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• W2069192199 abstract "Analysis of a transgene-induced mutation at the mouse med locus led to the identification of the novel voltage-gated sodium channel gene Scn8a (Burgess, D. L., Kohrman, D. C., Galt, J., Plummer, N. W., Jones, J. M., Spear, B., and Meisler, M. H. (1995) Nat. Genet. 10, 461-465). We now report the identification of splicing defects in two spontaneous mutations of Scn8a. The original med mutation was caused by insertion of a truncated LINE element into exon 2 of Scn8a. The med transcript is spliced from exon 1 to a cryptic acceptor site in intron 2. A 4-base pair deletion within the 5′ donor site of exon 3 in the medJ allele results in splicing from exon 1 to exon 4. Both mutant transcripts have altered reading frames with premature stop codons close to the N terminus of the protein. Loss of Scn8a expression results in progressive paralysis and early death. Intron 2 of Scn8a is flanked by minor class AT-AC splice sites. The observed splicing patterns of the med and medJ mutant transcripts provide the first evidence for preferential in vivo splicing between donor and acceptor sites of the same class. The apparent functional incompatibility may be a consequence of the different composition of spliceosomes bound to major and minor splice sites. Analysis of a transgene-induced mutation at the mouse med locus led to the identification of the novel voltage-gated sodium channel gene Scn8a (Burgess, D. L., Kohrman, D. C., Galt, J., Plummer, N. W., Jones, J. M., Spear, B., and Meisler, M. H. (1995) Nat. Genet. 10, 461-465). We now report the identification of splicing defects in two spontaneous mutations of Scn8a. The original med mutation was caused by insertion of a truncated LINE element into exon 2 of Scn8a. The med transcript is spliced from exon 1 to a cryptic acceptor site in intron 2. A 4-base pair deletion within the 5′ donor site of exon 3 in the medJ allele results in splicing from exon 1 to exon 4. Both mutant transcripts have altered reading frames with premature stop codons close to the N terminus of the protein. Loss of Scn8a expression results in progressive paralysis and early death. Intron 2 of Scn8a is flanked by minor class AT-AC splice sites. The observed splicing patterns of the med and medJ mutant transcripts provide the first evidence for preferential in vivo splicing between donor and acceptor sites of the same class. The apparent functional incompatibility may be a consequence of the different composition of spliceosomes bound to major and minor splice sites. Mutations at the mouse locus “motor endplate disease” (med) on distal chromosome 15 result in a recessive neuromuscular disorder (Green, 15Green M. Lyon M.F. Searle A.G. Catalog of Mutant Genes and Polymorphic Loci: Genetic Variants and Strains of the Laboratory Mouse. 2nd Ed. Oxford University Press, Oxford U. K.1989: 234Google Scholar). The original med mutation arose in Edinburgh in 1958, and the medJ allele was identified at The Jackson Laboratory a few years later (Sidman et al., 39Sidman R.L. Cowen J.S. Eicher E.M. Ann. N. Y. Acad. Sci. 1979; 317: 497-505Crossref PubMed Scopus (44) Google Scholar). Both mutations result in severe, progressive skeletal muscle atrophy that is lethal within the first month after birth (Duchen et al., 11Duchen L.W. Searle A.G. Strich S.J. J. Physiol. (Lond.). 1967; 189: 4Google Scholar; Sidman et al., 39Sidman R.L. Cowen J.S. Eicher E.M. Ann. N. Y. Acad. Sci. 1979; 317: 497-505Crossref PubMed Scopus (44) Google Scholar). Electrophysiological analyses demonstrated that the muscle atrophy in med and medJ mice is secondary to a loss of functional innervation (Duchen and Stefani, 10Duchen L.W. Stefani E. J. Physiol. (Lond.). 1971; 212: 535-548Crossref Scopus (72) Google Scholar; Angaut-Petit et al., 3Angaut-Petit D. McArdle J.J. Mallart A. Bournaud R. Pincon-Raymond M. Riegers F. Proc. R. Soc. Lond. B Biol. Sci. 1982; 215: 117-125Crossref PubMed Scopus (30) Google Scholar). The number of motor neurons in the spinal cord of mutant mice is not reduced, but transmission of the action potential across the neuromuscular junction is impaired. The med allele also produces a cerebellar ataxia associated with a loss of spontaneous electrical activity in cerebellar Purkinje cells (Dick et al., 8Dick D.J. Boakes R.J. Harris J.B. Neuropathol. Appl. Neurobiol. 