Copyright � 2008 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.094227

A Targeted Deleterious Allele of the Splicing FactorSCNM1 in the Mouse

Viive M. Howell,* Georgius de Haan,*,1 Sarah Bergren,* Julie M. Jones,* Cymbeline T. Culiat,†

Edward J. Michaud,† Wayne N. Frankel‡ and Miriam H. Meisler*,2

*Department of Human Genetics, University of Michigan, Ann Arbor, Michigan 48109-5618, †Oak Ridge NationalLaboratory, Oak Ridge, Tennessee 37831-6445 and ‡The Jackson Laboratory, Bar Harbor, Maine 04609

Manuscript received July 27, 2008Accepted for publication September 4, 2008

ABSTRACT

The auxiliary spliceosomal protein SCNM1 contributes to recognition of nonconsensus splice donorsites. SCNM1 was first identified as a modifier of the severity of a sodium channelopathy in the mouse.The most severely affected strain, C57BL/6J, carries the variant allele SCNM1R187X, which is defectivein splicing the mutated donor site in the Scn8amedJ transcript. To further probe the in vivo function ofSCNM1, we constructed a floxed allele and generated a mouse with constitutive deletion of exons 3–5.The SCNM1D3-5 protein is produced and correctly localized to the nucleus, but is more functionallyimpaired than the C57BL/6J allele. Deficiency of SCNM1 did not significantly alter other braintranscripts. We characterized an ENU-induced allele of Scnm1 and evaluated the ability of wild-typeSCNM1 to rescue lethal mutations of I-mfa and Brunol4. The phenotypes of the Scnm1D3-5 mutant confirmthe role of this splice factor in processing the Scn8amedJ transcript and provide a new allele of greaterseverity for future studies.

SODIUM channel modifier 1 (Scnm1) is an auxiliarysplice factor that was identified by its role in the

strain-specific lethality of the sodium channel mutationScn8amedJ (Buchner et al. 2003). A direct role for SCNM1in splicing the Scn8amedJ transcript was subsequentlydemonstrated in a cell culture assay (Howell et al.2007). The Scn8amedJ mutation is a 4-bp deletion in thesplice donor site of exon 3, nucleotides 15 to 18,generating a weak site with a C nucleotide at theconsensus 15G position (Kohrman et al. 1996). Inthe presence of a wild-type Scnm1 gene, only 10% of theScn8amedJ transcripts are correctly spliced and encode anactive channel, while �90% of the transcripts skip exon2 and exon 3. In the presence of the C57BL/6J-specificScnm1R187X allele, the amount of full-length transcript isreduced to 5% and the Scn8amedJ mice do not survive(Buchner et al. 2003). It is not clear from the earlierstudies whether Scnm1R187X is a null allele or retainspartial function.

SCNM1 is a 229-amino-acid protein with a bipartitenuclear localization signal near the N terminus and oneC2H2 zinc finger of the U1C type (Figure 1A). It isan accessory component of the U1 splicesome proteincomplex and interacts with the spliceosomal Sm andU1-70K proteins (Howell et al. 2007). Along with other

members of the U1C protein family, SCNM1 is thoughtto contribute to the recognition of different subsets ofnonconsensus splice donor sites (Roca et al. 2005). Byanalogy with the effect of Scnm1R187X on the splicingof Scn8amedJ, variants of these proteins in the humanpopulation could modify the severity of disorderscaused by splice donor site mutations.

LUC7L2 is a mammalian homolog of yeast Luc7p, acomponent of the U1 snRNP with a role in splicingnonconsensus splice donor sites (Fortes et al. 1999).SCNM1 interacts directly with LUC7L2, and the twoproteins cooperate to increase the splicing efficiency ofan Scn8amedJ minigene in cultured cells (Howell et al.2007).

Auxiliary splice factors can be used to restore theactivity of genes with splice-site mutations. For example,in cells from a cystic fibrosis patient with an intronicmutation that generates a cryptic splice donor site,overexpression of Htra2-b1 increased the level of cor-rectly spliced transcript and restored function (Nissim-Rafinia et al. 2004).

Inactivation of a splice factor itself may lead to disease.Germline inactivation of the SR proteins ASF/SF2, SC35,and SRp20 results in embryonic lethality (Xu et al. 2005).The neuron-specific auxiliary splice factor NOVA-1 andits paralog NOVA-2 regulate alternative splicing of 41synapse-related transcripts (Ule et al. 2006). Loss ofNOVA-1 due to autoimmune disease in cancer patientsleads to ataxia, seizures, and dementia. Inactivation ofmouse NOVA-1 is lethal, but inactivation of NOVA-2 is

1Present address: Broad Institute, MIT, Cambridge, MA 02142.2Corresponding author: Department of Human Genetics, 4909 Buhl,

University of Michigan Medical School, Ann Arbor, MI 48109-5618.E-mail: meislerm@umich.edu

Genetics 180: 1419–1427 (November 2008)

not (Huang et al. 2005). Patients with mutations in thesplice factors PRPF31, PRPF8, and PRPF3 developphotoreceptor degeneration, although the proteins areubiquitously expressed. They are associated with thespliceosomal complex U4/U6.U5 tri-snRNP, which isinvolved in the activation of the spliceosome for lariatformation and intron removal (Pacione et al. 2003).Mutation of PRPF31 inhibits pre-mRNA splicing ofRhodopsin, leading to retinal apoptosis and autosomaldominant retinitis pigmentosa (Yuan et al. 2005).

