Dysfunction of the Orleans reeler gene arising from exon skipping due to transposition of a full-length copy of an active L1 sequence into the skipped exon
Dysfunction of the Orleans reeler gene arising from exon skipping due to transposition of a full-length copy of an active L1 sequence into the skipped exonTokuei Takahara1,2, Tomoya Ohsumi1, Junro Kuromitsu1, Kazuhiro Shibata1, Nobuya Sasaki1, Yasushi Okazaki1, Hideo Shibata1, Shigeo Sato1, Atsushi Yoshiki1, Moriaki Kusakabe1, Masami Muramatsu1, Minoru Ueki2, Kiyoji Okuda2 and Yoshihide Hayashizaki1,*
1Genome Science Laboratory and Division of Experimental Animal Research, RIKEN Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan and 2Department of Obstetrics and Gynecology, Osaka Medical College, 2-7 Daigaku-chou, Takatsuki, Osaka 569, Japan
Received February 22, 1996;Revised and Accepted April 11, 1996GenBank accession no. D84391
We examined the genomic structure of the reeler gene in Orleans reelermouse mutant. Exon skipping of the reeler gene caused a 220 bp deletion in the transcript, resulting in a frame shift of the reeler gene which disrupts the 8th EGF-like motif of the reeler product. Surprisingly, the skipped exon was inserted by the 7104 bp L1 element which carried the full-length stretch of the mouse L1 sequence, consisting of a 212 bp F-type tandem repeat, open reading frame 1 (ORF1), ORF2, the polyadenylation signal and a poly A stretch. The transposed L1 sequence was flanked by 13 bp of the target sequence at both ends. ORF1 and ORF2 of this L1 repeat element are thought to encode a component of the RNP particle and the reverse transcriptase, respectively. Orleans reelerwas originally established by spontaneous mutation caused by L1 insertion, and this L1 sequence is considered to be potentially active for transposition in mouse genome.
The reeler mouse mutant shows typical neurological symptoms, such as ataxia and tremors, arising from brain malformations caused by defective neuroblast migration during neuronal development (1 ,2 ). Our group (3 ,4 ) and D'Arcangelo, G. et al. (5 ) have identified the cDNA responsible for the reeler phenotype using RLGS spot markers and SSLP markers. This gene can clearly account for the reeler phenotype, which is considered to be an extracellular matrix protein carrying eight EGF-like motifs which is expressed by the pioneer neurons (3 ,5 ). Two independent mutant alleles, Jackson reeler (rlJ)and Orleans reeler (rlOrl),were used in these studies. The 150 kb genomic deletion was found in rlJ (6 ) whereas the rlOrlallele was transcribed to produce mRNA carrying a 220 bp deletion (nucleotides 3314-3533 of GenBank accession number D63520, nucleotides 10049-10268 of No. U24703) (3 ,7 ), resulting in a frame shift which disrupted the 8th EGF-like motif. In the present study, we analyzed the reeler Orleans mutant allele. Interestingly, the 220 bp deletion is due to exon skipping caused by insertion of the full-length LINE 1 (L1) element which is considered to be active for transposition in the mouse genome. Up to 100 000 L1 copies are distributed in the mouse and human genome and the most 5' upstream region of these L1 elements is generally truncated (8 ). This is the first report to describe the full-length L1 sequence with two intact open reading frames (ORFs) that itself is actually retrotransposed in vivo, maintaining the intact L1 structure.
