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Lissencephaly-1 Protein
GENOMICS
47, 200–206 (1998) GE975121
ARTICLE NO.
Cloning and Characterization of cDNAs and the Gene Encoding the Mouse Platelet-Activating Factor Acetylhydrolase Ib a Subunit/Lissencephaly-1 Protein Miklo´s Pe´terfy,*,1Tibor Gyuris,* David Grosshans,† Christina C. Cuaresma,* and La´szlo´ Taka´cs* *Department of Biomedical Science and †Department of Computational Biology, Amgen, Inc., Thousand Oaks, California 91320 Received April 4, 1997; accepted November 12, 1997
Platelet-activating factor acetylhydrolases (PAFAHs) play an important role in the metabolism of PAF, a potent phospholipid mediator affecting various physiological processes. The heterotrimeric form of intracellular PAF-AH consists of two catalytic subunits (PAF-AH Ibb and PAF-AH Ibg) and a potential regulatory subunit (PAF-AH Iba). Hemizygous deletion of the gene encoding the a subunit has been implicated in two related neurological disorders: isolated lissencephaly sequence and Miller–Dieker syndrome. Here we report the isolation and characterization of mouse Pafaha/Lis1 cDNAs and the corresponding Pafaha/Lis1 gene. We have cloned five cDNAs representing alternatively polyadenylated messages. Northern blot analysis revealed that the various Pafaha/Lis1 mRNAs are differentially expressed in mouse tissues. The Pafaha/Lis1 gene spans a genomic region of more than 50 kb and consists of 12 exons, the first 2 of which are embedded in CpG islands. We have identified two sites of alternative splicing of Pafaha/Lis1: one affecting the length of the 5* untranslated region, the other potentially resulting in a truncated form of the encoded protein. q 1998 Academic Press
INTRODUCTION
Platelet-activating factor acetylhydrolases (PAFAHs) are calcium-independent phospholipases that hydrolyze phospholipids containing short-chain ester moieties at the sn-2 position (Stafforini et al., 1996a). The hydrolysis of PAF by these enzymes gives rise to a biologically inactive phospholipid, lyso-PAF (Imaizumi et al., 1995). In addition to PAF, PAF-AHs also inactivate PAF-like bioactive molecules generated by the oxidative fragmentation of various long-chain phosSequence data from this article have been deposited with the GenBank Data Library under Accession Nos. U95116–U95120. 1 To whom correspondence should be addressed at Amgen, Inc., M/S 8-1-D, Thousand Oaks, CA 91320. Telephone: (805) 447-6596. Fax: (805) 498-8674. E-mail: [email protected].
0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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pholipids (Patel et al., 1992). Thus, PAF-AHs play an important role in the regulation of diverse pathological processes, including inflammation (Tjoelker et al., 1995a), oxidative stress (Smiley et al., 1991), and atherogenesis (Watson et al., 1995). Although PAF-AH activity can be detected in blood and other tissues (Tjoelker et al., 1995b; Stafforini et al., 1991), it has become clear that a family of structurally unrelated proteins is responsible for catalyzing the same enzymatic reaction. The extracellular/plasma form of PAF-AH is a 45-kDa serine esterase secreted primarily by macrophages. This enzyme regulates PAF level in the blood and therefore it has anti-inflammatory (Tjoelker et al., 1995a) and antiallergenic (Stafforini et al., 1996b) effects. The intracellular forms of PAF-AHs show a great degree of molecular heterogeneity. The erythrocyte-type enzyme is a homodimer of two 25-kDa subunits (Stafforini et al., 1993). Brain, liver, and kidney tissues contain three isoforms of PAH-AH: a 40-kDa monomeric serine esterase (PAFAH II) is the dominant isoform in liver, while a 60-kDa heterodimer (PAF-AH Ia) and a 100-kDa heterotrimer (PAF-AH Ib) are found primarily in brain (Hattori et al., 1995). Interestingly, two of the subunits (30-kDa PAF-AH Ibb and 29-kDa PAF-AH Ibg) of the latter enzyme possess catalytic activity, while the 45-kDa PAF-AH Iba subunit may have a regulatory role (Hattori et al., 1996). The importance of the PAF-AH Iba subunit was highlighted by the recent discovery that it was identical to the product of LIS1, the proposed disease-associated gene of two related neuronal migration disorders: isolated lissencephaly sequence (ILS) and Miller–Dieker syndrome (MDS) (Hattori et al., 1994; Reiner et al., 1993). The LIS1 gene is located in HSA17p13.3, a chromosomal region showing frequent hemizygous deletions in ILS and MDS patients (Chong et al., 1997). Furthermore, LIS1 was found to contain point mutations in cytogenetically normal patients (Lo Nigro et al., 1997). Whereas in ILS only the LIS1 gene is affected, MDS was proposed to be a contiguous gene syn-
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drome in which additional genes are also deleted or disrupted, resulting in the complex phenotype observed (Dobyns et al., 1993; Chong et al., 1997). The mouse ortholog of the PAF-AH Iba/LIS1 gene (Pafaha/Lis1) has been mapped to a region of MMU11 showing syntenic conservation with HSA17p13.3 (Pe´terfy et al., 1995; Kurtz and Zimmer, 1995; Hirotsune et al., 1997), and partial cDNAs have been cloned (Pe´terfy et al., 1994; Reiner et al., 1995). In this paper we report the characterization of full-length Pafaha/Lis1 cDNAs and the determination of the exon–intron structure of the corresponding gene. MATERIALS AND METHODS Isolation of cDNA clones. Clones (4 1 105) of a mouse brain cDNA library in l-phage vector (Stratagene) were screened using a PCRamplified (5*-GCTTGACATTACAGCCAAGATG-3*, 5*-AAGCTCAATCTACATCCAGAAT-3*) probe containing the entire coding region of the human LIS1 cDNA (Reiner et al., 1993). Duplicate filters (Hybond; Amersham) were hybridized in 51 SSC, 0.1% SDS, 51 Denhardt’s, denatured salmon sperm DNA (100 mg/ml) overnight at 607C and washed four times in 21 SSC, 0.1% SDS for 15 min each at room temperature and once in 11 SSC, 0.1% SDS for 15 min at 607C. Filters were exposed to Kodak XAR autoradiography film at 0807C using intensifying screens. Hybridizing phage clones were plaque purified and converted to pBluescript plasmid clones by in vivo excision, and the inserts were sequenced using cycle sequencing with Taq polymerase. 