To further characterize the function of SCNM1, weexamined two new alleles, an internal deletion of 92residues generated by gene targeting in embryonic stem(ES) cells (Figure 1A) and an N-ethyl-N-nitrosourea(ENU)-induced missense mutation identified by screen-ing a DNA repository (Michaud et al. 2005). Weexamined the effect of the deletion allele using expres-sion arrays and the role of Scnm1R187X in the strain-specific lethality of two mouse mutants.

MATERIALS AND METHODS

Generation of a floxed allele of Scnm1: The targeted alleleof Scnm1 was constructed as shown in Figure 1. Two genomicfragments (5.0 and 4.4 kb) containing Scnm1 and flankingregions were amplified from the C57BL/6J BAC clone RP23-11G21 (http://bacpac.chori.org) and cloned into pSP72(Promega, Madison, WI). Restriction sites were engineeredby Quikchange multi-site mutagenesis (Stratagene, La Jolla,CA) and used to incorporate two loxP sites, two Flp recombi-nase (FRT) sites, and a PGKneo cassette from plox-2FRT-PGKneo (kindly provided by David Gordon, University ofColorado).

The targeting construct was electroporated into C57BL/6JES cell line BL/6-III (Schuster-Gossler et al. 2001) by theUniversity of Michigan Transgenic Animal Model Core (T.Saunders; http://www.med.umich.edu/tamc). Correct target-ing of the construct was evaluated in neomycin-resistant ES cellclones by PCR and Southern blotting. The 59 region of thetargeting construct was amplified using a forward primerupstream of the targeting construct and a reverse primerdownstream of the first loxP site (Figure 1B, primers 1 and 2),which generated a 1.8-kb PCR product after correct integra-tion (Figure 1C). The 39 region was evaluated by Southern blothybridization of BamHI-digested DNA using probe HP2 todetect a wild-type product of 8.5 kb and a targeted product of10.4 kb (Figure 1D).

Two euploid clones were expanded, micro-injected intoalbino C57BL/6J-Tyrc-2J blastocysts, and implanted into pseu-dopregnant C57BL/6J females. Male chimeric founders withpatches of white hair were mated with C57BL/6J females.Germline transmission of the targeted allele was detected byPCR of genomic DNA using primers 3 and 2 flanking the firstloxP site (Figure 1B), generating a 187-bp wild-type productand a 220-bp product from the targeted allele. FRT sites wereremoved in vivo by crossing male Scnm1TAR/1 mice with astrain expressing the FLPE recombinase [B6:SJL-Tg(ACTFL-Pe)9205Dym/J, Jackson Laboratory stock 003800]. The floxedallele was detected by PCR using primers 4 and 5 flanking thesecond loxP site (Figure 1B), which generated a 220-bp wild-type product and a 305-bp floxed allele product.

Generation of the Scnm1D3-5 allele: Scnm1flox/1 mice werecrossed with mice carrying a ubiquitously expressed EIIa-CRE

transgene [B6.FVB-Tg(EIIa-cre)C5379Lmgd/J, Jackson Labo-ratory stock 003724] (Lakso et al. 1996). The deleted allele(D3-5) was detected by PCR using primers 2, 3, and 5 (Figure1B), which generate a 380-bp wild-type product and 330 bp fromthe deleted allele (Figure 1E). All experimental procedureswere approved by the University of Michigan Committee forthe Use and Care of Animals. Animals were housed and caredfor in accordance with National Institutes of Health guidelines.

ENU mutant alleles of Scnm1: DNA and cDNA samplesfrom the Cryopreserved Mutant Mouse Bank, derived from4000 G1 male offspring of ENU-treated B6 mutant mice(Michaud et al. 2005), were screened by temperature-gradientcapillary electrophoresis to detect variants in the exons ofScnm1 at the Oak Ridge National Laboratory. Mice wererecovered from cryopreserved sperm by intracytoplasmic sperminjection, as previously described (Michaud et al. 2005).

Culture and immunocytochemistry of Scnm1D3-5 fibroblasts:Skin fibroblasts were cultured from tail biopsies taken fromhomozygous Scnm1D3-5 mice. Cells were isolated in the presenceof collagenase type II (Worthington Biochemical, Lakewood,NJ) in RPMI-1640 (Invitrogen, Carlsbad, CA) supplementedwith 15% fetal calf serum (Invitrogen) and incubated at 37�, 5%CO2. Immunocytochemistry was performed as previously de-scribed (Howell et al. 2007).