In order to determine whether or not the deleted 220 bp fragment on the rlOrl cDNA corresponds to a single exon, we performed PCR amplification of genomic DNA using the primer sets DF1 and DR1 which are positioned at both sites of the 220 bp fragment (Fig. 1 a, 3 a and 3 b). The 212 bp fragment can be expected if the deleted region corresponds to a single exon. As shown in Figure 1 a (lane 1), a signal of the expected size appeared on wild-type genomic DNA, indicating that the 212 bp is located in a single exon. On the other hand, no DNA fragments were amplified from rlOrl genomic DNA using the same primers (lane 2 in Fig. 1 a), indicating the presence of an abnormal DNA structure on this exon sequence. As the next step, to examine the exon-intron border flanking the exon, two primer sets, DIF1-DIR1 and DIF2-DIR2, were designed to amplify the flanking genomic region of the exon (Fig. 3 a and 3 b). The resulting products are shown in lanes 1-4 in Figure 1 b. DIF1-DIR1 and DIF2-DIR2, produced 2.3 kbp and 2.0 kbp fragments, although the expected lengths of these PCR products from the reeler cDNA were 53 bp and 59 bp, respectively (Fig. 3 a). Since these discrepancies were considered to be caused by the introns flanking the 220 bp sequence, the 2.3 and 2.0 kbp PCR fragments were sequenced. The data showed that the exon-intron junctions are consistent with the 5' and 3' borders of the 220 bp DNA fragment which is deleted in the Orleans reeler transcript (Fig. 3 a). Further precise sequencing analysis of the PCR-amplified intron revealed no mutations in the intronic sequence of rlOrlcompared with the wild-type (BALB/c), including the splicing consensus sequence, such as the GT-AG intron-exon border sequence and the branching region of a lariat formation (data not shown). Thus, we concluded that the 220 bp fragment is missed out in the RNA splicing (exon skipping), due to the putative abnormal structure on the skipped exon.
For further analysis of the mechanism of exon skipping, we examined the exon of the Orleans reeler mutant gene by Southern hybridization, using the 212 bp PCR product amplified by the primer set DF1-DR1. The result is shown in Figure 2 . The wild-type genomic DNA produced 3.5 kbp and 0.9 kbp fragments (lane 1 in Fig. 2 ), whereas an 8.0 kbp fragment was detected in the Orleans reeler gene in place of the 0.9 kbp fragment (lane 2 in Fig. 2 ). These data imply that the skipped exon sequence carries an additional sequence with an estimated length of 7.1 kbp. This result is consistent with the fact that no PCR products were amplified on rlOrl genomic DNA under the condition used, with primers DF1-DR1 (lane 2 in Fig. 1 a).
Next, two cosmid libraries of reeler Orleans mouse and wild-type inbred strain, BALB/c, were constructed with the partially digested MboIgenomic DNA fragments. We screened 3*105 colonies using the 212 bp PCR product as a probe and isolated two clones which cover the genomic region of the skipped exon. For further sequencing analysis, two positive cosmid clones were subcloned by pBluescript KS(+) vector and all the subclones were sequenced. The results showed the 7.1 kbp inserted sequence to be an L1 repetitive element (Fig. 3 a and 3 b, GenBank accession number D84391). The structure of the L1 sequence consists of typical full-length L1 retroposon components. The five-time repetition of the 212 bp tandem repeat sequence situated at the 5' upstream site, is classified as an F-type. These five repeating units showed very significant homology (93.4%) with each other. This sequence is followed by two intact ORFs, ORF1 and ORF2. As shown in Fig. 3 b, the 1152 bp stretch of ORF1 is located 5' upstream from ORF2 which is 3900 bp long, using a different codon frame from that of ORF1. The 14 bp region of these two ORFs overlap each other as shown in Figure 3 b. The polyadenylation signal lies at the 3' downstream region, followed by the polyA stretch. The entire inserted fragment was bordered at both ends by a direct repeat of the 13 bp exon sequence originating from the target site of the reeler gene, and this type of DNA insertion is specific for retroposon integration. In combination with the fact that the rlOrl mutation was produced spontaneously, these data indicate that the inserted sequence is a full-length copy of the L1 element and this L1 sequence is active for transposition in the mouse genome.