3* RACE was performed on a Marathon-Ready mouse brain cDNA template (Clontech), using the Expand High Fidelity PCR system (Boehringer Mannheim) according to the manufacturer’s instructions. PCR fragments were cloned using the TA cloning kit (Invitrogen) and sequenced. Northern blot analysis. Various fragments of the mouse Pafaha/Lis1 cDNA were used to probe Northern blots (Clontech). Probe A was generated with the primers used for cDNA library screening (see above), probe B was obtained by isolating the 1.5kb fragment after XbaI digestion of cDNA10, and probe C was PCR amplified with the primers 5*-GGCACTTGAAAAGGTTACTCTTCCT-3* and 5*-TATATAGGCATTTAATAGTTTACCA-3* (Fig. 1A). To verify the alternative splicing of exon 2, an exon 2a-specific antisense RNA probe was used after in vitro transcription (MAXIscript; Ambion) of a PCR-generated (5*-ATGCTAATACGACTCACTATAGGGAGGCTAGAGAGGGTAAATAGT-3*, 5*-GGCGTCGGGTTCTCCGCTTGTCCTTA-3*), T7 promoter-containing DNA fragment (Fig. 3A). Hybridizations were carried out in Express Hybridization solution (Clontech) according to the manufacturer’s instructions. Cloning of the Pafaha/Lis1 gene. Clones (4 1 105) of a mouse genomic library (a gift from Dr. A. Nagy, Mount Sinai Hospital, Toronto) in a l-phage vector (DASH II; Stratagene) were screened with the probe used for cDNA library screening as described above. Positive phage clones were mapped with restriction enzymes and subjected to Southern hybridization to identify exon-containing subfragments. Hybridizing fragments were subcloned into pBluescript vector and sequenced. All manipulations were performed using standard protocols. Regions of the gene that were not covered by the phage clones were identified by a combination of interexon PCR and genomic walking, using the Promoter Finder DNA template (Clontech). Long-range PCR was performed with the Expand Long Template System (Boehringer Mannheim) according to the manufacturer’s instructions. Amplified fragments were cloned and sequenced as described above. Database searches were performed using the GCG software package (Wisconsin OpenVMS V8.1, Madison, WI). PCR analysis of alternative splicing. To verify the alternative splicing of exon 2, PCR was performed on Marathon-Ready mouse brain cDNA (Clontech) using the Expand High Fidelity PCR System (Boehringer Mannheim) according to the manufacturer’s in-
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structions. The following primers were used (Fig. 3A): primer A, 5*-GGCCCTGCGGAGGCGGTGGTGCA-3 *; primer B, 5*-GCTATCCACGCCCTTCCCACCCAAGA-3 *; primer C, 5*-GGCGTCGGGTTCTCCGCTTGTCCTTA-3 *; and primer D, 5*-TGGATTTGAATTAAATATGCCACTATGTGGCTTCCACAG-3 *. PCR products were analyzed on 6% polyacrylamide gels (Novex), and the nucleotide sequences of the amplified fragments were determined by direct sequencing.
RESULTS
Pafaha/Lis1 Is Alternatively Polyadenylated Since only partial Pafaha/Lis1 cDNAs have been reported so far, we set out to determine the sequence of the full-length cDNA. Screening of a mouse brain cDNA library using a human PAF-AH Iba/LIS1 cDNA probe resulted in multiple phage clones. Two of these (lcDNA-9 and lcDNA-15) contained the entire coding region and had poly(A) tails at nearby nucleotide positions (2204 and 2247, respectively), and a third clone (lcDNA-10) had no poly(A) (Fig. 1A). Extension of the 3* UTR by 3* RACE indicated that there were three more sites of polyadenylation at nucleotide positions 5143 (3* RACE-2), 5224 (3* RACE-11), and 5810 (3* RACE-4). The longest cDNA clone probably represents the full-length message, as shown by the ubiquitous Ç6.0-kb major band on a Northern blot hybridized with a probe (probe A) covering the coding region of the cDNA (Figs. 1A and 1B). An Ç5.4-kb message in brain and, to a lesser extent, in kidney and liver, was detectable, as was an Ç4.4-kb heart-specific and an Ç2.3kb ubiquitous message most abundantly expressed in testis. To test if the alternatively polyadenylated cDNAs represent the various transcripts detected on the Northern blot, we reprobed the blot with probes corresponding to different regions of the 3* UTR (Fig. 1A). Probe B, which lies 0.2–1.7 kb downstream of the translation stop codon, hybridizes to both the Ç6.0and the Ç5.4-kb messages. However, probe C, which is specific for the longest cDNA, only detects the Ç6.0kb transcript. These results confirm that, with the exception of the Ç4.4-kb heart-specific message, the various Pafaha/Lis1 transcripts observed on the Northern blot arise by differential polyadenylation. Genomic Organization of the Pafaha/Lis1 Gene To determine the exon–intron organization of the Pafaha/Lis1 gene, we screened a mouse genomic library with a cDNA-derived probe and obtained several lambda clones. Exon-containing genomic fragments of two phage inserts (l4/1 and l12/2) (Fig. 2) were identified by Southern blotting, subcloned, and sequenced. Since multiple attempts to extend the phage contig by conventional library screening were unsuccessful, we pursued a PCR-based walking approach using adaptorligated genomic DNA. The Pafaha/Lis1 gene consists of 12 exons and spans more than 50 kb of genomic DNA. The accurate size of the genomic region could not be determined, because of a gap in the fragment contig
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FIG. 1. Alternative polyadenylation and expression pattern of the Pafaha/Lis1 gene. (A) The lambda clones represent three Pafaha/ Lis1 cDNAs isolated from a mouse brain cDNA library. lcDNA-10 was extended by 3 * RACE. The solid box indicates the coding region, and the positions of hybridization probes are shown. (B) Northern blot analysis using the probes shown in (A). 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; and 8, testis. Each lane contains Ç2 mg poly(A)/ RNA (Clontech).
in the second intron of the gene (Fig. 2). The first two exons are untranslated, and the third exon contains the rest of the 5* UTR and the ATG translation initiation codon. Two CpG islands (Gardiner-Garden and Frommer, 1987) were identified in the 5* region of the gene: one including part of exon 1 and possibly ex-
tending to intron 1, the other covering exon 2 (Fig. 2). The last exon contains 75 bp of translated sequence, the TGA translation termination site, and 3714 bp of 3* UTR. All exon sequences, including exon 12 with the alternative polyadenylation sites, are identical to those of the cDNA. All exon–intron boundaries were
FIG. 2. Genomic organization of the mouse Pafaha/Lis1 gene. Exons are boxed and numbered. Open and solid boxes represent noncoding and coding exons, respectively. Hatched boxes represent longer forms of exons 2 and 11 (2a and 11a), resulting from alternative SD site usage. Translation initiates on exon 3 and terminates on exon 12. Lambda phage clones and PCR fragments are shown under the gene. The positions of CpG islands are indicated.