Gene expression and splicing profiling: RNA was extractedfrom fresh tissue using TRIzol reagent (Invitrogen) andpurified using RNeasy mini kit (Qiagen, Valencia, CA)according to the manufacturers’ protocols. RNA was preparedfrom two Scnm1D3-5 homozygotes and two Scnm1R187X homozy-gous littermates. Gene expression microarray analysis usingAffymetrix Mouse 430 2.0 GeneChips (Affymetrix, SantaClara, CA) was carried out at the University of MichiganComprehensive Cancer Center Affymetrix and MicroarrayCore. Three arrays for each genotype were analyzed usingAffymetrix and Limma software packages of Bioconductor.Splicing microarrays that interrogate 1339 genes for 1639splicing events (Srinivasan et al. 2005) were analyzed inManny Ares’ lab at the Center for Molecular Biology of RNA,University of California, Santa Cruz. The Mouse G-ProteinCoupled Receptor Splicearray (469 genes) and Ion ChannelSplicearray (330 genes) (44K format) were analyzed byExonHit Therapeutics (Gaithersburg, MD).

Quantitation of correctly spliced Scn8a transcripts: RNAwas extracted as described above from brain of Scn8amed

homozygous mice carrying various Scnm1 alleles. The controlwas brain RNA from wild-type C3HeB/FeJ mice, genotypeScn8a1/1, Scnm11/1. Five micrograms of total RNA was treatedwith DNase 1 (Invitrogen) and reverse transcribed in a 20-mlreaction using the SuperScript first-strand synthesis system(Invitrogen) according to the manufacturer’s protocol. Cor-rectly spliced Scn8a transcripts were specifically amplified with aforward primer in exon 1 (59-CCG ACA GTT TCA AGC CTTTCA CCC-39) and a reverse primer in exon 2 (59-AGG ACT TAGAAT GTA CAA GGC AGG-39). Only correctly spliced Scn8atranscripts are amplified with these primers as the exon 3 splice-site mutation in Scn8amedJ results in a majority of incorrectlyspliced transcripts lacking exon 2 and exon 3 (Kohrman et al.1996). RT–PCR products were examined on agarose gels.

To quantitate the correctly spliced Scn8a transcript in eachcDNA, we used the ABI PRISM 7900HT sequence detec-tion system (Applied Biosystems, Foster City, CA). Reactionswere carried out in a 96-well plate (Applied Biosystems)with a final reaction volume of 30 ml. The Taqman probeMm01300417_m1 (Applied Biosystems) that spans the exon1–2 junction was used according to manufacturer’s protocol.TATA box binding protein (Tbp) transcript was measured fornormalization (Mm00446971_m1, Applied Biosystems). Sam-ples were assayed in triplicate or quadruplicate. The fold

1420 V. M. Howell et al.

change in Scn8a gene expression was calculated as 2�DDCt,where DDCt ¼ (Ct, Scn8a � Ct, Tbp)Scn8a

medJ =medJsample �

(Ct, Scn8a � Ct, Tbp)wild-type sample.Protein interaction and yeast two-hybrid screen: The yeast

two-hybrid screen of a mouse embryo cDNA library was carriedout as previously described (Howell et al. 2007). Wild-typeScnm1 cDNA from strain C3HeB/FeJ was cloned into themammalian expression vector pCMVTnT (Promega). Full-length I-mfa cDNA from C57BL/6J brain was cloned intopCMV-MYC (Clontech Laboratories, Mountain View, CA).Transfection in COS7 cells, immunoprecipitation, and Westernblotting were carried out as previously described (Howell et al.2007). Protein was extracted 48 hr after transfection using RIPAbuffer (Sigma-Aldrich, St. Louis) containing 1 mm phenyl-methylsulfonyl fluoride (Roche Diagnostics, Indianapolis).

RESULTS

Targeted disruption of Scnm1: To generate a targetedallele on a homogenous C57BL/6J background for

subsequent phenotypic analysis, targeting was carriedout in ES cell line BL/6-III, and the targeting constructwas derived from a C57BL/6J BAC clone. To avoiddisrupting the expression of the nearby upstream anddownstream genes in this gene-rich region, we intro-duced the loxP sites into intron 2 and intron 5 of Scnm1(Figure 1B). This produced a target for CRE recom-binase that results in removal of most of the codingsequence of SCNM1 (Figure 1A). Correct targeting ofES cells was detected by PCR for the 59 flank (Figure 1C)and by Southern blotting for the 39 flank (Figure 1D).Germline transmission of the targeted construct wasobtained from two chimeric founders derived from thesame ES cell clone. The PGK-neo cassette was removedby crossing male Scnm1TAR/1 mice with an FLPE trans-genic mouse to generate the Scnm1flox allele (Figure 1B).

Floxed mice were crossed with an EIIa-CRE transgenicline expressing Cre recombinase in the zygote (Lakso