1985; 11: 141-147Crossref PubMed Scopus (26) Google Scholar). A mild allele, medjo, exhibits cerebellar ataxia only (Harris and Pollard, 19Harris J.B. Pollard S.L. J. Neurol. Sci. 1986; 76: 239-253Abstract Full Text PDF PubMed Scopus (25) Google Scholar; Harris et al., 21Harris J.B. Boakes R.J. Court J.A. J. Neurol. Sci. 1992; 110: 186-194Abstract Full Text PDF PubMed Scopus (20) Google Scholar). We recently identified a new allele of med that was generated by nontargeted transgene insertion (Kohrman et al., 28Kohrman D.C. Plummer N.W. Schuster T. Jones J.M. Jang W. Burgess D.L. Galt J. Spear B.T. Meisler M.H. Genomics. 1995; 26: 171-177Crossref PubMed Scopus (38) Google Scholar). Analysis of the transgene insertion site led to isolation of a novel voltage-gated sodium channel gene designated Scn8a (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar). Loss of expression of Scn8a in the transgenic mice is responsible for the neuromuscular disorder. Scn8a encodes a 1732-amino acid member of the voltage-gated sodium channel alpha subunit gene family. These channels mediate the influx of sodium ions in response to changes in membrane potential in electrically excitable cells such as neurons and muscle, and are responsible for the rising phase of the action potential (Catterall, 7Catterall W.A. Physiol. Rev. 1992; 72: S15-S48Crossref PubMed Google Scholar). Expression of Scn8a is limited to brain and spinal cord (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar). The rat ortholog of Scn8a is expressed in both neurons and glia and is widely distributed in many regions of the brain including cerebellar granule cells and pyramidal and granule cells of the hippocampus (Schaller et al., 36Schaller K.L. Krzemien D.M. Yarowsky P.J. Krueger B.K. Caldwell J.H. J. Neurosci. 1995; 15: 3231-3242Crossref PubMed Google Scholar). The human SCN8A gene has been mapped to a conserved linkage group on chromosome 12q13 (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar) and is a candidate gene for inherited neuromuscular disease. To determine the molecular defect in the spontaneous mutants med and medJ, we analyzed the structure and expression of Scn8a in the mutant mice. Both mutations described in this report interfere with splicing of the Scn8a transcript and are predicted to encode nonfunctional proteins. Analysis of the unusual pattern of exon skipping in the mutants identified Scn8a as a member of a small group of genes containing introns with nonstandard AT-AC splice sites (Jackson, 25Jackson I.J. Nucleic Acids Res. 1991; 19: 3795-3798Crossref PubMed Scopus (277) Google Scholar; Hall and Padgett, 17Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (184) Google Scholar). It has recently been demonstrated that AT-AC introns are processed by alternative splicing machinery that includes U11 and U12 snRNPs (Tarn and Steitz, 42Tarn W.Y. Steitz J.A. Cell. 1996; 84: 801-811Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar; Hall and Padgett, 18Hall S.L. Padgett R.A. Science. 1996; 271: 1716-1718Crossref PubMed Scopus (168) Google Scholar). The med allele arose as a spontaneous mutation in a multiple recessive stock (Duchen et al., 11Duchen L.W. Searle A.G. Strich S.J. J. Physiol. (Lond.). 1967; 189: 4Google Scholar) and has been maintained at the University of Newcastle since 1970. The medJ allele arose on a stock carrying the caracul mutation (Sidman et al., 39Sidman R.L. Cowen J.S. Eicher E.M. Ann. N. Y. Acad. Sci. 1979; 317: 497-505Crossref PubMed Scopus (44) Google Scholar) and was obtained in 1994 from the frozen embryo repository of The Jackson Laboratory (Bar Harbor, ME). Poly(A)+ RNA, 2 µg, was electrophoresed through 1.2% agarose in the presence of 6% formaldehyde and transferred to a Zetaprobe GT filter (Bio-Rad) in 20× standard sodium citrate. The filter was rinsed in 2× standard sodium citrate, baked at 80°C for 30 min, and prehybridized at 65°C for 30 min in 20 ml of 0.25 M Na2HPO4, pH 7.2, containing 7% SDS and 1 mM EDTA. An RT-PCR 1The abbreviations used are: RT-PCRreverse transcription-polymerase chain reactionbpbase pair(s). product corresponding to nucleotides 1 through 771 of the Scn8a cDNA was gel-purified and labeled to a specific activity of 1 × 109 cpm/µg using the Readi-Prime random hexamer labeling kit (Amersham). The filter was hybridized with denatured probe (5 × 106 cpm/ml) overnight at 65°C in 10 ml of prehybridization solution and washed at 65°C to a final stringency of 0.2× standard sodium citrate. The filter was exposed at −70°C to Kodak XAR-5 film with an intensifying screen. reverse transcription-polymerase chain reaction base pair(s). Fourteen primers