Leptin Receptor Isoform 219.1: An Example of Protein Evolution by LINE-1–Mediated Human-Specific Retrotransposition of a Coding SVA Element
http://www.100md.com
分子生物学进展 2004年第4期
Paul-Ehrlich-Institut, Langen, Germany
E-mail: loero@pei.de.
Abstract
Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.
Key Words: SVA ? SINE-R ? leptin receptor ? retrotransposition ? HERV-K ? L1
Introduction
Retrotransposons contribute to expansion, flexibility, and evolution of genomes. Ono, Kawakami, and Takezawa (1987) reported a new member of the family of short interspersed nuclear elements (SINE) in humans and named it SINE-R. More recently, the term SVA (SINE-VNTR-Alu) (Shen et al. 1994) was introduced to describe these elements. SVAs are composite retroposons consisting of several degraded copies of Alus, a GC-rich 49-bp unit tandem repeat and a 3' part identical to the 3' part of transcripts of the human/primate endogenous retrovirus HERV-K. Notably, the HERV-K homologous part features an extensive deletion that leads to the destruction of the Rec responsive element (RcRE) located within the U3 region of HERV-K transcripts. This deletion prevents binding of the HERV-K–encoded RNA export adaptor Rec, which mediates nuclear export of unspliced and incompletely spliced HERV-K transcripts (Magin, L?wer, and L?wer 1999; Magin-Lachmann et al. 2001). Prevention of export of unspliced primary transcripts of cellular genes is vital to avoid deleterious effects on cell metabolism.
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/SVA–type 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.
Materials and Methods
Database Analysis
Localization of the leptin receptor alternative exons 20 on human genomic sequence was performed using the Blast program at (www.ncbi.nlm.nih.gov). RepeatMasker analysis was performed using the University of Washington ftp server at (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker). Descriptions of specific repetitive elements (Hal-1 and SINE-R/SVA) were obtained from Repbase Update (Repbase release 7.2).
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 [L?wer 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-1–specific 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-1–specific probe, the 3' part of the Hal-1 element extending from the XbaI site was used.
Results and Discussion
With the availability of sequences derived from the human genome project, mapping of the alternative C-terminal exons of the leptin receptor was possible simply by database analysis. We could localize the 3' part of an SVA element in intron 19 of the human leptin receptor gene (GenBank accession number NT_004636, nt 228197 to nt 228430), constituting the alternative exon 20 of the leptin receptor 219.1 isoform (figs. 1A and 2). The splice acceptor signal and the 219.1 exon 20 coding sequence are derived from the HERV-K homologous part of the SVA only. Figure 1B illustrates the region of homology to the SVA (SINE-R11 [Ono, Kawakami, and Takezawa 1987]) present on chromosome 9. A 5' truncation is clearly evident. RepeatMasker analysis (Jurka 2000) indicated that the leptin receptor SVA sequence was inserted into a Hal-1/LINE (Repbase Update [Repbase release 7.2]) sequence (GenBank accession number NT_004636, nt 228012 to nt 229098). The presence of a contiguous 5' truncated SVA sequence in intron 19 of the human LEPR locus disproves the hypothesis published by Kapitonov and Jurka (1999), who postulated the presence of a solitary HERV-K LTR in this region and reported that the 219.1 exon 20 was the result of a double splicing event within the LTR. The fact that the 219.1 exon 20 shows up to 99% homology to SVAs annotated on chromosomes 3, 4, and 7, but at maximum 94% and 88% (5' and 3' of the RcRE, respectively) homology to HERV-K LTRs, strongly supports the notion that insertion of the SVA into the leptin receptor locus is the result of SVA and not LTR or HERV-K retrotransposition.