Figure 1.—Targeting of Scnm1.(A) Full-length SCNM1(NP_081289) and the region tar-geted for deletion. N, nuclearlocalization signal; ZNF, zinc fin-ger; A, acidic domain. (B) The tar-geting construct contains loxPsites that flank Scnm1 exons 3–5and a neomycin resistance cas-sette flanked by FRT sites in in-tron 5. The 59 homologous armis 2 kb in length and includesexon 1 of the upstream geneAK011517 (open box). The 39 ho-mologous arm is 5.6 kb in lengthand contains exons 5–7 of Scnm1,as well as exons 4–9 of the adja-cent downstream gene Tmod4(open boxes). B, BamHI sites;HP2, Southern blotting probe.Arrows 1–5 indicate primer loca-tions. (C) Amplification of embry-onic stem cell DNA using primers1 and 2 to detect correct targetingof the 59 arm of the construct. (D)Southern blot of BamHI-digestedembryonic stem cell DNA usingprobe HP2 to detect correct tar-geting of the 39 arm of the con-struct. (E) Genotyping ofScnm1D3-5 mice using primers 2,3, and 5. (F) Endogenous SCNM1proteins in brain nuclear extractsfrom C5BL/6J wild-type andScnm1D3-5 homozygous mice de-tected by Western blot usinganti-SCNM1 antiserum (Howell

et al. 2007). (G) Immunocyto-chemistry of Scnm1D3-5 homozy-gous fibroblasts demonstratesnuclear localization of the trun-cated SCNM1 protein detectedin F. DNA is stained with DAPI.Bar, 20 um.

New Allele of Splicing Factor Scnm1 1421

et al. 1996). Chimeric offspring were intercrossed toobtain mice with germline deletion of exons 3–5 ofScnm1, which were identified by genotyping as shown inFigure 1E.

Phenotype of Scnm1D3-5 mice with ubiquitous deletionof exons 3–5: Normal Mendelian inheritance wasobserved in crosses between Scnm1D3-5/1 heterozygotes(Table 1). In the first two generations, the body weightof Scnm1D3-5 homozygotes was 50% of littermates. How-ever, this effect disappeared when the CRE transgenesegregated away from Scnm1D3-5. Other examples of CREtransgenes that cause reduced body weight were re-cently described (Naiche and Papaioannou 2007).Homozygous Scnm1D3-5 mice were viable and fertile, withnormal grip strength and swimming and beam-walkingability. No visible abnormalities were detected in miceafter aging .1 year (data not shown).

Western blots from Scnm1D3-5 homozygotes demon-strated that the mutant protein is stable in vivo, but ispresent at reduced levels (Figure 1F). The mutant pro-tein migrates more slowly than predicted by its 10.3-kDacalculated molecular weight as previously reported forother SCNM1 isoforms (Howell et al. 2007). Immuno-histochemistry of cultured fibroblasts from Scnm1D3-5

homozygotes demonstrated that the mutant protein,which retains the nuclear localization signal (Figure1A), is correctly localized to the nucleus (Figure 1G).

Viability of Scnm1D3-5 homozygotes on other strainbackgrounds: Scnm1D3-5/1 mice were crossed to strains129S6 and C3H, and homozygous F2 mice were gener-ated. In both crosses, homozygotes were born in thepredicted Mendelian ratios (Table 1), and no abnormalphenotypes were observed.

The Scnm1D3-5 allele reduces splicing of the Scn8amedJ

transcript: To determine the functional effect of thetargeted deletion allele, we compared the amount ofcorrectly spliced Scn8a transcript in brains of Scn8amedJ

homozygous mice carrying various Scnm1 alleles. F2

mice with the indicated genotypes were obtained byintercrossing Scn8amedJ/1, Scnm1D3-5/R187X double heterozy-gous C57BL/6J mice (Table 2). All genotypes were recov-ered in the predicted Mendelian ratios (data not shown).Correctly spliced transcripts were quantitated with aTaqman assay using a probe spanning the cDNA junction

between exon 1 and exon 2 as described in materials

and methods.Scn8amedJ/medJ mice carrying the wild-type Scnm11/1

allele produced �8.1% of correctly spliced transcriptrelative to wild-type mice, consistent with previousestimates using other assays (Table 2). This was reducedby another 50% to give 3.8% correctly spliced transcriptin mice homozygous for the B6 allele, Scnm1R187X, and asimilar level in individuals heterozygous for Scnm1R187X

and the D3-5 allele (Table 2). In mice homozygous forthe targeted allele Scnm1D3-5, there was a further re-duction to 1.5% correctly spliced Scn8amedJ transcript(Table 2 and Figure 2A). The data demonstrate that theScnm1D3-5 allele is more severe than the previouslydescribed alleles and indicate that the Scnm1R187X alleleretains partial function in splicing of the Scn8amedJ

transcript. It is not clear whether Scnm1D3-5 is a completenull or retains a low level of residual activity, but thisallele is significantly less active than the Scnm1R187X allelepreviously described in strain C57BL/6J (Buchner et al.2003).

Exacerbation of the Scn8amedJ movement disorder bythe Scnm1D3-5 allele: The Scn8a gene encodes sodiumchannel Nav1.6, the major sodium channel at nodes ofRanvier in myelinated neurons of adult mice. Theneurological phenotype of homozygotes for the Scn8a-medJ mutation is modified by the Scnm1 genotype, with adirect correlation between the percentage of correctlyspliced Scn8amedJ transcripts and clinical severity (re-viewed in Meisler et al. 2004). In the presence of thewild-type allele of Scnm1, Scn8amedJ homozygotes have anormal life span with a movement disorder that in-cludes ataxic gait, visible tremor, and chronic dystonia.In the Scnm1R187X/R187X background of C57BL/6J mice,Scn8amedJ homozygotes survive for only 1 month, withsignificant muscle weakness but retention of hind-limbfunction. To determine the clinical effect of the reducedScn8amedJ splicing described above, we generated doublehomozygous mice with the genotype Scn8amedJ/medJ,Scnm1D3-5/D3-5 (Table 3). The eight double homozygotesall developed hind-limb paralysis that was not observedin littermates who were Scnm1D3-5/R187X or homozygousfor Scnm1R187X (Table 3 and Figure 2B). These datademonstrate that the residual 1.4% of full-length Scn8a