corresponding to Scn8a cDNA sequence (GenBank™) or genomic sequence from a P1 clone (GenBank™) were used in this study. Primer sequences (5′ to 3′) are: a, ACAGA TCTCA CGAGA TGAGA AGATG GCAGC G; b, TTTCA TCTAC GGGGA CATCC C; c, GGAGC AAGGT TCTAG GCAGC TTTAA GTGTG; d, CAGAT TTAGC GCCAC TCCTG CCTTG; e, GGACT TAGAA TGTAC AAGGC AGGAG; f, GTCAA AGCCC CGGAC GTGCA CACTC ATTCC; g, CAGAT GGGAT GTCCA GGGCT TTACC TTGTG; h, GTCTT CAGCA TGATC ATCAT GTGCA CCATC; i, CATAA ATACA CAGTT GGTCA AAATN GTGCA CAT; j, CTGTG GAGCA ACAGA ACCAG ACTCT ATGCG; k, TCTAT GCAGA AACCT CTGGC; l, CTAGG CTATA TGGTG AGTTC AAGGA CAACC; m, GTCTG ACAGC TTCTT CACAG ACTGG; n, TCTCC AGATA GCTCT CGTTG AAGTT TATGG. Primer a contains a 10-bp BglII linker at the 5′ end. Genomic DNA was prepared from spleens of mice homozygous for the med or medJ mutation by protease digestion and phenol/chloroform extraction. Genomic DNA from strains C57BL/6J-Ca sl and AKR.C3H-Ca hm sl carrying the caracul mutation was purchased from The Jackson Laboratory. Total RNA was prepared from brains of 10-15-day-old animals using the Trizol reagent (Life Technologies, Inc.). First strand cDNA was synthesized from 10 µg of RNA with a random hexamer primer using the Superscript RT Preamplification System (Life Technologies, Inc.) according to the manufacturer's instructions. First strand cDNA was used as template in 50-µl PCR reactions containing 50 mM KCl, 0.01% gelatin, 1.5 mM MgCl2, 0.01% Nonidet P-40, 0.01% Tween 20, 200 µM dNTP, and 0.5 µM primers in 10 mM Tris, pH 8.3. After 5 min at 97°C, 2 units of Taq DNA polymerase were added, and reactions were incubated for 30 cycles at 94°C for 30 s, 60-65°C for 30 s, and 72°C for 1-3 min. The same conditions were used for PCR of genomic DNA, with 100 ng of DNA as template. P1 clone 2206 containing the Scn8a gene (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar) and the overlapping P1 clone 3F2-4A were digested with SacI (Boehringer Mannheim) and cloned in the vector pGEM-5zf (Promega) in Escherichia coli strain DH5α. Ampicillin-resistant colonies were arrayed in microtiter dishes, replicated onto nylon filters (Hybond N, Amersham), and hybridized with an RT-PCR product containing nucleotides 1-895 of the Scn8a cDNA (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar) to isolate subclones containing exons 1-4. After gel electrophoresis and visualization by ethidium bromide fluorescence, PCR products were excised and purified with QIAEX beads (QIAGEN) according to the manufacturer's instructions. The purified DNA was sequenced directly by dideoxynucleotide incorporation using Taq DNA polymerase and analyzed with an ABI373A automated sequencer. Public sequence data bases were searched using the BLAST algorithm (Altschul et al., 2Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (68368) Google Scholar) via a WWW server. MacVector 3.5 software (Kodak/IBI) was used for sequence alignment. Abnormal Scn8a Transcripts in med and medJ—Northern analysis of poly(A)+ RNA from brain was carried out using an Scn8a cDNA probe. Two transcripts of ∼10 and 12 kilobases are present in wild-type C57BL/6J brain (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar) (Fig. 1A). Transcripts of similar length and abundance were detected in brain RNA from both mutants (Fig. 1A). To detect small differences in transcript length, six overlapping RT-PCR products spanning the coding region of the Scn8a mRNA were amplified from mutant and wild-type brain RNA, as described previously (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar). Mutant and wild-type RT-PCR products were compared by electrophoresis on 1.5% agarose gels. All of the products were of normal length except those amplified with primers a plus m. These primers amplify a 771-bp product from wild-type RNA (Fig. 1B) that corresponds to nucleotides 1-771 of the cDNA sequence. Two products of 756 and 351 bp were obtained from med RNA, and a single product of 572 bp from medJ RNA (Fig. 1B). These data suggest that the med and medJ mutations are located within the 5′ portion of the gene. Intron/Exon Structure of Scn8a To define the intron/exon structure of the 5′ region of Scn8a, genomic sequence from mouse P1 clones was aligned with the wild-type cDNA sequence (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar) Exons 1 through 4 were analyzed by sequencing P1 subclones using the primers b, d, g, i, k, and vector primers (Fig. 2.) The predicted splice sites (Table I) correspond well to standard consensus sites, with the