Jackson reeler (rlJ) and Orleans reeler (rlOrl)are independent and spontaneous mutant alleles which show the reeler phenotype. The rlJallele carries a 150 kb genomic deletion (6 ), whereas the rlOrl allele produces a transcript with a 220 bp deletion (3 ). However, it was not known what caused the mutation for the 220 bp deletion in the rlOrl transcript. In this study, we examined the genomic structure of the rlOrl reeler gene and found that the deleted 220 bp corresponded to a single exon which is missed out during the RNA splicing (exon skipping). Further analyses revealed that a 7.1 kbp fragment was inserted into the skipped exon, although no mutations were found in the intron sequence flanking the skipped exon, including the splicing consensus sequence. This additional fragment which expands the length of the skipped exon can account for the exon skipping phenomenon, in agreement with the previous report that for normal mRNA splicing, the length of the exon sequence must be appropriate for concerted recognition of the 3' and 5' splice site boundaries for splice site recognition factors and that an exon longer than 300 nucleotides will not be correctly recognized (9 ). Thus, the skipped exon (about 7.3 kb) might be too large to be recognized as an exon.
Nucleic acid homology search of the insertional fragment using GenBank databases showed that the fragment was one type of the L1 element. L1 families are found in all mammalian genomes and belong to a subclass of retrotransposons (class II) which are amplified by a process that includes an RNA intermediate (8 ). During mammalian evolution, the mechanism of retrotrans- position is thought to have increased the number of L1 repeating sequences to result in up to 100 000 copies in the mouse and human haploid genomes (8 ,10 ).
In the process of L1 retrotransposition, insertional mutagenesis by L1 elements has occurred in both somatic and germ cells. In the case of somatic cells, L1 insertions at the myc locus in a human breast carcinoma (11 ) and the APC gene in a human colon cancer (12 ) have been reported. Some human and mouse mutants, such as the human hereditary diseases of hemophilia A (13 ) and muscular dystrophy (14 ,15 ) and spastic mouse (16 ,17 ), are caused by insertional mutagenesis of L1 transposition into germ cells. Aberrant splicing was found in the patient with Duchenne muscular dystrophy caused by L1 insertions into exon 44 of the dystrophin gene (14 ). Another similar example was reported in the spastic mouse mutation carrying Ll transposition into intron 5 of the glycine receptor [beta]-subunit (Glrb) gene (16 ,17 ). In these L1 insertions, most of the L1 elements are truncated at various sites from the 3' end, although the consensus L1 element is 6-7 kb long (8 ). Generally, the consensus structure of L1 elements in mice consists of a series of direct tandem repeats at the 5' end, ORF1, ORF2, and a polyadenylation signal followed by an A-rich region. Moreover, the L1 sequence is usually flanked by target site duplications (8 ). ORF1 encodes a product that may form a complex with L1 RNA (18 ) although its function is not known. ORF2 also encodes a product homologous to the reverse transcriptase (RT) (19 ) and ORF2 protein of the human L1 element (L1.2A) has been shown to have reverse transcriptase activity (20 ). The L1 element discovered in this study carried all these components, including ORF1 containing three tandemly repeating blocks of 42 bp (21 ) and ORF2 encoding all seven canonical boxes, such as an F-X-D-D box, which were highly conserved in RTs (22 ). Moreover, the rlOrl was spontaneously generated, implying that our full-length L1 element should be active for transposition.
Based on the 5' tandem repeat sequence, mouse L1 families are classified into A or F-types (23 ). The A-type L1 elements have been considered to be active for transposition from the following evidence. First, the A-type repeat has the promoter activity (24 ). Second, full-length A-type L1 copies with intact ORFs have been isolated from both genomic DNA and cDNA (23 ,25 ,26 ), especially as transcripts in the mouse F9 cell line (18 ,21 ). However, the 5' tandem repeat sequence of the L1 element inserted into the reeler gene is highly homologous to F-type elements (L1Md-F11) (27 ), but not to the A-type repeat. The 5' direct repeat of our Ll element showed a highly significant homology to that of the L1 sequence inserted into the Glrb gene in spastic mouse (95% nucleotide identity) (16 ,17 ). This L1 sequence in the Glrb gene was a full-length element and recently it was reported that the ORF2 encoded substantial RT activity using an in vivo assay in S.cerevisiae (28 ). As far as reported for mouse, only the F-type L1 element is active for retrotransposition.