TABLE 1

Strain background does not influence survival of homozygous Scnm1D3-5 mice

No. of F2 offspring with the indicated genotypeLikelihood of Mendelian

segregation (P-value)Cross 1/1 D3-5/1 D3-5/D3-5

B6.Scnm1D3-5/1 3 B6 21 44 22 0.98B6.Scnm1D3-5/1 3 129S6 12 28 17 0.63B6.Scnm1D3-5/1 3 C3H 22 45 13 0.19

F2 litters were obtained by crossing heterozygous Scnm1D3-5/1 F1 mice. Scnm1D3-5 homozygotes were recovered atthe expected frequency of 25% from crosses with three inbred strains. P-values for agreement with Mendelianratios were calculated by x2 test for goodness of fit. B6, C57BL/6J; 129S6, 129S6/SvEvTac; C3H, C3HeB/FeJ.

1422 V. M. Howell et al.

transcripts in Scn8amedJ/medJ, Scnm1D3-5/D3-5 mice is insuffi-cient for normal hind-limb innervation, and confirmsthe greater severity of the D3-5 allele. The relationshipbetween hind-limb phenotype and transcript level issummarized in Figure 2C.

Expression array analysis of Scnm1D3-5 tissues: Toidentify additional transcripts with altered expressioncaused by the Scnm1D3-5 allele, we analyzed brain RNAfrom Scnm1D3-5 homozygotes and wild-type controls byhybridization with Affymetrix Mouse 430 2.0 GeneChips.The greatest change observed was a 3.8-fold reduction inthe Scnm1 transcript itself, which may reflect reducedstability of the Scnm1D3-5 transcript. Decreased transcriptabundance was confirmed by RT–PCR (data not shown)and is likely to contribute to the reduced amount ofmutant protein (Figure 1F). The 20 genes with the largest

changes are listed in Table 4; they include 8 genes withnucleic-acid-binding domains and two splice factors. Inview of the small magnitude of the observed changes,confirmation of biological significance would requirefurther study.

To determine whether the Scnm1D3-5 mutationchanges the ratios of alternative transcripts from geneswith weak donor splice sites, we interrogated brain andtestis RNA using microarrays with probes to exonjunctions (Srinivasan et al. 2005). Experiments in-cluded two Scnm1D3-5 homogygotes and two littermatecontrols, with three replicates for each genotype anddye-swap controls. No statistically significant changeswere identified (data not shown). RNA samples werealso hybridized to microarrays containing exon junctionprobes for 799 G-coupled protein receptors and ion

Figure 2.—Correlation of molecular and neu-rological phenotypes for the indicated Scnm1genotypes in combination with the Scn8amedJ/medJ

mutation. (A) Semiquantitative RT–PCR of cor-rectly spliced Scn8amedJ transcripts is consistentwith the quantitative RT–PCR data in Table 2.100% is defined as the transcript level in wild-typeScnm11/1 Scn8a1/1 mice. Numerical values abovethe gel are from Table 2. (B) Hind-limb paralysisin the Scn8amedJ/medJ, Scnm1D3-5/D3-5 double homo-zygote producing 1.4% of normal transcriptabundance (left). Some hind-limb function is re-tained in the Scn8amedJ/medJ, Scnm1D3-5/R187X animalwith one copy of the partially functional R187 al-lele and 3.4% of normal transcript abundance(right). (C) Summary of transcript levels andphenotypes of Scn8amedJ homozygotes with variousScnm1 genotypes.

TABLE 2

Effect of the Scnm1D3-5 targeted allele on splicing of the Scn8amedJ transcript

GenotypeNo. ofmice

% correctly splicedScn8a transcripts

Previousestimates (%)

Scn8a1/1 3 100 6 16 (12) 100 by definitionScn8amedJ/medJ, Scnm11/1 1 8.1 6 0.7 (4) 12a; 10b

Scn8amedJ/medJ, ScnmR187X/R187X 2 3.8 6 0.4 (8) 6.4a; 5b

Scn8amedJ/medJ, Scnm1D3-5/R187X 1 3.4 6 0.3 (4) NAScn8amedJ/medJ, Scnm1D3-5/D3-5 2 1.4 6 0.1 (8) NA

Mice were generated from intercrosses of (B6.Scn8a1medJ/1, Scnm1D3-5/R187X) mice and of (B6.Scn8a1medJ/1,Scnm1R187X/1) mice. Scn8a transcripts in brain RNA were quantitated with a Taqman assay; the probe spannedthe exon1/exon 2 junction to specifically detect correctly spliced Scn8a transcripts. Values represent 2(�DDCt) 3100, mean 6 SD (n), where n is the number of replicate assays (four per mouse). NA, not applicable.

a Kearney et al. (2002).b Buchner et al. (2003).