FIG. 1. Structure and organization of the SVA insertion into the LEPR locus. (A) Sequence comparison of the LEPR loci of humans and nonhuman primates. Only the relevant region of intron 19 is shown. Hal-1 homologous sequences are in boldface or italicized (SVA microhomology). A putative target-site duplication (AAA) is underlined. The SVA insertion is only present in human DNA. (B) Alignment of the LEPR SVA to the SVA (SINE-R11 [Ono, Kawakami, and Takezawa 1987]) present on chromosome 9 (GenBank accession numbers ac 006952 and nt 175026 to nt 176644). The first codon (ATG) and stop codon (TGA) of the LEPR exon 20, as well as the polyadenylation signal and differences to the SVA sequence on chromosome 9, are boldface and underlined. The proposed microhomology to the primate LEPR insertion site is boldface and italicized. TSD = target site duplication
FIG. 2. Southern blot analysis of the leptin receptor intron 19 in different primate species. Genomic fragments were amplified using primers located in exons 19 and 20 (long isoform of LEPR), digested with Xba I, separated, and blotted. Blots were hybridized with a SVA-specific probe (left panel) and subsequently with a Hal-1–specific (right panel) probe. A schematic representation of the human leptin receptor intron 19 is given on top. Locations of probes, restriction sites and the Hal-1 element are indicated. Alternative exons (219.1, 219.2, and 219.3) are named according to Cioffi et al. (1996)
Because SINE-Rs/SVAs are found in all hominoid primates (Kim, Takenaka, and Crow 1999), we next wanted to investigate whether insertion of the sequence used as LEPR alternative exon 20 is human specific. Genomic DNA of chimpanzee, orangutan, and gorilla were analyzed for the presence of the SVA and surrounding Hal-1 sequences. The respective regions were amplified using primers flanking the human Hal-1 element. Cloning and sequencing of the PCR fragments revealed absence of the SVA in nonhuman primates, whereas the Hal-1 element was present in all three species investigated. Sequences of the Hal-1 elements are more than 90% identical in all fragments tested (fig. 1A).
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 endonuclease–dependent "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-1–specific 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 retrovirus–derived 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.
Fig. 1. (Continued)
Acknowledgements
We thank R.R. T?njes for the gift of chimpanzee DNA, C. Coulibaly and R. Plesker from the central animal facility of the Paul-Ehrlich-Institute for providing whole blood of different primates, and U. Held for technical assistance. We are especially grateful to G. Schumann for helpful and stimulating discussions.
Literature Cited
Bennett, B. D., G. P. Solar, J. Q. Yuan, J. Mathias, G. R. Thomas, and W. Matthews. 1996. A role for leptin and its cognate receptor in hematopoiesis. Curr. Biol. 6:1170-1180.
Cioffi, J. A., A. W. Shafer, T. J. Zupancic, J. Smith-Gbur, A. Mikhail, D. Platika, and H. R. Snodgrass. 1996. Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nat. Med. 2:585-589.
Dewannieux, M., C. Esnault, and T. Heidmann. 2003. LINE-mediated retrotransposition of marked Alu sequences. Nat. Genet. 35:41-48.
Esnault, C., J. Maestre, and T. Heidmann. 2000. Human LINE retrotransposons generate processed pseudogenes. Nature Genet. 24:363-367.
Gilbert, N., S. Lutz-Prigge, and J. V. Moran. 2002. Genomic deletions created upon LINE-1 retrotransposition. Cell 110:315-325.
Jurka, J. 2000. Repbase update: a database and an electronic journal of repetitive elements. Trends Genet. 16:418-420.
Kajikawa, M., and N. Okada. 2002. LINEs mobilize SINEs in the eel through a shared 3' sequence. Cell 111:433-444.
Kapitonov, V. V., and J. Jurka. 1999. The long terminal repeat of an endogenous retrovirus induces alternative splicing and encodes an additional carboxy-terminal sequence in the human leptin receptor. J. Mol. Evol. 48:248-251.
Kim, H. S., and O. Takenaka. 2001. Phylogeny of SINE-R retroposons in Asian apes. Mol. Cells 12:262-266.
Kim, H. S., O. Takenaka, and T. J. Crow. 1999. Cloning and nucleotide sequence of retroposons specific to hominoid primates derived from an endogenous retrovirus (HERV-K). AIDS Res. Hum. Retroviruses 15:595-601.
L?wer, R., J. L?wer, H. Frank, R. Harzmann, and R. Kurth. 1984. Human teratocarcinomas cultured in vitro produce unique retrovirus-like viruses. J. Gen. Virol. 65:887-898.
Magin, C., R. L?wer, and J. L?wer. 1999. cORF and RcRE, the Rev/Rex and RRE/RxRE homologues of the human endogenous retrovirus family HTDV/HERV-K. J. Virol. 73:9496-9507.
Magin-Lachmann, C., S. Hahn, H. Strobel, U. Held, J. L?wer, and R. L?wer. 2001. Rec (formerly Corf) function requires interaction with a complex, folded RNA structure within its responsive element rather than binding to a discrete specific binding site. J. Virol. 75:10359-10371.
Moran, J. V., R. J. De Berardinis, and H. H. Kazazian, Jr. 1999. Exon shuffling by L1 retrotransposition. Science. 283:1530-1534.
Morrish, T. A., N. Gilbert, J. S. Myers, B. J. Vincent, T. D. Stamato, G. E. Taccioli, M. A. Batzer, and J. V. Moran. 2002. DNA repair mediated by endonuclease-independent LINE-1 retrotransposition. Nat. Genet. 31:159-165.