Our data demonstrate that the intact L1 sequence has been transposed into the exon that encodes the 8th EGF-like motif of the reeler gene, resulting in exon skipping which causes a frame shift. Consequently, the phenotype of Orleans reeler mutants have arisen from de novo L1 retrotransposition. Moreover, this F-type L1 sequence transposed into the reeler gene is thought to be active for retrotransposition.
Orleans reeler mouse was originally provided by Kyushyu University (Japan) and bred in the Division of Experimental Animal Research (RIKEN Tsukuba Life Science Center, Japan). Orleans reeler and wild-type BALB/c strain mice were used in this study.
Sequences of primers used are as follows: DF1, 5'-CGACTGCTCTGTCTTCAGTCACGAG-3'; DR1, 5'-CTCGAGTGAGGTCCAGTGGCTT-3'; DIF 1, 5'-GGCCGTCTGCATCTGCGATG A- A-3'; DIR1, 5'-TGAAGACAGAGCAGTCGTCAC3'; DIF2, 5'-CACTGGACCTCACTCGAGCAA-3'; DIR2, 5'-GGCTGGGCTCCCAATTTGCAA-3'. PCR amplification was performed as follows: for DF1-DR1, 30 cycles of 94oC for 1 min, 64oC for 1 min, 72oC for 1 min, for DIF1-DIR1 and DIF2-DIR2, 30 cycles of 94oC for 1 min, 62oC for 1 min, 72oC for 3 min. The PCR product of DF1-DIR1 primers was subjected to 6% poly- acrylamide gel electrophoresis. The products of DIF1-DIR1 and DIF2-DIR2 were subjected to 0.8% agarose gel electrophoresis.
Genomic DNA was isolated from BALB/c and rlOrl liver tissues. Southern blot analysis was performed by standard techniques as described elsewhere (29 ). The radio-labeled probe with [[alpha]-32P]dCTP using BcaBESTTM Labeling Kit (Takara) was the 212 bp PCR product of genomic DNA from BALB/c that was generated by primers, DF1-DR1. Hybridization was carried out in 10% PEG, 7% SDS at 65oC. Filters were washed in 0.5* SSC, 1% SDS at 65oC.
Genomic DNA from rlOrland BALB/c were partially digested with MboIand ligated to the BamHI site of pWE15 cosmid vector (Stratagene). The library was screened with the 212 bp PCR product of genomic DNA from BALB/c that was generated by primers, DF1-DR1. Positive cosmid clones derived from rlOrl were digested with PvuII and PstI. Thereafter two positive PvuII-PstI fragments, 3 kb and 5 kb which contained the whole insertional region were identified by hybridization and subcloned into the pBluescript KS(+) (Stratagene)
For DNA sequencing of the inserted fragment, nested deletion sets were constructed in both orientations with a Kilobase Deletion Kit (Takara). For DNA sequencing of PCR products, PCR fragments were subcloned into pCR II vector (Invitrogen). Sequencing was performed using a Dye Primer Cycle Sequencing Kit with Ampli Taq DNA polymerase FS(+) (Applied Biosystems) and analyzed using an ABI 377 automated sequencer.
We thank Dr O. Sugimoto for his excellent advice. We also thank Ms. Owa for her technical assistance. This study was supported by Special Coordination Funds and a Research Grant for the Genome Exploration Research Project of the Science and Technology Agency of the Japanese Government, and by a Grant-in-Aid for a Creative Basic Research (Human Genome Program), a Grant-in-Aid for Scientific Research on Priority Areas and a Grant-in-Aid for Developmental Scientific Research (B) on Priority Areas from The Ministry of Education and Culture, Japan to Y.H. This work was also supported by a Grant for Research on Aging and Health, a Grant-in-Aid for a Second Term Comprehensive 10-Year Strategy for Cancer Control and a grant for cardiomyopathy study from the Ministry of Health and Welfare of Japan, and special funds subsidized by J.R.A. to Y.H.
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