exceptions of the donor site downstream of exon 2 and the acceptor site upstream of exon 3. These sites belong to a newly recognized, minor class of splicing signals (Hall and Padgett, 17Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (184) Google Scholar; Tarn and Steitz, 42Tarn W.Y. Steitz J.A. Cell. 1996; 84: 801-811Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). The effect of these nonstandard sites on processing of the mutant transcripts is discussed below.Table IPartial intron/exon structure of Scn8αExonLengthSplice acceptorSplice donorT Q K1276ACGCAGAAAgagtxggaT F VI H S2119aatggtttctctctctctttctccacACTTTTGTAATACATTCatccttttV F SN V E390cctcgctcgccccttaactcctctctAGTCTTCAGCAATGTGGAaagtaacaY T FM M A4129taacctcagctgtccgcatccctgacGTACACATTCATGATGGCgaggtccc Open table in a new tab The partial structure of Scn8a is shown in Fig. 2. The positions of the identified splice junctions are identical with those described for the human muscle sodium channel gene SCN4A (McClatchey et al., 31McClatchey A.I. Lin C.S. Wang J. Hoffman E.P. Rojas C. Gusella J.F. Hum. Mol. Genet. 1992; 1: 521-527Crossref PubMed Scopus (48) Google Scholar; George et al., 13George A.L. Iyer G.S. Kleinfield R. Kallen R.G. Barchi R.L. Genomics. 1993; 15: 598-606Crossref PubMed Scopus (58) Google Scholar). The 756- and 351-bp RT-PCR products from med brain (Fig. 1B) were isolated and sequenced. Alignment with wild-type cDNA and genomic sequences demonstrated that the 756-bp product lacks exon 2 and results from splicing between the normal donor site of exon 1 and a cryptic acceptor site in intron 2 (Fig. 3). The intron sequence preceding the cryptic site conforms to the consensus for standard acceptor sites, including the canonical AG. The resulting out-of-frame transcript contains a stop codon derived from exon 3 and is predicted to encode a severely truncated protein of 127 residues. RNase protection analysis confirmed that the 756-bp RT-PCR product corresponds to the most abundant Scn8a transcript in med brain (not shown). The 351-bp RT-PCR product lacks exons 2, 3, 4, and 5 and results from splicing between the normal donor site of exon 1 and the normal acceptor site of exon 6. The transcript contains a stop codon derived from exon 6 and is predicted to encode a truncated protein of 93 residues. This product was also detected at low levels in several samples of wild-type brain RNA. To determine the basis for omission of exon 2 from the major med transcript, exon 2 was amplified from med/med genomic DNA using the primers shown in Fig. 2. Primers c plus e amplified a product of ∼280 bp in wild-type DNA and 460 bp in med DNA (Fig. 4, lanes 1 and 2). Primers c plus f amplified a product of ∼190 bp in the wild type and 370 bp in med (Fig. 4, lanes 4 and 5). Primers d plus f amplified the same product of 130 bp from wild-type and med DNA (Fig. 4, lanes 7 and 8). The data are consistent with the insertion of ∼180 bp of DNA into the 5′ half of exon 2. The PCR product obtained from med DNA with primers c plus f was sequenced. Comparison with wild-type exon 2 identifed a 180-bp insert containing 72 bp from the 3′-untranslated region of an L1 repetitive element, followed by a poly(A) tail of ∼110 bp that corresponds to a polyuridine tract in the primary transcript (Fig. 5). The transcriptional orientation of the truncated L1 element is opposite to that of Scn8a. The insertion is flanked by a 12-bp repeat, a characteristic feature that is believed to originate from staggered breaks in genomic DNA at the site of L1 integration (Hutchison et al., 24Hutchison III, C.A. Hardies S.C. Loeb D.D. Shehee W.R. Edgell M.H. Mobile Genetic Elements. Academic Press, New York1983: 593Google Scholar). Other polypyrimidine insertions interfere with splice site cleavage (Furdon and Kole, 12Furdon P.J. Kole R. Mol. Cell. Biol. 1988; 8: 860-866Crossref PubMed Scopus (41) Google Scholar). The splice sites flanking exons 2 and 3 in med genomic DNA were identical with the wild type. The 572-bp RT-PCR product from medJ brain RNA (Fig. 1B) was gel purified and sequenced. Alignment with wild type cDNA demonstrated that the medJ product lacks exons 2 and 3 and is the product of splicing from the normal donor site of exon 1 to the normal acceptor site of exon 4 (Fig. 6). The aberrant splicing alters the reading frame so that the transcript contains a stop codon derived from exon 4 and encodes a predicted protein of only 101 residues. RNase protection analysis confirmed that the 572-bp RT-PCR product is derived from