New Allele of Splicing Factor Scnm1 1423

channel genes (ExonHit Therapeutics). No substantialchanges were observed. It thus appears that reducedexpression of Scnm1 alone does not produce majorchanges in transcript processing.

Can SCNM1 rescue defective splicing of a Brunol4null allele? The spontaneous neurological mutantfrequent-flyer (Ff) was caused by a transgene insertion inintron 1 of Bruno-like 4 (Brunol4), which encodes anRNA-binding protein involved in RNA processing.There is no detectable Brunol4 transcript in frequent-flyerhomozygous mice, apparently due to disrupted splicing

by the inserted transgene (Yang et al. 2007). Brunol4Ff

homozygotes are not viable on strain C57BL/6J but havehigher rates of survival on other inbred strains (Yang

et al. 2007). To determine whether impaired splicingby the Scnm1R187X allele accounts for the greater lethal-ity on C57BL/6J, we generated B6.Brunol4Ff homozy-gous mice carrying a wild-type SCNM1 cDNA transgene.This transgene, under the regulation of the chickenb-actin promoter, rescued the lethality of Scn8amedJ/medJ,Scnm1R187X/R187X mice (Buchner et al. 2003). Brunol4Ff/Ff

mice were born at the expected overall frequency of25% (11/41) (Table 5). The 11 homozygotes included5 transgenic and 6 nontransgenic mice, indicating thatthe transgene did not increase prenatal survival. Fourof the five transgenic homozygotes died by postnatalday 4, as did 5 of the 6 nontransgenic homozygotes, andthe 2 survivors did not live beyond weaning, indicatingthat the presence of the transgene did not contributeto postnatal survival. Thus, impaired splicing bySCNM1R187X is not responsible for the lethality of thefrequent flyer mutation of Brunol4 on strain C57BL/6J.

SCNM1 interacts with I-MFA, a transcriptional re-pressor: I-MFA (Inhibitor of MyoD Family isoform A) isa transcriptional regulator active during myogenesis.In the course of screening a mouse embryo yeast two-hybrid cDNA library with SCNM1 as bait (Howell et al.2007), we recovered two overlapping cDNA clonescontaining the cysteine-rich region of I-MFA (Figure3A). Cysteine-rich domains are known to interact withC2H2 zinc-finger domains (Chen et al. 1996; Mizugishi

TABLE 4

The 20 transcripts with greatest differences in abundance between RNAfrom Scnm1R187X/R187X mice and from Scnm1D3-5/D3-5 mice

Gene Fold change Effect of D3-5 t-statistic P-value

Sodium channel modifier 1 (Scnm1) 3.8 Decrease �15.29 0.0001RIKEN cDNA B230343J05 gene 2.4 Increase 15.31 0.0001Glucocorticoid induced transcript 1 2.1 Increase 7.01 0.0022Synaptotagmin binding, cytoplasmic RNA interacting 2.1 Increase 7.63 0.0016Eph receptor A5 2.0 Increase 7.82 0.0014ELOVL family member 7, elongation of long chain fatty acids (yeast) 2.0 Decrease �7.72 0.0015RIKEN cDNA E030025D05 gene 1.9 Increase 7.52 0.0017Gap junction membrane channel protein a-12 1.9 Decrease �10.55 0.0005Endothelial differentiation, sphingolipid G-protein-coupled receptor, 8 1.9 Decrease �10.88 0.0004SRY-box containing gene 4 1.8 Increase 7.54 0.0017IQ motif and WD repeats 1 1.8 Increase 8.62 0.0010Myelin-associated oligodendrocytic basic protein 1.8 Decrease �16.86 ,0.0001Splicing factor, arginine/serine-rich 7 1.7 Increase 9.03 0.0008RIKEN cDNA 4632409L22 gene 1.7 Increase 10.68 0.0004RNA-binding motif protein 3 1.7 Decrease �11.27 0.0004Immunoglobulin heavy chain 6 (heavy chain of IgM) 1.7 Decrease �7.35 0.0018UDP galactosyltransferase 8A 1.7 Decrease �12.56 0.0002Gelsolin 1.7 Decrease �15.77 ,0.0001Gelsolin 1.7 Decrease �24.15 ,0.0001CD38 antigen 1.7 Decrease �9.1 0.0008

RNA was hybridized to Affymetrix Mouse 430 2.0 GeneChips. Three replicates for each genotype were assayed, including twobiological replicates. The decreased abundance of Scnm1 may reflect instability of the D3-5 transcript.

TABLE 3

Hind-limb paralysis is observed only in Scn8amedJ homozygotesthat are also homozygous for Scnm1D3-5

medJ/medJ

R187X/R187X

R187X/D3-5

D3-5/D3-5 P-value

Cross 1 9 20 5 0.37Cross 2 NE 2 3 0.57Total 9 22 8Hind-limb paralysis 0 0 8

P-values are for agreement with Mendelian predictions.Scnm1 genotypes are indicated for all Scn8amedJ homozygotesfrom both crosses. Hind-limb paralysis was observed in allScnm1D3-5, Scn8amedJ double homozygotes and not in mice withother genotypes. Cross 1, (Scnm1D3-5/R187X 3 Scn8amedJ/1)F2; cross2, (Scnm1D3-5/ D3-5, Scn8amedJ/1) 3 (Scnm1D3-5/R187X, Scn8amedJ/1). NE,none expected.