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TABLE 1 Exon–Intron Organization of the Pafaha/Lis1 Gene Exona 1 2 2a 3 4 5 6 7 8 9 10 11 11ae 12
cDNA positionb
Exon size (bp)
1–242 243–681 682–891 892–976 977–1051 1052–1258 1259–1427 1428–1530 1531–1759 1760–1861 1862–2018
242 439 1237 210 85 75 207 169 103 229 102 157
2019–5810
3792
Splice acceptor c
aggccag aggccag tttttag ctttcag ctcctag tctcaag ttgacag gttttag ttgacag ttaatag ctttaag ctttaag ctttcag
GCCCTCC GCCCTCC GTGGAAT A AAT CGA AAT GAA G GTA ATG G GTG TGG G GT CAC GA C TAC TGT ACT AAA A GTG GGT C GTG GGT C AT TTC CA
Splice donor c GCCGCGG CGCTCCG CTCCCAG AT GAA CT A GAT ATG A AAA AAG A ATT AAG ATG CAC G AA ACT GG A TCT GAG G ACT CTT TCC TTG G
ND gtaaggc gtgagaa gtaagtt gtatgct gtaacta gcaagtt gtaaggt gtaagta gtactgt gtaagtt gtatgta
Intron size (kb)d 10.0* ú16 ú16 8.3** 0.238 1.026 2.691 1.362 0.713 1.135 2.621 1.716
a
Suffixes refer to alternative exons resulting from alternative splicing. The longer forms of exon 2 (2a) and exon 11 (11a) are not included in the numbering. c Exon and intron sequences are in uppercase and lowercase letters, respectively. Splice site consensus nucleotides conforming to the ag/ gt rule are in boldface. ND, not determined. d Intron sizes were determined by PCR (*), restriction mapping (**), or sequencing. The minimum size estimated for intron 2 is based on available intron sequences. e Exon 11a was identified based on a cDNA (L25109) described elsewhere (Reiner et al., 1995). Since this cDNA has no poly(A) tail, the size of exon 11a is unknown. b
sequenced except for the splice donor (SD) site of exon 1. The splice sites conform to the AG/GT rule except for the SD site of intron 7, which begins with the rarely occurring consensus sequence GCAAG (Shapiro and Senapathy, 1987) (Table 1). Although the human gene has only 11 exons, the overall exon–intron organization is highly conserved between the two species (Lo Nigro et al., 1997). Alternative Splicing of Pafaha/Lis1 The Pafaha/Lis1 cDNA sequence shows very high overall similarity to the corresponding human and bovine sequences (Pe´terfy et al., 1994). It was unexpectedly found, however, that the similarity between the bovine (GenBank Accession No. D30615) and the mouse cDNAs abruptly changes at the exon 2/exon 3 boundary (data not shown). While there is over 80% nucleotide identity (including UTRs) downstream of exon 2, the first 655 bp of the bovine cDNA does not show any homology to exon 1 or exon 2 sequences. This finding raised the possibility that instead of the bovine homolog of mouse exon 2, an alternative exon is utilized in the bovine brain. This hypothesis was supported by the identification of a mouse genomic sequence 175 bp downstream of exon 2 showing significant similarity (75%) to the 5* end of the bovine cDNA. Moreover, a consensus SD site was found in the mouse genomic sequence at the end of the homology region (data not shown). These results predicted a novel mouse exon not represented in the mouse cDNA clones obtained by our initial library screening. To determine whether the predicted new SD site was
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functional, we performed PCR on mouse brain cDNA. Two primer pairs were used to detect splicing of the putative novel exon to exon 2 and exon 3 (Fig. 3A). The 390-bp product amplified with primers C and D verified the splicing between the predicted SD site and the splice acceptor site of exon 3. The nucleotide sequence of the 410-bp PCR product obtained with primers A and B was identical to the genomic sequence, which indicated that no splicing occurred in this genomic region. To confirm alternative splicing of exon 2 further, we hybridized a Northern blot with a probe specific for the alternative exon (Fig. 3A). An Ç2.3-kb message was detected in testis (Fig. 3B), corresponding in size to the smallest transcript observed with the coding region probe (probe A; Fig. 1B). A weaker band of Ç1.4 kb was also detected in heart. It should be noted that the hybridization signal obtained with the exon 2-specific probe was significantly weaker than that detected with probe A, suggesting that only a fraction of the Ç2.3-kb message contains the alternatively spliced exon (see Discussion). Taken together, an alternative splice variant of the Pafaha/ Lis1 message was identified, resulting in an extended form of exon 2 (exon 2a) (Fig. 2 and Table 1). Database searches with Pafaha/Lis1 genomic sequences revealed an additional alternative splicing site at exon 11. We found that intron 11 was not spliced out in a mouse Pafaha/Lis1 cDNA clone (GenBank Accession No. L25109; Reiner et al., 1995) isolated from a brain cDNA library, indicating that the SD site of exon 11 was unrecognized by the splicing machinery in the corresponding transcript (Fig. 2). As a result,
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FIG. 3. Alternative splicing of the Pafaha/Lis1 gene. (A) Verification of alternative splicing of exon 2 by RT-PCR. Open boxes indicate exons (exon 2 and 3) that are represented in the Pafaha/Lis1 cDNAs obtained by library screening. The hatched box indicates the mouse genomic region showing homology to a bovine cDNA (D30615). The positions of PCR primers A, B, C, and D are indicated by arrows and the hybridization probe by a black bar. The predicted SD site (SDP) was confirmed by sequence analysis of the 390-bp PCR product. (B) Northern blot analysis of alternative splicing using the probe shown in (A).