the major Scn8a transcript in medJ brain (not shown). To determine the basis for the absence of exons 2 and 3 in the medJ transcript, the splice sites adjacent to exons 2, 3, and 4 were sequenced. Genomic DNA from a homozygous medJ animal was amplified with primers c plus l (Fig. 2). The 2.3-kilobase PCR product containing exons 2, 3, and 4 was gel purified and sequenced with primers c, g, and k. Comparison of the splice donor site of exon 3 from medJ with the wild-type sequence identified a 4-bp deletion that results in replacement of the G at the +5 position with a C (Fig. 7). The +5 G is highly conserved in eukaryotic splice donor sites (Shapiro and Senapathy, 38Shapiro M.B. Senapathy P. Nucleic Acids Res. 1987; 15: 7155-7174Crossref PubMed Scopus (1951) Google Scholar), and mutations at this position cause aberrant splicing in mammalian genes (Nakai and Sakamoto, 33Nakai K. Sakamoto H. Gene (Amst.). 1994; 141: 171-177Crossref PubMed Scopus (255) Google Scholar). Normal sequences were present at the exon 2 donor and acceptor sites and the acceptor sites for exons 3 and 4 of the medJ allele. The 4-bp deletion is thus likely to be responsible for the abnormal splicing of the medJ transcript. The medJ mutation arose on a chromosome carrying the mutant caracul allele Ca (Sidman et al., 39Sidman R.L. Cowen J.S. Eicher E.M. Ann. N. Y. Acad. Sci. 1979; 317: 497-505Crossref PubMed Scopus (44) Google Scholar). ca and med have not recombined in the intervening 40 years because the two loci are very closely linked, at a distance of <0.2 centimorgan (Beechy and Searle, 4Beechy C.V. Searle A.G. Mouse News Lett. 1984; 71: 28Google Scholar). To test the presumed ancestral chromosome for the splice site deletion, genomic DNA samples from two independent strains carrying the same Ca allele were amplified with primers h plus j, and the products were sequenced. Both strains had the wild-type donor site (Fig. 7), indicating that the 4-bp deletion was not present on the progenitor Ca chromosome. The deletion was also absent in eight other inbred strains tested (data not shown). Although the sequences of most 5′ and 3′ splice sites are highly conserved throughout eukaryotic genomes, a minor class of introns containing nonstandard splice sites has recently been recognized (Shapiro and Senapathy, 38Shapiro M.B. Senapathy P. Nucleic Acids Res. 1987; 15: 7155-7174Crossref PubMed Scopus (1951) Google Scholar; Jackson, 25Jackson I.J. Nucleic Acids Res. 1991; 19: 3795-3798Crossref PubMed Scopus (277) Google Scholar; Hall and Padgett, 17Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (184) Google Scholar). The minor introns, which were identified in three mammalian genes and one Drosophila gene, lack the standard GT and AG dinucleotides at splice donor and acceptor sites and are similar in organization to introns from yeast. Consensus sequences for the standard and nonstandard splice sites are compared in Table II. The functional compatibility of standard and nonstandard sites has not been experimentally tested, but they have recently been shown to bind different snRNAs and proteins (Tarn and Steitz, 42Tarn W.Y. Steitz J.A. Cell. 1996; 84: 801-811Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar).Table IINonstandard splice sites in intron 2&of Scn8aSplice donorBranch siteSplice acceptorStandard&AAGTTNCTRACYYYYYYYYYYYYNCNonstandardATATCCTTTCCTTAACYCCACMouse Scn8a……..C…….CTTAACTCCTC..T..Human SCN8A……..C….G..CTTGACTCTTC..T..Human SCN4A…….G…..G..CTTGACCCTGC….. Open table in a new tab Comparison of the unusual splice sites from Scn8a (Table I) with the consensus sequences indicates that intron 2 of Scn8a is a member of this minor class of introns (Table II). The Scn8a sequences are identical at 8 of 8 nucleotides with the invariant nonstandard splice donor site, and at 7 of 8 and 4 of 5 nucleotides with the branch site and splice acceptor sites of the nonstandard class, respectively. The nonstandard splice sites are conserved in the human ortholog, SCN8A, and in the more distantly related human muscle sodium channel gene SCN4A (Table II), indicating that the nonstandard intron predates the divergence of brain and muscle sodium channel genes. We have identified the molecular defects in two spontaneous mutant alleles of the Scn8a locus, med and medJ. Normal levels of mRNA are present in both mutants, but exon skipping results in altered reading frames with premature stop codons. The predicted proteins are truncated within the first of the four transmembrane domains of the sodium channel α subunit and are unlikely to retain channel function. med and medJ can therefore be classified as null alleles of Scn8a, like the transgene-induced allele medtg. The loss of expression of the Scn8a sodium channel in spinal motor neurons results in progressive failure of neuromuscular transmission, with muscle atrophy as a secondary effect. This conclusion is supported by a large body of experimental data on the pathophysiology of the med and medJ mutants. The skeletal muscle fibers of med/med animals are responsive to direct electrical stimulation (Duchen and Stefani, 10Duchen L.W. Stefani E. J. Physiol. (Lond.). 