1424 V. M. Howell et al.

et al. 2004). To verify the interaction between SCNM1and I-MFA, we cotransfected COS7 cells with full-lengthcDNAs for myc-tagged I-MFA and SCNM1. The twoproteins were coprecipitated from the COS7 cell ex-tracts by anti-SCNM1 and by anti-myc antisera (Figure 3,B and C). The interaction with I-MFA suggests thatSCNM1 might have a second role in transcriptionalregulation.

To test the biological significance of this interaction,we tested the ability of wild-type Scnm1 to rescue atargeted null allele of I-MFA, which exhibits embryoniclethality on strain C57BL/6J but not on strain 129/Sv(Kraut et al. 1998). As described above, we crossed theI-MFA (Mdfi) null mice with C57BL/6J mice carryingthe wild-type Scnm1 transgene Tg580 (Buchner et al.2003). Among 156 offspring of an F2 cross, only twohomozygous null mice were recovered, both of whichwere transgenic (Table 5). Since complete rescue of

lethality by the transgene is predicted to generate 12%rescued mice (18/156), it appears that Scnm1R187X is notresponsible for lethality of the I-MFA null genotype onthe C57BL/6J background.

An ENU-induced allele of Scnm1: We screenedstored, mutagenized genomic DNA from the Cryopre-served Mutant Mouse Bank at the Oak Ridge NationalLaboratory to identify new variants of SCNM1. DNAwas analyzed by heteroduplex analysis as previouslydescribed (Michaud et al. 2005). The nonsynonymousvariants I112T, S167S, and L228S and the intron sub-stitution IVS5 1 62T . C were identified in genomicDNA from mutagenized mice. We selected I112T forin vivo analysis because of the evolutionary conservationof residue isoleucine 112. Sperm carrying this mutationwere thawed and used for in vitro fertilization by intra-cytoplasmic sperm injection. Heterozygous Scnm1I112T

mice were recovered and intercrossed to generate I112Thomozygotes, which were viable and fertile. We thengenerated Scnm1I112T/I112T, Scn8amedJ/medJ double homozy-gotes. These mice did not survive beyond weaning, butthey did not exhibit the hind-limb paralysis describedabove for Scnm1D3-5. Thus, I112T does not reduceSCNM1 function as severely as Scnm1D3-5.

DISCUSSION

Analysis of an allele series can often provide addi-tional insights into gene function beyond that availablefrom a single allele (Oliver et al. 2007). We previouslydescribed a naturally occurring variant of the splicefactor SCNM1 in C57BL/6J and related inbred strains ofmice. It was not clear from previous work whether theC57BL/6J allele, Scnm1R187X, causes partial or completeloss of function. In this work, we sought to generate anull allele of Scnm1 by targeted deletion of exons 3–5.This deletion removes a further 92 residues, includingthe zinc-finger domain, from the 187-residue proteincharacteristic of C57BL/6J mice. The deleted proteinreduced the correct Scn8amedJ splicing from �4% ofnormal in Scnm1R187X homozygotes to ,2% in Scnm1D3-5

homozygotes. This result demonstrates that SCNM1R187X

retains partial function and that SCNM1D3-5 is a moresevere allele. Because so much of the protein has beendeleted, it seems likely that SCNM1D3-5 is a completenull. The residual 1.4% of correctly spliced Scn8amedJ

transcript in Scnm1D3-5 homozygotes would then beproduced by other proteins with overlapping activitytoward nonconsensus splice donor sites.

RNA from Scnm1D3-5 homozygotes was analyzed tosearch for additional transcripts dependent on splicingby SCNM1. Expression arrays have been successfullyused to identify substrates of splice factors such asNOVA-1 and NOVA-2 (Ule et al. 2005). However,hybridization of several different splicing arrays didnot uncover additional splicing events that dependheavily on the function of SCNM1. To date, the Scn8amedJ

Figure 3.—I-MFA interacts with SCNM1. (A) Two overlap-ping clones from the cysteine-rich region of I-MFA, yc1 andyc2, were recovered in a yeast two-hybrid screen with SCNM1as bait, indicating that the two proteins can interact. (B andC) Confirmation of protein interaction by co-immunoprecip-itation from lysates of transfected COS7 cells. (B) The MYC/I-MFA immunoprecipitate contains SCNM1, but the IgGnegative control does not. SCNM1 was detected on the West-ern blot with anti-SCNM1 antiserum. (C) The anti-SCNM1immunoprecipitate contains MYC/I-MFA, but the preim-mune rabbit serum does not.

New Allele of Splicing Factor Scnm1 1425

mutant splice site is the only site at which SCNM1function can be measured.

Another approach that we applied to investigateSCNM1 function was transgene rescue of mutantphenotypes in vivo. We tested the ability of wild-typeScnm1 to rescue C57BL/6J-specific phenotypes associ-ated with null alleles of I-MFA, a binding partner ofSCNM1, and BRUNOL4, an RNA-binding protein.Phenotypic rescue was not observed in either case,indicating that the C57BL/6J-specific lethality of thesemutants cannot be attributed to Scnm1R187X.