translation is expected to be terminated by an in-frame stop codon in exon 11a, giving rise to a truncated form of the Pafaha/Lis1 polypeptide. DISCUSSION
The PAF-AH Iba/LIS1 gene codes for the 45-kDa noncatalytic subunit of the brain-type intracellular PAF acetylhydrolase. Defects in this gene have been proposed to be responsible for isolated lissencephaly sequence and to contribute to Miller–Dieker syndrome, two devastating neurological disorders (Lo Nigro et al., 1997). In an effort to characterize the mouse ortholog of the PAF-AH Iba/LIS1 gene, we report the cloning of several mouse Pafaha/Lis1 cDNAs and the structural organization of the mouse Pafaha/Lis1 gene. The five forms of cDNA obtained from a mouse brain cDNA library result from the utilization of different polyadenylation signals by the nuclear machinery (Fig. 1). Alternative polyadenylation was also observed in human PAF-AH Iba/LIS1 cDNAs (Lo Nigro et al., 1997). Interestingly, the expression pattern of the different Pafaha/ Lis1 mRNA species is markedly different in the tissues tested (Fig. 1B). Certain messages (e.g., Ç4.4-kb mRNA in heart) are tissue specific, while others (e.g., Ç6.0-kb mRNA) are ubiquitously expressed. Although the exact role of the differential polyadenylation of the Pafaha/Lis1 transcript is not known, the presence or absence of cis-acting elements in the 3* UTR may influence the structural and/or functional characteristics of the message in a tissue-specific manner (Decker and Parker, 1995). We used a PCR-based approach to extend genomic phage clones obtained by conventional library screening, because we could not isolate overlapping clones
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covering the entire Pafaha/Lis1 gene. Interestingly, the 5* part of the human gene also proved to be unclonable in cosmids (Chong et al., 1997). The analysis of the Pafaha/Lis1 gene revealed that it spans over 50 kb of genomic DNA and contains 12 exons, ranging in size from 75 (exon 5) to 3792 bp (exon 12). The large size (9.6 and ú16 kb) of the 5* introns is fairly typical; the presence of two 5* noncoding exons, however, is uncommon among vertebrate genes (Hawkins, 1988). Despite a highly similar exon–intron organization, the human gene has only 11 exons, lacking a homologue of the first noncoding exon of the mouse (Lo Nigro et al., 1997). In accordance with the ubiquitous expression of the Pafaha/Lis1 gene, a CpG island was found to cover the predicted transcriptional start site, and a separate CpG island was located on exon 2 (Fig. 2). As a result, the 5* UTR of the mature transcript is highly GC rich (74% GC content in the first 680 nucleotides). Although most 5* CpG islands extend past the translation start site (Gardiner-Garden and Frommer, 1987), no CpG-rich sequences were associated with exon 3, containing the ATG translation initiator. Sequence comparison between intron 2 of the mouse gene and a bovine PAF-AH Iba/LIS1 cDNA showed significant sequence similarity and predicted that an alternative SD site, 799 bp downstream of exon 2, may be utilized, resulting in a longer form of this exon (exon 2a) (Fig. 2). Alternative splicing of exon 2 was verified by RT-PCR and Northern blot analysis (Fig. 3). Although exon 2a splicing was detectable in brain by PCR, testis is the most significant site of expression of the alternative transcript, as shown by Northern blot hybridization (Fig. 3B). Despite the presence of the longer form of exon 2, the size of the alternative transcript is similar (Ç2.3 kb) to those containing the
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shorter form of exon 2, suggesting additional alternative splicing or the existence of an alternative transcriptional start site in testis. Supporting the latter possibility is the considerable length of exon 2a (1237 bp), which strongly argues against it being an internal exon (Berget, 1995; Hawkins, 1988). Therefore, we predict the presence of an alternative promoter in exon 2 of the mouse gene. The relatively high degree of sequence conservation (over 75% identity) of 5* UTRs in the mouse, bovine, and human cDNAs points to the functional importance of this region. Various features of the leader sequences of mRNAs are known to influence the efficiency of translation (Kozak, 1991a). In particular, 5* UTRs with multiple upstream AUG codons (‘‘AUG-burdened leader sequences’’) impair translation of the authentic ORF, due to the inefficient reinitiation by the ribosomes after translating a short 5* ORF (Kozak, 1991b). Sequence analysis of the alternatively spliced exon 2a reveals that it contains two AUG codons in favorable contexts followed by in-frame translation stop codons within 144 and 27 bp. Therefore, alternative splicing of exon 2 may be a mechanism for regulating the efficiency of translation of the Pafaha/Lis1 message in response to certain developmental or environmental stimuli. Supporting the possibility of such a posttranscriptional control mechanism is the recent demonstration that the extension by alternative splicing of one of the 5* untranslated exons of the GM-CSFRa message reduces in vitro translation efficiency (Chopra et al., 1996). Recently, a mouse cDNA was described (Lis3-4; GenBank Accession No. L25109), which was identical to the Pafaha/Lis1 cDNA sequence at its 5* end, but showed no similarity at its 3* end. The authors concluded that the corresponding transcript derived from a gene different from Pafaha/Lis1 (Reiner et al., 1995). Here we show that the 3* nonhomologous end of Lis34 is identical to intron 11 sequences of the Pafaha/ Lis1 gene. This suggests that Lis3-4 is derived from the Pafaha/Lis1 locus by alternative splicing of intron 11, resulting in a longer form of exon 11 (exon 11a) (Fig. 2). Since Lis3-4 has no poly(A) tail, we do not know whether the entire intron 11 or only part of it is present in this message. Due to an in-frame stop codon in exon 11a, the resulting ORF is 57 bp shorter than the one including exon 12 sequences. The deduced protein encoded by the alternatively spliced transcript is not only shorter than the longer form by 19 amino acids, but also contains five C-terminal residues not present in the full-length product. Thus, this truncation results in the loss of most of the last GH-WD motif of the seven repeat units in the Pafaha/Lis1 protein (Neer et al., 1993). The functional consequences of the truncation remain to be determined. In conclusion, we determined the exon–intron structure of the mouse Pafaha/Lis1 gene and showed that the primary transcript undergoes alternative splicing and polyadenylation in a tissue-specific manner. These
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data will facilitate functional studies of the Pafaha/ Lis1 gene. ACKNOWLEDGMENTS We thank Dr. F. K. Lin and Dr. L. G. Ko˝mu¨ves for useful comments on the manuscript.