Ono, M., M. Kawakami, and T. Takezawa. 1987. A novel human nonviral retroposon derived from an endogenous retrovirus. Nucleic Acids Res. 15:8725-8737.
Ostertag, E. M., and H. H. Kazazian, Jr. 2001. Biology of mammalian L1 retrotransposons. Annu. Rev. Genet. 35:501-538.
Shen, L., L. C. Wu, S. Sanlioglu, R. Chen, A. R. Mendoza, A. W. Dangel, M. C. Carroll, W. B. Zipf, and C. Y. Yu. 1994. Structure and genetics of the partially duplicated gene RP located immediately upstream of the complement C4A and the C4B genes in the HLA class III region: molecular cloning, exon-intron structure, composite retroposon, and breakpoint of gene duplication. J. Biol. Chem. 269:8466-8476.
Symer, D. E., C. Connelly, S. T. Szak, E. M. Caputo, G. J. Cost, G. Parmigiani, and J. D. Boeke. 2002. Human l1 retrotransposition is associated with genetic instability in vivo. Cell 110:327-338.
Zhu, Z. B., S. L. Hsieh, D. R. Bentley, R. D. Campbell, and J. E. Volanakis. 1992. A variable number of tandem repeats locus within the human complement C2 gene is associated with a retroposon derived from a human endogenous retrovirus. J. Exp. Med. 175:1783-1787.(Annette Damert, Johannes )
E-mail: loero@pei.de.
Abstract
Phylogenetically new insertions of repetitive sequences may contribute to genome evolution by altering the function of preexisting proteins. One example is the SVA sequence, which forms the C-terminal coding exon of the human leptin receptor isoform 219.1. Here, we report that the SVA insertion into the LEPR locus has occurred after divergence of humans and chimpanzees. The SVA element was inserted into a Hal-1/LINE element present in all monkeys and apes tested. Structural features point toward an integration event that was mediated by the L1 protein machinery acting in trans. Thus, our findings add evidence to the hypothesis that retrotransposition events are a driving force in genomic evolution and that the presence or absence of specific retroelements are one distinguishing feature that separates humans from chimpanzees.
Key Words: SVA ? SINE-R ? leptin receptor ? retrotransposition ? HERV-K ? L1
Introduction
Retrotransposons contribute to expansion, flexibility, and evolution of genomes. Ono, Kawakami, and Takezawa (1987) reported a new member of the family of short interspersed nuclear elements (SINE) in humans and named it SINE-R. More recently, the term SVA (SINE-VNTR-Alu) (Shen et al. 1994) was introduced to describe these elements. SVAs are composite retroposons consisting of several degraded copies of Alus, a GC-rich 49-bp unit tandem repeat and a 3' part identical to the 3' part of transcripts of the human/primate endogenous retrovirus HERV-K. Notably, the HERV-K homologous part features an extensive deletion that leads to the destruction of the Rec responsive element (RcRE) located within the U3 region of HERV-K transcripts. This deletion prevents binding of the HERV-K–encoded RNA export adaptor Rec, which mediates nuclear export of unspliced and incompletely spliced HERV-K transcripts (Magin, L?wer, and L?wer 1999; Magin-Lachmann et al. 2001). Prevention of export of unspliced primary transcripts of cellular genes is vital to avoid deleterious effects on cell metabolism.
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/SVA–type 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.
Materials and Methods
Database Analysis
Localization of the leptin receptor alternative exons 20 on human genomic sequence was performed using the Blast program at (www.ncbi.nlm.nih.gov). RepeatMasker analysis was performed using the University of Washington ftp server at (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker). Descriptions of specific repetitive elements (Hal-1 and SINE-R/SVA) were obtained from Repbase Update (Repbase release 7.2).
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 [L?wer 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-1–specific 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-1–specific probe, the 3' part of the Hal-1 element extending from the XbaI site was used.