1971; 212: 535-548Crossref Scopus (72) Google Scholar) and develop normally when transplanted into wild-type mice (Zacks and Sheff, 44Zacks S.I. Sheff M.F. Lab. Invest. 1977; 36: 303-309PubMed Google Scholar). The number and morphology of spinal motor neurons is normal (Harris and Pollard, 19Harris J.B. Pollard S.L. J. Neurol. Sci. 1986; 76: 239-253Abstract Full Text PDF PubMed Scopus (25) Google Scholar; Goldstein et al., 14Goldstein L.A. Porter J.D. Kasarskis E.J. Spear B.T. Exp. Neurol. 1996; 139: 328-334Crossref PubMed Scopus (13) Google Scholar) but the conduction velocity of motor axons is reduced, consistent with a sodium channel defect (Angaut-Petit et al., 3Angaut-Petit D. McArdle J.J. Mallart A. Bournaud R. Pincon-Raymond M. Riegers F. Proc. R. Soc. Lond. B Biol. Sci. 1982; 215: 117-125Crossref PubMed Scopus (30) Google Scholar). Spontaneous endplate potentials have been recorded in nerve/muscle preparations from homozygous med mice, demonstrating that the components of the neurotransmitter release machinery in the presynaptic nerve terminal are functional (Duchen and Stefani, 10Duchen L.W. Stefani E. J. Physiol. (Lond.). 1971; 212: 535-548Crossref Scopus (72) Google Scholar; Angaut-Petit et al., 3Angaut-Petit D. McArdle J.J. Mallart A. Bournaud R. Pincon-Raymond M. Riegers F. Proc. R. Soc. Lond. B Biol. Sci. 1982; 215: 117-125Crossref PubMed Scopus (30) Google Scholar). The failure of transmission across the neuromuscular junction has been directly demonstrated by the lack of evoked potentials from muscle fibers of med mutants after electrical stimulation of the nerve (Duchen and Stefani, 10Duchen L.W. Stefani E. J. Physiol. (Lond.). 1971; 212: 535-548Crossref Scopus (72) Google Scholar; Harris and Ward, 20Harris J.B. Ward M.R. Exp. Neurol. 1974; 42: 169-180Crossref PubMed Scopus (38) Google Scholar; Weinstein, 43Weinstein S.P. J. Physiol. (Lond.). 1980; 307: 453-464Crossref Scopus (16) Google Scholar). The phenotype of the null mutants demonstrates that Scn8a is essential for propagation of action potentials into the neuromuscular junction. This required role could be related to a unique site of subcellular localization or to intrinsic channel properties; additional experiments will be required to determine the basis for this requirement. Purkinje cells appear to be particularly sensitive to loss of Scn8a (Dick et al., 8Dick D.J. Boakes R.J. Harris J.B. Neuropathol. Appl. Neurobiol. 1985; 11: 141-147Crossref PubMed Scopus (26) Google Scholar). In view of the broad distribution of Scn8a expression in brain (Schaller et al., 36Schaller K.L. Krzemien D.M. Yarowsky P.J. Krueger B.K. Caldwell J.H. J. Neurosci. 1995; 15: 3231-3242Crossref PubMed Google Scholar), it seems likely that additional effects of the channel deficiency will be identified in the future. LINE element insertion is a well recognized source of mutation in mammals, and two active human L1 elements capable of generating transposable copies have been isolated (Dombroski et al., 9Dombroski B.A. Mathias S.L. Nanthakumar E. Scott A.F. Kazazian Jr., H.H. Science. 1991; 254: 1805-1808Crossref PubMed Scopus (351) Google Scholar; Holmes et al., 23Holmes S.E. Dombroski B.A. Krebs C.M. Boehm C.D. Kazazian Jr., H.H. Nat. Genet. 1994; 7: 143-148Crossref PubMed Scopus (193) Google Scholar). The mouse mutation spastic is caused by L1 insertion into an intron of the glycine receptor β subunit gene (Mulhardt et al., 32Mulhardt C. Fischer M. Gass P. Simon-Chazottes D. Guenet J.L. Kuhse J. Betz H. Becker C.M. Neuron. 1994; 13: 1003-1015Abstract Full Text PDF PubMed Scopus (170) Google Scholar; Kingsmore et al., 27Kingsmore S.F. Giros B. Suh D. Bieniarz M. Caron M.G. Seldin M.F. Nat. Genet. 1994; 7: 136-141Crossref PubMed Scopus (190) Google Scholar), and independent insertions of large L1 elements into exons of the human factor VIII gene have been identified in two patients with severe hemophilia (Kazazian et al., 26Kazazian Jr., H.H. Wong C. Youssoufian H. Scott A.F. Phillips D.G. Antonarakis S.E. Nature. 