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(1992). Novel leukocyte agonists are released by endothelial cells exposed to peroxide. J. Biol. Chem. 267: 15168–15175. Pe´terfy, M., Gyuris, T., Basu, R., and Taka´cs, L. (1994). Lissencephaly-1 is one of the most conserved proteins between mouse and human: A single amino-acid difference in 410 residues. Gene 150: 415–416. Pe´terfy, M., Hozier, J. C., Hall, B., Gyuris, T., Pe´terfy, K., and Taka´cs, L. (1995). Localization of the mouse lissencephaly-1 gene to mouse chromosome 11B3, in close proximity to D11Mit65. Somatic Cell Mol. Genet. 21: 345–349. Reiner, O., Carrozzo, R., Shen, Y., Wehnert, M., Faustinella, F., Dobyns, W. B., Caskey, C. T., and Ledbetter, D. H. (1993). Isolation of a Miller–Dieker lissencephaly gene containing G protein b-subunit-like repeats. Nature 364: 717–721. Reiner, O., Albrecht, U., Gordon, M., Chianese, K. A., Wong, C., GalGerber, O., Sapir, T., Siracusa, L. D., Buchberg, A. M., Caskey, C. T., and Eichele, G. (1995). Lissencephaly gene (LIS1) expression in the CNS suggests a role in neuronal migration. J. Neurosci. 15: 3730–3738. Shapiro, M. B., and Senapathy, P. (1987). RNA splice junctions of different classes of eukaryotes: Sequence statistics and functional implications in gene expression. Nucleic Acids Res. 15: 7155–7174. Smiley, P. L, Stremler, K. E., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1991). Oxidatively fragmented phosphatidylcholines activate human neutrophils through the receptor for plateletactivating factor. J. Biol. Chem. 266: 11104–11110. Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre,
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T. M. (1991). Platelet-activating factor acetylhydrolase activity in human tissues and blood cells. Lipids 26: 979–985. Stafforini, D. M., Rollins, E. N., Prescott, S. M., and McIntyre, T. M. (1993). The platelet-activating factor acetylhydrolase from human erythrocytes. J. Biol. Chem. 268: 3857–3865. Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1996a). Mammalian platelet-activating factor acetylhydrolases. Biochim. Biophys. Acta 1301: 161–173. Stafforini, D. M., Satoh, K., Atkinson, D. L., Tjoelker, L. W., Eberhardt, C., Yoshida, H., Imaizumi, T., Takamatsu, S., Zimmerman, G. A., McIntyre, T. M., Gray, P. W., and Prescott, S. M. (1996b). Platelet-activating factor acetylhydrolase deficiency. J. Clin. Invest. 97: 2784–2791. Tjoelker, L. W., Wilder, C., Eberhardt, C., Stafforini, D. M., Dietsch, G., Schimpf, B., Hooper, S., Trong, H. L., Cousens, L. S., Zimmerman, G. A., Yamada, Y., McIntyre, T. M., Prescott, S. M., and Gray, P. W. (1995a). Anti-inflammatory properties of a platelet-activating factor acetylhydrolase. Nature 374: 549–552. Tjoelker, L. W., Eberhardt, C., Unger, J., Trong, H. L., Zimmerman, G. A., McIntyre, T. M., Stafforini, D. M., Prescott, S. M., and Gray, P. W. (1995b). Plasma platelet- activating factor acetylhydrolase is a secreted phospholipase A2 with a catalytic triad. J. Biol. Chem. 270: 25481–25487. Watson, A. D., Navab, M., Hama, S. Y., Sevanian, A., Prescott, S. M., Stafforini, D. M., McIntyre, T. M., La Du, B. N., Fogelman, A. M., and Berliner, J. A. (1995). Effect of platelet activating factor-acetylhydrolase on the formation and action of minimally oxidized low density lipoprotein. J. Clin. Invest. 95: 774–782.
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ARTICLE NO.
Cloning and Characterization of cDNAs and the Gene Encoding the Mouse Platelet-Activating Factor Acetylhydrolase Ib a Subunit/Lissencephaly-1 Protein Miklo´s Pe´terfy,*,1Tibor Gyuris,* David Grosshans,† Christina C. Cuaresma,* and La´szlo´ Taka´cs* *Department of Biomedical Science and †Department of Computational Biology, Amgen, Inc., Thousand Oaks, California 91320 Received April 4, 1997; accepted November 12, 1997
Platelet-activating factor acetylhydrolases (PAFAHs) play an important role in the metabolism of PAF, a potent phospholipid mediator affecting various physiological processes. The heterotrimeric form of intracellular PAF-AH consists of two catalytic subunits (PAF-AH Ibb and PAF-AH Ibg) and a potential regulatory subunit (PAF-AH Iba). Hemizygous deletion of the gene encoding the a subunit has been implicated in two related neurological disorders: isolated lissencephaly sequence and Miller–Dieker syndrome. Here we report the isolation and characterization of mouse Pafaha/Lis1 cDNAs and the corresponding Pafaha/Lis1 gene. We have cloned five cDNAs representing alternatively polyadenylated messages. Northern blot analysis revealed that the various Pafaha/Lis1 mRNAs are differentially expressed in mouse tissues. The Pafaha/Lis1 gene spans a genomic region of more than 50 kb and consists of 12 exons, the first 2 of which are embedded in CpG islands. We have identified two sites of alternative splicing of Pafaha/Lis1: one affecting the length of the 5* untranslated region, the other potentially resulting in a truncated form of the encoded protein. q 1998 Academic Press
INTRODUCTION
Platelet-activating factor acetylhydrolases (PAFAHs) are calcium-independent phospholipases that hydrolyze phospholipids containing short-chain ester moieties at the sn-2 position (Stafforini et al., 1996a). The hydrolysis of PAF by these enzymes gives rise to a biologically inactive phospholipid, lyso-PAF (Imaizumi et al., 1995). In addition to PAF, PAF-AHs also inactivate PAF-like bioactive molecules generated by the oxidative fragmentation of various long-chain phosSequence data from this article have been deposited with the GenBank Data Library under Accession Nos. U95116–U95120. 1 To whom correspondence should be addressed at Amgen, Inc., M/S 8-1-D, Thousand Oaks, CA 91320. Telephone: (805) 447-6596. Fax: (805) 498-8674. E-mail: [email protected].
0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.
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pholipids (Patel et al., 1992). Thus, PAF-AHs play an important role in the regulation of diverse pathological processes, including inflammation (Tjoelker et al., 1995a), oxidative stress (Smiley et al., 1991), and atherogenesis (Watson et al., 1995). Although PAF-AH activity can be detected in blood and other tissues (Tjoelker et al., 1995b; Stafforini et al., 1991), it has become clear that a family of structurally unrelated proteins is responsible for catalyzing the same enzymatic reaction. The extracellular/plasma form of PAF-AH is a 45-kDa serine esterase secreted primarily by macrophages. This enzyme regulates PAF level in the blood and therefore it has anti-inflammatory (Tjoelker et al., 1995a) and antiallergenic (Stafforini et al., 1996b) effects. The intracellular forms of PAF-AHs show a great degree of molecular heterogeneity. The erythrocyte-type enzyme is a homodimer of two 25-kDa subunits (Stafforini et al., 1993). Brain, liver, and kidney tissues contain three isoforms of PAH-AH: a 40-kDa monomeric serine esterase (PAFAH II) is the dominant isoform in liver, while a 60-kDa heterodimer (PAF-AH Ia) and a 100-kDa heterotrimer (PAF-AH Ib) are found primarily in brain (Hattori et al., 1995). Interestingly, two of the subunits (30-kDa PAF-AH Ibb and 29-kDa PAF-AH Ibg) of the latter enzyme possess catalytic activity, while the 45-kDa PAF-AH Iba subunit may have a regulatory role (Hattori et al., 1996). The importance of the PAF-AH Iba subunit was highlighted by the recent discovery that it was identical to the product of LIS1, the proposed disease-associated gene of two related neuronal migration disorders: isolated lissencephaly sequence (ILS) and Miller–Dieker syndrome (MDS) (Hattori et al., 1994; Reiner et al., 1993). The LIS1 gene is located in HSA17p13.3, a chromosomal region showing frequent hemizygous deletions in ILS and MDS patients (Chong et al., 1997). Furthermore, LIS1 was found to contain point mutations in cytogenetically normal patients (Lo Nigro et al., 1997). Whereas in ILS only the LIS1 gene is affected, MDS was proposed to be a contiguous gene syn-
