis of Genomic Fragments
Genomic DNA was prepared from either whole blood (lower primates and chimpanzees), established lymphoblast cell lines (orangutan and gorilla: European Collection of Animal Cell Cultures, numbers 89072703 and 89072705), or a teratocarcinoma cell line (GH [Löwer et al. 1984]) using the Qiagen kit according to the manufacturer's instructions. Fragments encompassing the SVA sequence and Hal-1 element were PCR amplified using the oligonucleotides Hal-1 FW 5'-AGCCATCACTCTAAACTTTCCC-3' and Hal-1 REV 5'-TCATCTGGCCAGAACCCTGC-3' (annealing temperature 60°C). The resulting fragments were gel purified and cloned into pGEM-T easy (Promega) according to the manufacturer's instructions. Sequencing was done using dye-labeled dideoxynucleotides. Long-range PCR for amplification of intron 19 was performed using the Expand Long Template PCR System (Roche) and the primers Exon 19 UP2 5'-GGAAGATGTTCCGAACCCCAAGAATTG-3' and Intron19/20B 5'-GTATGCTTGATAAAAAGATGCTCAAACGTTTCTGG-3' according to the manufacturer's instructions. Resulting fragments were digested with XbaI, separated on a 0.8% agarose gel, blotted onto Hybond N+ (Amersham), and hybridized with SVA and Hal-1specific probes. The SVA probe was obtained by amplification of the SVA sequence on the human Hal-1 element (primers used were SINE-R FW 5'-ATGCTTGAAGGCAGCATG-3' and SINE-R REV 5'-TCATTCTTGGGTGTTTCT-3', annealing temperature 50°C) and subcloning into pGEM-T easy. As Hal-1specific probe, the 3' part of the Hal-1 element extending from the XbaI site was used.
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Results and Discussion |
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Insertion of the SVA occured into a T/A-rich region of the Hal-1 element. Sequence analysis revealed that the insertion displays characteristic features of a LINE (L1)mediated retrotransposition event (Ostertag and Kazazian 2001): a specific endonuclease cleavage site (3'-AA/TTTT-5'), presence of a poly A tail at the 3' end of the insertion immediately downstream of a polyadenylation signal, incomplete reverse transcription leading to substantial 5' truncation of the retrotransposed element, and a short (AAA [underlined in figure 1A]) target-site duplication flanking the integrated sequence. These features strongly suggest the involvement of the L1 protein machinery in retrotransposition of this SVA sequence. RepeatMasker analysis (Jurka 2000) also showed that sequence homology to SVA retroposons extends into the region 5' of the insertion (italicized in figure 1A), indicating that the second strand synthesis might have been initiated by the presence of microhomologies at the site of insertion, as has been proposed by Symer et al. (2002). Meeting the specific requirements of L1 mediated retrotransposition, however, does not completely exclude less-specific integration events such as an L1 endonucleasedependent "blunt insertion" (Gilbert, Lutz-Prigge, and Moran 2002) or an endonuclease-independent mechanism (Morrish et al. 2002). Although retrotransposition of SINEs has been assumed to be mediated by the L1 machinery for a couple of years, only recently experimental proof has been obtained on LINE-mediated SINE transposition. Kajikawa and Okada (2002) described the mobilization in the eel genome of a tRNA-derived SINE by LINEs through a shared 3' sequence. Evidence for LINE-mediated retrotransposition of Alus has been provided by Dewannieux, Esnault, and Heidmann (2003).
To determine when the insertion of the Hal-1 element has occurred in evolution, long-range PCR of the leptin receptor intron 19 was performed on a number of primate DNAs. Subsequent Southern blotting with a Hal-1specific probe revealed that the Hal-1 element was present in all primates tested (figure 2, lower right panel). As expected, only the amplification product from human genomic DNA hybridized with the SVA-specific probe (figure 2, lower left panel).
In conclusion, we can state that retrotransposition of the SVA sequence into the leptin receptor locus is human specific and has occurred in a Hal-1 element inserted earlier in evolution. This insertion of a repetitive endogenous retrovirusderived element into a cellular gene resulted in a newly acquired protein-coding sequence, with possible consequences for its function in leptin-induced signal transduction. Thus, retrotransposition events mediated by L1 acting in trans might have contributed to protein evolution not only by exon shuffling (Moran, De Berardinis, and Kazazian 1999) but also by combining endogenous retroviral sequences with coding sequences of the host.
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Acknowledgements |
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Footnotes |
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Literature Cited |
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