1988; 332: 164-166Crossref PubMed Scopus (626) Google Scholar). Exon skipping has been associated with two L1 insertions in the dystrophin gene, a 2-kilobase insertion into exon 48 (Holmes et al., 23Holmes S.E. Dombroski B.A. Krebs C.M. Boehm C.D. Kazazian Jr., H.H. Nat. Genet. 1994; 7: 143-148Crossref PubMed Scopus (193) Google Scholar) and a 600-bp insertion into exon 44 (Narita et al., 34Narita N. Nishio H. Kitoh Y. Ishikawa Y. Ishikawa Y. Minami R. Nakamura H. Matsuo M. J. Clin. Invest. 1993; 91: 1862-1867Crossref PubMed Scopus (173) Google Scholar). In both cases, exon skipping could be attributed to the increase in exon length beyond the usual limit of 400 bp, which is thought to interfere with the exon definition step of splicing (Berget, 5Berget S.M. J. Biol. Chem. 1995; 270: 2411-2414Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar). The 180-bp L1 insertion in exon 2 of Scn8a results in an exon length of 300 bp, which is within the normal range. Skipping of exon 2 in the med transcript is therefore more likely to be related to the poly(U) sequence of the L1 element, since insertion of polypyrimidine tracts inhibits splicing (Furdon and Kole, 12Furdon P.J. Kole R. Mol. Cell. Biol. 1988; 8: 860-866Crossref PubMed Scopus (41) Google Scholar). Splice Site Mutation in medJ—Exon skipping in medJ results from a 4-bp deletion within the 5′ donor site of exon 3. The deleted nucleotides are part of a short direct repeat, consistent with a mutational mechanism in which DNA polymerase “slips” during replication of repeated sequences (Krawczak and Cooper, 29Krawczak M. Cooper K.N. Hum. Genet. 1991; 86: 425-441Crossref PubMed Scopus (460) Google Scholar). Site-directed mutagenesis has been used to demonstrate that the +5 G residue is essential for efficient splicing (Zhuang and Weiner, 45Zhuang Y. Weiner A.M. Cell. 1986; 46: 827-835Abstract Full Text PDF PubMed Scopus (459) Google Scholar; Seraphin et al., 37Seraphin B. Kretzner L. Rosbash M. EMBO J. 1988; 7: 2533-2538Crossref PubMed Scopus (243) Google Scholar; Siliciano and Guthrie, 40Siliciano P.G. Guthrie C. Genes Dev. 1988; 2: 1258-1267Crossref PubMed Scopus (209) Google Scholar). The high degree of evolutionary conservation of the +5 G residue (Shapiro and Senapathy, 38Shapiro M.B. Senapathy P. Nucleic Acids Res. 1987; 15: 7155-7174Crossref PubMed Scopus (1951) Google Scholar) and its frequent mutation in mammalian disease genes with aberrant splicing (Nakai and Sakamoto, 33Nakai K. Sakamoto H. Gene (Amst.). 1994; 141: 171-177Crossref PubMed Scopus (255) Google Scholar) are consistent with its critical role in splice site selection. The presence of the wild-type splice site sequence on chromosomes carrying the Ca mutation provides additional evidence that the splice site deletion did not precede the origin of medJ. Splice Site Incompatibility May Account for the Unusual Pattern of Exon Skipping in the med and med J Mutations Exon skipping is a common consequence of splice site mutations (Nakai and Sakamoto, 33Nakai K. Sakamoto H. Gene (Amst.). 1994; 141: 171-177Crossref PubMed Scopus (255) Google Scholar), but in most cases only one exon is missing from the transcript (Berget, 5Berget S.M. J. Biol. Chem. 1995; 270: 2411-2414Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar). We therefore would have predicted that the med transcript would be spliced from exon 1 to exon 3 (Fig. 8, top, dotted line) and that the medJ transcript would be spliced from exon 2 to exon 4 (Fig. 8, bottom, dotted line). Both predictions would require splicing between one standard site (S) and one of the nonstandard AT-AC sites (N) in intron 2. Instead, both observed transcripts result from splicing between two standard sites, avoiding the N sites (Fig. 8, solid lines). In the med transcript, a cryptic, standard acceptor site in intron 2 is utilized in preference to the AC acceptor site. These data provide experimental evidence that splicing between sites of the same class is favored in vivo. The splice sites of intron 2 belong to the minor class of sites previously recognized in only four genes (Table II) (Hall and Padgett, 17Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (184) Google Scholar). Divergence between major and minor splice sites is most notable at the dinucleotides bordering the intron, where the nearly invariant GT and AG of standard 5′ and 3′ splice sites are replaced