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drome in which additional genes are also deleted or disrupted, resulting in the complex phenotype observed (Dobyns et al., 1993; Chong et al., 1997). The mouse ortholog of the PAF-AH Iba/LIS1 gene (Pafaha/Lis1) has been mapped to a region of MMU11 showing syntenic conservation with HSA17p13.3 (Pe´terfy et al., 1995; Kurtz and Zimmer, 1995; Hirotsune et al., 1997), and partial cDNAs have been cloned (Pe´terfy et al., 1994; Reiner et al., 1995). In this paper we report the characterization of full-length Pafaha/Lis1 cDNAs and the determination of the exon–intron structure of the corresponding gene. MATERIALS AND METHODS Isolation of cDNA clones. Clones (4 1 105) of a mouse brain cDNA library in l-phage vector (Stratagene) were screened using a PCRamplified (5*-GCTTGACATTACAGCCAAGATG-3*, 5*-AAGCTCAATCTACATCCAGAAT-3*) probe containing the entire coding region of the human LIS1 cDNA (Reiner et al., 1993). Duplicate filters (Hybond; Amersham) were hybridized in 51 SSC, 0.1% SDS, 51 Denhardt’s, denatured salmon sperm DNA (100 mg/ml) overnight at 607C and washed four times in 21 SSC, 0.1% SDS for 15 min each at room temperature and once in 11 SSC, 0.1% SDS for 15 min at 607C. Filters were exposed to Kodak XAR autoradiography film at 0807C using intensifying screens. Hybridizing phage clones were plaque purified and converted to pBluescript plasmid clones by in vivo excision, and the inserts were sequenced using cycle sequencing with Taq polymerase. 3* RACE was performed on a Marathon-Ready mouse brain cDNA template (Clontech), using the Expand High Fidelity PCR system (Boehringer Mannheim) according to the manufacturer’s instructions. PCR fragments were cloned using the TA cloning kit (Invitrogen) and sequenced. Northern blot analysis. Various fragments of the mouse Pafaha/Lis1 cDNA were used to probe Northern blots (Clontech). Probe A was generated with the primers used for cDNA library screening (see above), probe B was obtained by isolating the 1.5kb fragment after XbaI digestion of cDNA10, and probe C was PCR amplified with the primers 5*-GGCACTTGAAAAGGTTACTCTTCCT-3* and 5*-TATATAGGCATTTAATAGTTTACCA-3* (Fig. 1A). To verify the alternative splicing of exon 2, an exon 2a-specific antisense RNA probe was used after in vitro transcription (MAXIscript; Ambion) of a PCR-generated (5*-ATGCTAATACGACTCACTATAGGGAGGCTAGAGAGGGTAAATAGT-3*, 5*-GGCGTCGGGTTCTCCGCTTGTCCTTA-3*), T7 promoter-containing DNA fragment (Fig. 3A). Hybridizations were carried out in Express Hybridization solution (Clontech) according to the manufacturer’s instructions. Cloning of the Pafaha/Lis1 gene. Clones (4 1 105) of a mouse genomic library (a gift from Dr. A. Nagy, Mount Sinai Hospital, Toronto) in a l-phage vector (DASH II; Stratagene) were screened with the probe used for cDNA library screening as described above. Positive phage clones were mapped with restriction enzymes and subjected to Southern hybridization to identify exon-containing subfragments. Hybridizing fragments were subcloned into pBluescript vector and sequenced. All manipulations were performed using standard protocols. Regions of the gene that were not covered by the phage clones were identified by a combination of interexon PCR and genomic walking, using the Promoter Finder DNA template (Clontech). Long-range PCR was performed with the Expand Long Template System (Boehringer Mannheim) according to the manufacturer’s instructions. Amplified fragments were cloned and sequenced as described above. Database searches were performed using the GCG software package (Wisconsin OpenVMS V8.1, Madison, WI). PCR analysis of alternative splicing. To verify the alternative splicing of exon 2, PCR was performed on Marathon-Ready mouse brain cDNA (Clontech) using the Expand High Fidelity PCR System (Boehringer Mannheim) according to the manufacturer’s in-
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structions. The following primers were used (Fig. 3A): primer A, 5*-GGCCCTGCGGAGGCGGTGGTGCA-3 *; primer B, 5*-GCTATCCACGCCCTTCCCACCCAAGA-3 *; primer C, 5*-GGCGTCGGGTTCTCCGCTTGTCCTTA-3 *; and primer D, 5*-TGGATTTGAATTAAATATGCCACTATGTGGCTTCCACAG-3 *. PCR products were analyzed on 6% polyacrylamide gels (Novex), and the nucleotide sequences of the amplified fragments were determined by direct sequencing.
RESULTS
Pafaha/Lis1 Is Alternatively Polyadenylated Since only partial Pafaha/Lis1 cDNAs have been reported so far, we set out to determine the sequence of the full-length cDNA. Screening of a mouse brain cDNA library using a human PAF-AH Iba/LIS1 cDNA probe resulted in multiple phage clones. Two of these (lcDNA-9 and lcDNA-15) contained the entire coding region and had poly(A) tails at nearby nucleotide positions (2204 and 2247, respectively), and a third clone (lcDNA-10) had no poly(A) (Fig. 1A). Extension of the 3* UTR by 3* RACE indicated that there were three more sites of polyadenylation at nucleotide positions 5143 (3* RACE-2), 5224 (3* RACE-11), and 5810 (3* RACE-4). The longest cDNA clone probably represents the full-length message, as shown by the ubiquitous Ç6.0-kb major band on a Northern blot hybridized with a probe (probe A) covering the coding region of the cDNA (Figs. 1A and 1B). An Ç5.4-kb message in brain and, to a lesser extent, in kidney and liver, was detectable, as was an Ç4.4-kb heart-specific and an Ç2.3kb ubiquitous message most abundantly expressed in testis. To test if the alternatively polyadenylated cDNAs represent the various transcripts detected on the Northern blot, we reprobed the blot with probes corresponding to different regions of the 3* UTR (Fig. 1A). Probe B, which lies 0.2–1.7 kb downstream of the translation stop codon, hybridizes to both the Ç6.0and the Ç5.4-kb messages. However, probe C, which is specific for the longest cDNA, only detects the Ç6.0kb transcript. These results confirm that, with the exception of the Ç4.4-kb heart-specific message, the various Pafaha/Lis1 transcripts observed on the Northern blot arise by differential polyadenylation. Genomic Organization of the Pafaha/Lis1 Gene To determine the exon–intron organization of the Pafaha/Lis1 gene, we screened a mouse genomic library with a cDNA-derived probe and obtained several lambda clones. Exon-containing genomic fragments of two phage inserts (l4/1 and l12/2) (Fig. 2) were identified by Southern blotting, subcloned, and sequenced. Since multiple attempts to extend the phage contig by conventional library screening were unsuccessful, we pursued a PCR-based walking approach using adaptorligated genomic DNA. The Pafaha/Lis1 gene consists of 12 exons and spans more than 50 kb of genomic DNA. The accurate size of the genomic region could not be determined, because of a gap in the fragment contig
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FIG. 1. Alternative polyadenylation and expression pattern of the Pafaha/Lis1 gene. (A) The lambda clones represent three Pafaha/ Lis1 cDNAs isolated from a mouse brain cDNA library. lcDNA-10 was extended by 3 * RACE. The solid box indicates the coding region, and the positions of hybridization probes are shown. (B) Northern blot analysis using the probes shown in (A). 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; and 8, testis. Each lane contains Ç2 mg poly(A)/ RNA (Clontech).