Results and Discussion
With the availability of sequences derived from the human genome project, mapping of the alternative C-terminal exons of the leptin receptor was possible simply by database analysis. We could localize the 3' part of an SVA element in intron 19 of the human leptin receptor gene (GenBank accession number NT_004636, nt 228197 to nt 228430), constituting the alternative exon 20 of the leptin receptor 219.1 isoform (figs. 1A and 2). The splice acceptor signal and the 219.1 exon 20 coding sequence are derived from the HERV-K homologous part of the SVA only. Figure 1B illustrates the region of homology to the SVA (SINE-R11 [Ono, Kawakami, and Takezawa 1987]) present on chromosome 9. A 5' truncation is clearly evident. RepeatMasker analysis (Jurka 2000) indicated that the leptin receptor SVA sequence was inserted into a Hal-1/LINE (Repbase Update [Repbase release 7.2]) sequence (GenBank accession number NT_004636, nt 228012 to nt 229098). The presence of a contiguous 5' truncated SVA sequence in intron 19 of the human LEPR locus disproves the hypothesis published by Kapitonov and Jurka (1999), who postulated the presence of a solitary HERV-K LTR in this region and reported that the 219.1 exon 20 was the result of a double splicing event within the LTR. The fact that the 219.1 exon 20 shows up to 99% homology to SVAs annotated on chromosomes 3, 4, and 7, but at maximum 94% and 88% (5' and 3' of the RcRE, respectively) homology to HERV-K LTRs, strongly supports the notion that insertion of the SVA into the leptin receptor locus is the result of SVA and not LTR or HERV-K retrotransposition.
FIG. 1. Structure and organization of the SVA insertion into the LEPR locus. (A) Sequence comparison of the LEPR loci of humans and nonhuman primates. Only the relevant region of intron 19 is shown. Hal-1 homologous sequences are in boldface or italicized (SVA microhomology). A putative target-site duplication (AAA) is underlined. The SVA insertion is only present in human DNA. (B) Alignment of the LEPR SVA to the SVA (SINE-R11 [Ono, Kawakami, and Takezawa 1987]) present on chromosome 9 (GenBank accession numbers ac 006952 and nt 175026 to nt 176644). The first codon (ATG) and stop codon (TGA) of the LEPR exon 20, as well as the polyadenylation signal and differences to the SVA sequence on chromosome 9, are boldface and underlined. The proposed microhomology to the primate LEPR insertion site is boldface and italicized. TSD = target site duplication
FIG. 2. Southern blot analysis of the leptin receptor intron 19 in different primate species. Genomic fragments were amplified using primers located in exons 19 and 20 (long isoform of LEPR), digested with Xba I, separated, and blotted. Blots were hybridized with a SVA-specific probe (left panel) and subsequently with a Hal-1–specific (right panel) probe. A schematic representation of the human leptin receptor intron 19 is given on top. Locations of probes, restriction sites and the Hal-1 element are indicated. Alternative exons (219.1, 219.2, and 219.3) are named according to Cioffi et al. (1996)
Because SINE-Rs/SVAs are found in all hominoid primates (Kim, Takenaka, and Crow 1999), we next wanted to investigate whether insertion of the sequence used as LEPR alternative exon 20 is human specific. Genomic DNA of chimpanzee, orangutan, and gorilla were analyzed for the presence of the SVA and surrounding Hal-1 sequences. The respective regions were amplified using primers flanking the human Hal-1 element. Cloning and sequencing of the PCR fragments revealed absence of the SVA in nonhuman primates, whereas the Hal-1 element was present in all three species investigated. Sequences of the Hal-1 elements are more than 90% identical in all fragments tested (fig. 1A).
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 endonuclease–dependent "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-1–specific 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 retrovirus–derived 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.
Fig. 1. (Continued)
Acknowledgements
We thank R.R. T?njes for the gift of chimpanzee DNA, C. Coulibaly and R. Plesker from the central animal facility of the Paul-Ehrlich-Institute for providing whole blood of different primates, and U. Held for technical assistance. We are especially grateful to G. Schumann for helpful and stimulating discussions.
Literature Cited
Bennett, B. D., G. P. Solar, J. Q. Yuan, J. Mathias, G. R. Thomas, and W. Matthews. 1996. A role for leptin and its cognate receptor in hematopoiesis. Curr. Biol. 6:1170-1180.
Cioffi, J. A., A. W. Shafer, T. J. Zupancic, J. Smith-Gbur, A. Mikhail, D. Platika, and H. R. Snodgrass. 1996. Novel B219/OB receptor isoforms: possible role of leptin in hematopoiesis and reproduction. Nat. Med. 2:585-589.
Dewannieux, M., C. Esnault, and T. Heidmann. 2003. LINE-mediated retrotransposition of marked Alu sequences. Nat. Genet. 35:41-48.
Esnault, C., J. Maestre, and T. Heidmann. 2000. Human LINE retrotransposons generate processed pseudogenes. Nature Genet. 24:363-367.
Gilbert, N., S. Lutz-Prigge, and J. V. Moran. 2002. Genomic deletions created upon LINE-1 retrotransposition. Cell 110:315-325.
Jurka, J. 2000. Repbase update: a database and an electronic journal of repetitive elements. Trends Genet. 16:418-420.
Kajikawa, M., and N. Okada. 2002. LINEs mobilize SINEs in the eel through a shared 3' sequence. Cell 111:433-444.
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