by AT and AC in the rare introns. Either of these substitutions alone significantly reduces the splice site efficiency of standard introns (Aebi et al., 1Aebi M. Hornig H. Padgett R.A. Reiser J. Weissmann C. Cell. 1986; 47: 555-565Abstract Full Text PDF PubMed Scopus (245) Google Scholar). Splicing occurs by a complex pathway involving protein and RNA factors in the spliceosomal complex that interact with each other and with the splice sites of the substrate pre-mRNA (Green, 16Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (552) Google Scholar). Their unique sequences suggested that different spliceosome factors may be required for recognition of nonstandard splice sites (Hall and Padgett, 17Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (184) Google Scholar). Two of these factors have recently been identified. Using an in vitro splicing system, Tarn and Steitz (42Tarn W.Y. Steitz J.A. Cell. 1996; 84: 801-811Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar) demonstrated that U11 small nuclear ribonucleoprotein replaces U1 and U12 small nuclear ribonucleoprotein replaces U2 in the nonstandard spliceosome. Mutations in the branch site of the AT-AC introns block splicing in cultured cells, and co-expression of a U12 small nuclear RNA with compensatory mutations restores splicing activity (Hall and Padgett, 18Hall S.L. Padgett R.A. Science. 1996; 271: 1716-1718Crossref PubMed Scopus (168) Google Scholar). These studies demonstrate that dual mechanisms for splicing major and minor introns are present in mammalian cells. Joining of adjacent exons is thought to be mediated by interactions between splicing factors bound to the intervening intron (for review, see Green (16Green M.R. Annu. Rev. Cell Biol. 1991; 7: 559-599Crossref PubMed Scopus (552) Google Scholar) and Berget (5Berget S.M. J. Biol. Chem. 1995; 270: 2411-2414Abstract Full Text Full Text PDF PubMed Scopus (855) Google Scholar)). Our results indicate that factors bound to standard and nonstandard sites do not interact efficiently across an intron. Further biochemical studies will be required to completely characterize the components of the minor spliceosome and the basis for its incompatibility with the major splicesome during exon joining. In addition to the genes analyzed by Hall and Padgett (17Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (184) Google Scholar), the voltage-gated sodium channel α subunits are a fifth example of AT-AC intron-containing genes. In the Drosophila prospero gene, alternation between major and minor splice sites may contribute to developmental regulation; but in the other examples, there is no evidence for a regulatory role of minor sites. Nonstandard sites are present in the 3′-terminal intron of the human and chicken cartilage matrix protein genes, indicating an origin prior to the divergence of avian and mammalian lineages roughly 300 million years ago (Hall and Padgett, 17Hall S.L. Padgett R.A. J. Mol. Biol. 1994; 239: 357-365Crossref PubMed Scopus (184) Google Scholar). The nonstandard intron 2 of Scn8a evidently arose prior to divergence from the muscle sodium channel SCN4A. The Drosophila sodium channel gene para lacks an intron at the site corresponding to intron 2 of Scn8a and SCN4A (Loughney et al., 30Loughney K. Kreber R. Ganetzky B. Cell. 1989; 58: 1143-1154Abstract Full Text PDF PubMed Scopus (405) Google Scholar), indicating either that the nonstandard intron was introduced after the divergence of invertebrates and vertebrates or that it has been lost from para. The voltage-gated sodium channel α subunits comprise a highly conserved gene family with more than a dozen paralogous genes in the mammalian genome as well as closely related genes in lower vertebrates and invertebrates (Burgess et al., 6Burgess D.L. Kohrman D.C. Galt J. Plummer N.W. Jones J.M. Spear B. Meisler M.H. Nat. Genet. 1995; 10: 461-465Crossref PubMed Scopus (253) Google Scholar). Analysis of the intron 2 splice sites of additional family members in a variety of species would contribute to our understanding of the evolutionary origin and fate of the minor introns. The recently published structure of the human cardiac sodium channel gene SCN5A demonstrates conservation of the AT-AC intron (Wang, Q., Li, Z., Shen, J., and Keating, M. P. (1996) Genomics 34, 9-16." @default.
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• W2069192199 title "Mutation Detection in the and Alleles of the Sodium Channel" @default.
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