in the second intron of the gene (Fig. 2). The first two exons are untranslated, and the third exon contains the rest of the 5* UTR and the ATG translation initiation codon. Two CpG islands (Gardiner-Garden and Frommer, 1987) were identified in the 5* region of the gene: one including part of exon 1 and possibly ex-
tending to intron 1, the other covering exon 2 (Fig. 2). The last exon contains 75 bp of translated sequence, the TGA translation termination site, and 3714 bp of 3* UTR. All exon sequences, including exon 12 with the alternative polyadenylation sites, are identical to those of the cDNA. All exon–intron boundaries were
FIG. 2. Genomic organization of the mouse Pafaha/Lis1 gene. Exons are boxed and numbered. Open and solid boxes represent noncoding and coding exons, respectively. Hatched boxes represent longer forms of exons 2 and 11 (2a and 11a), resulting from alternative SD site usage. Translation initiates on exon 3 and terminates on exon 12. Lambda phage clones and PCR fragments are shown under the gene. The positions of CpG islands are indicated.
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TABLE 1 Exon–Intron Organization of the Pafaha/Lis1 Gene Exona 1 2 2a 3 4 5 6 7 8 9 10 11 11ae 12
cDNA positionb
Exon size (bp)
1–242 243–681 682–891 892–976 977–1051 1052–1258 1259–1427 1428–1530 1531–1759 1760–1861 1862–2018
242 439 1237 210 85 75 207 169 103 229 102 157
2019–5810
3792
Splice acceptor c
aggccag aggccag tttttag ctttcag ctcctag tctcaag ttgacag gttttag ttgacag ttaatag ctttaag ctttaag ctttcag
GCCCTCC GCCCTCC GTGGAAT A AAT CGA AAT GAA G GTA ATG G GTG TGG G GT CAC GA C TAC TGT ACT AAA A GTG GGT C GTG GGT C AT TTC CA
Splice donor c GCCGCGG CGCTCCG CTCCCAG AT GAA CT A GAT ATG A AAA AAG A ATT AAG ATG CAC G AA ACT GG A TCT GAG G ACT CTT TCC TTG G
ND gtaaggc gtgagaa gtaagtt gtatgct gtaacta gcaagtt gtaaggt gtaagta gtactgt gtaagtt gtatgta
Intron size (kb)d 10.0* ú16 ú16 8.3** 0.238 1.026 2.691 1.362 0.713 1.135 2.621 1.716
a
Suffixes refer to alternative exons resulting from alternative splicing. The longer forms of exon 2 (2a) and exon 11 (11a) are not included in the numbering. c Exon and intron sequences are in uppercase and lowercase letters, respectively. Splice site consensus nucleotides conforming to the ag/ gt rule are in boldface. ND, not determined. d Intron sizes were determined by PCR (*), restriction mapping (**), or sequencing. The minimum size estimated for intron 2 is based on available intron sequences. e Exon 11a was identified based on a cDNA (L25109) described elsewhere (Reiner et al., 1995). Since this cDNA has no poly(A) tail, the size of exon 11a is unknown. b
sequenced except for the splice donor (SD) site of exon 1. The splice sites conform to the AG/GT rule except for the SD site of intron 7, which begins with the rarely occurring consensus sequence GCAAG (Shapiro and Senapathy, 1987) (Table 1). Although the human gene has only 11 exons, the overall exon–intron organization is highly conserved between the two species (Lo Nigro et al., 1997). Alternative Splicing of Pafaha/Lis1 The Pafaha/Lis1 cDNA sequence shows very high overall similarity to the corresponding human and bovine sequences (Pe´terfy et al., 1994). It was unexpectedly found, however, that the similarity between the bovine (GenBank Accession No. D30615) and the mouse cDNAs abruptly changes at the exon 2/exon 3 boundary (data not shown). While there is over 80% nucleotide identity (including UTRs) downstream of exon 2, the first 655 bp of the bovine cDNA does not show any homology to exon 1 or exon 2 sequences. This finding raised the possibility that instead of the bovine homolog of mouse exon 2, an alternative exon is utilized in the bovine brain. This hypothesis was supported by the identification of a mouse genomic sequence 175 bp downstream of exon 2 showing significant similarity (75%) to the 5* end of the bovine cDNA. Moreover, a consensus SD site was found in the mouse genomic sequence at the end of the homology region (data not shown). These results predicted a novel mouse exon not represented in the mouse cDNA clones obtained by our initial library screening. To determine whether the predicted new SD site was
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functional, we performed PCR on mouse brain cDNA. Two primer pairs were used to detect splicing of the putative novel exon to exon 2 and exon 3 (Fig. 3A). The 390-bp product amplified with primers C and D verified the splicing between the predicted SD site and the splice acceptor site of exon 3. The nucleotide sequence of the 410-bp PCR product obtained with primers A and B was identical to the genomic sequence, which indicated that no splicing occurred in this genomic region. To confirm alternative splicing of exon 2 further, we hybridized a Northern blot with a probe specific for the alternative exon (Fig. 3A). An Ç2.3-kb message was detected in testis (Fig. 3B), corresponding in size to the smallest transcript observed with the coding region probe (probe A; Fig. 1B). A weaker band of Ç1.4 kb was also detected in heart. It should be noted that the hybridization signal obtained with the exon 2-specific probe was significantly weaker than that detected with probe A, suggesting that only a fraction of the Ç2.3-kb message contains the alternatively spliced exon (see Discussion). Taken together, an alternative splice variant of the Pafaha/ Lis1 message was identified, resulting in an extended form of exon 2 (exon 2a) (Fig. 2 and Table 1). Database searches with Pafaha/Lis1 genomic sequences revealed an additional alternative splicing site at exon 11. We found that intron 11 was not spliced out in a mouse Pafaha/Lis1 cDNA clone (GenBank Accession No. L25109; Reiner et al., 1995) isolated from a brain cDNA library, indicating that the SD site of exon 11 was unrecognized by the splicing machinery in the corresponding transcript (Fig. 2). As a result,
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FIG. 3. Alternative splicing of the Pafaha/Lis1 gene. (A) Verification of alternative splicing of exon 2 by RT-PCR. Open boxes indicate exons (exon 2 and 3) that are represented in the Pafaha/Lis1 cDNAs obtained by library screening. The hatched box indicates the mouse genomic region showing homology to a bovine cDNA (D30615). The positions of PCR primers A, B, C, and D are indicated by arrows and the hybridization probe by a black bar. The predicted SD site (SDP) was confirmed by sequence analysis of the 390-bp PCR product. (B) Northern blot analysis of alternative splicing using the probe shown in (A).
translation is expected to be terminated by an in-frame stop codon in exon 11a, giving rise to a truncated form of the Pafaha/Lis1 polypeptide. DISCUSSION
The PAF-AH Iba/LIS1 gene codes for the 45-kDa noncatalytic subunit of the brain-type intracellular PAF acetylhydrolase. Defects in this gene have been proposed to be responsible for isolated lissencephaly sequence and to contribute to Miller–Dieker syndrome, two devastating neurological disorders (Lo Nigro et al., 1997). In an effort to characterize the mouse ortholog of the PAF-AH Iba/LIS1 gene, we report the cloning of several mouse Pafaha/Lis1 cDNAs and the structural organization of the mouse Pafaha/Lis1 gene. The five forms of cDNA obtained from a mouse brain cDNA library result from the utilization of different polyadenylation signals by the nuclear machinery (Fig. 1). Alternative polyadenylation was also observed in human PAF-AH Iba/LIS1 cDNAs (Lo Nigro et al., 1997). Interestingly, the expression pattern of the different Pafaha/ Lis1 mRNA species is markedly different in the tissues tested (Fig. 1B). Certain messages (e.g., Ç4.4-kb mRNA in heart) are tissue specific, while others (e.g., Ç6.0-kb mRNA) are ubiquitously expressed. Although the exact role of the differential polyadenylation of the Pafaha/Lis1 transcript is not known, the presence or absence of cis-acting elements in the 3* UTR may influence the structural and/or functional characteristics of the message in a tissue-specific manner (Decker and Parker, 1995). We used a PCR-based approach to extend genomic phage clones obtained by conventional library screening, because we could not isolate overlapping clones
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covering the entire Pafaha/Lis1 gene. Interestingly, the 5* part of the human gene also proved to be unclonable in cosmids (Chong et al., 1997). The analysis of the Pafaha/Lis1 gene revealed that it spans over 50 kb of genomic DNA and contains 12 exons, ranging in size from 75 (exon 5) to 3792 bp (exon 12). The large size (9.6 and ú16 kb) of the 5* introns is fairly typical; the presence of two 5* noncoding exons, however, is uncommon among vertebrate genes (Hawkins, 1988). Despite a highly similar exon–intron organization, the human gene has only 11 exons, lacking a homologue of the first noncoding exon of the mouse (Lo Nigro et al., 1997). In accordance with the ubiquitous expression of the Pafaha/Lis1 gene, a CpG island was found to cover the predicted transcriptional start site, and a separate CpG island was located on exon 2 (Fig. 2). As a result, the 5* UTR of the mature transcript is highly GC rich (74% GC content in the first 680 nucleotides). Although most 5* CpG islands extend past the translation start site (Gardiner-Garden and Frommer, 1987), no CpG-rich sequences were associated with exon 3, containing the ATG translation initiator. Sequence comparison between intron 2 of the mouse gene and a bovine PAF-AH Iba/LIS1 cDNA showed significant sequence similarity and predicted that an alternative SD site, 799 bp downstream of exon 2, may be utilized, resulting in a longer form of this exon (exon 2a) (Fig. 2). Alternative splicing of exon 2 was verified by RT-PCR and Northern blot analysis (Fig. 3). Although exon 2a splicing was detectable in brain by PCR, testis is the most significant site of expression of the alternative transcript, as shown by Northern blot hybridization (Fig. 3B). Despite the presence of the longer form of exon 2, the size of the alternative transcript is similar (Ç2.3 kb) to those containing the
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shorter form of exon 2, suggesting additional alternative splicing or the existence of an alternative transcriptional start site in testis. Supporting the latter possibility is the considerable length of exon 2a (1237 bp), which strongly argues against it being an internal exon (Berget, 1995; Hawkins, 1988). Therefore, we predict the presence of an alternative promoter in exon 2 of the mouse gene. The relatively high degree of sequence conservation (over 75% identity) of 5* UTRs in the mouse, bovine, and human cDNAs points to the functional importance of this region. Various features of the leader sequences of mRNAs are known to influence the efficiency of translation (Kozak, 1991a). In particular, 5* UTRs with multiple upstream AUG codons (‘‘AUG-burdened leader sequences’’) impair translation of the authentic ORF, due to the inefficient reinitiation by the ribosomes after translating a short 5* ORF (Kozak, 1991b). Sequence analysis of the alternatively spliced exon 2a reveals that it contains two AUG codons in favorable contexts followed by in-frame translation stop codons within 144 and 27 bp. Therefore, alternative splicing of exon 2 may be a mechanism for regulating the efficiency of translation of the Pafaha/Lis1 message in response to certain developmental or environmental stimuli. Supporting the possibility of such a posttranscriptional control mechanism is the recent demonstration that the extension by alternative splicing of one of the 5* untranslated exons of the GM-CSFRa message reduces in vitro translation efficiency (Chopra et al., 1996). Recently, a mouse cDNA was described (Lis3-4; GenBank Accession No. L25109), which was identical to the Pafaha/Lis1 cDNA sequence at its 5* end, but showed no similarity at its 3* end. The authors concluded that the corresponding transcript derived from a gene different from Pafaha/Lis1 (Reiner et al., 1995). Here we show that the 3* nonhomologous end of Lis34 is identical to intron 11 sequences of the Pafaha/ Lis1 gene. This suggests that Lis3-4 is derived from the Pafaha/Lis1 locus by alternative splicing of intron 11, resulting in a longer form of exon 11 (exon 11a) (Fig. 2). Since Lis3-4 has no poly(A) tail, we do not know whether the entire intron 11 or only part of it is present in this message. Due to an in-frame stop codon in exon 11a, the resulting ORF is 57 bp shorter than the one including exon 12 sequences. The deduced protein encoded by the alternatively spliced transcript is not only shorter than the longer form by 19 amino acids, but also contains five C-terminal residues not present in the full-length product. Thus, this truncation results in the loss of most of the last GH-WD motif of the seven repeat units in the Pafaha/Lis1 protein (Neer et al., 1993). The functional consequences of the truncation remain to be determined. In conclusion, we determined the exon–intron structure of the mouse Pafaha/Lis1 gene and showed that the primary transcript undergoes alternative splicing and polyadenylation in a tissue-specific manner. These
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data will facilitate functional studies of the Pafaha/ Lis1 gene. ACKNOWLEDGMENTS We thank Dr. F. K. Lin and Dr. L. G. Ko˝mu¨ves for useful comments on the manuscript.
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