SCNM1 interacts directly with the recently identifiedsplice factor LUC7L2 (Howell et al. 2007). To evaluatetheir functional interaction in vivo, we generated twolines of mice carrying gene trap alleles of LUC7L2.Unfortunately, both alleles retained �25% of wild-typeexpression (data not shown). Array analysis of RNAfrom double mutants homozygous for a LUC7L2 genetrap allele and for Scnm1D3-5 did not detect alteredtranscripts (our unpublished observations). For futurestudies, we plan to generate a true null allele of Luc7l2by gene targeting and to examine transcripts in doublehomozygotes with Scnm1D3-5.

Splice-site mutations compose �15% of all disease-causing mutations, and a similar number occur in spliceenhancer and suppressor sequences (Nissim-Rafinia

and Kerem 2005). Splice-site mutations are oftenassociated with clinical heterogeneity within families,as described for Duchenne and Becker muscular dys-trophy, cardiac sodium channelopathy (SCN5A), famil-ial adenomatous polyposis (APC), severe combinedimmunodeficiency ( JAK3), and neurofibromatosis type2 (NF2) (Kluwe et al. 1998; Frucht et al. 2001;Mohamed et al. 2003; Rossenbacker et al. 2005;Gurvich et al. 2008). There is evidence that the clinicalseverity of these disorders is influenced by splicingefficiency. Therapeutic strategies that target the correc-tion of splicing defects are under development forspinal muscular atrophy, familial dysautonomia, andcystic fibrosis (Nissim-Rafinia et al. 2004; Hims et al.

2007; Hua et al. 2008). The molecular basis for individualvariation in splicing efficiency is largely unknown, butidentification of the responsible factors could facilitatethe development of therapies. The Scn8amedJ mutantutilized in our work is a sensitive reporter for splicingof nonconsensus splice donor sites, since a twofolddecrease in splicing results in a visible change in pheno-type. Scn8amedJ could be used for in vivo discovery ofsplicing modifier genes or for testing small-moleculetherapeutics for splicing deficiencies.

In conclusion, we have generated a new, severe allelefor dissection of the splicing function of SCNM1 and themechanism of splice donor site choice. Future work willfocus on combining this allele with mutations of otheraccessory splice factors. Cultured cells from Scnm1D3-5

mice will also be useful for defining the structuralrequirements of SCNM1 splice-site substrates and theeffects of knockdown of other factors on minigenesplicing. From the clinical variability for Scn8amedJ micecaused by variants of Scnm1, we predict that geneticvariants of human SCNM1 and other trans-acting splicefactors may be responsible for some of the variability ofsplicing-related disorders.

We are grateful to Susannah Cheek, Darcy Butts, Kian Preston-Suni,and Connie Mahaffey for technical assistance. We thank ChristiPreston, Lily Shiue, and Manny Ares for analysis using their printedoligonucleotide alternative splicing arrays (supported by R24GM070857 to M. Ares, D. Black, and X.-D. Fu). We acknowledge thecontributions of Elizabeth Hughes and Virginia Zawistowski of theUniversity of Michigan Transgenic Animal Model Core for ES celltargeting, with Core support from the Center for Organogenesis. Wethank Lauren Snider and Stephen Tapscott (Fred Hutchinson CancerResearch Center) for the gift of I-MFA knockout mice. We thankCarmen Foster for the rederivation of Scnm1I112T mice. Affymetrix geneexpression array analysis was carried out at the University of MichiganMicroarray Core with support from the University of Michigan CancerCenter Support grant 5 P30 CA46592. The production and screeningof the Cryopreserved Mutant Mouse Bank and rederivation of ENU-induced mutant mice from cryopreserved sperm were supported bythe U. S. Department of Energy at Oak Ridge National Laboratory,managed by UT-Battelle under contract DE-AC05-00OR22725. Thiswork was supported by National Institutes of Health grants R01GM24872 to M.H.M. and R01 NS31348 to W.N.F. V.M.H. acknowledges

TABLE 5

The wild-type SCNM1 transgene Tg580 does not rescue the lethality of B6.Brunol4Ff/Ff (null) mice or B6.Imfa�/� null mice

Genotype at mutation

Total

�/�Tg1 1/1 1/� Tg1 Tg� P-value

Cross 1: Brunol4Ff/1 3 Brunol4Ff/1,Tg1 41 20 10 20 5 6 0.95Cross 2: Imfa1/� 3 Imfa1/�,Tg1 156 80 52 102 2 0 0.65

Cross 1 was genotyped on postnatal day 1. None of the homozygous mice survived beyond weaning, and mostdid not survive beyond P4. Cross 2 was genotyped on postnatal day 14 (P14). In both crosses, there was nosignificant difference between the number of null offspring that inherited the Scnm1 transgene (Tg) and thosethat did not (5 vs. 6 and 2 vs. 0, P-values indicated). The Scnm1 wild-type transgene failed to rescue the postnatallethality of Bruno14Ff/Ff homozygotes and the prenatal lethality of Imfa�/� homozygotes.

1426 V. M. Howell et al.

the National Health and Medical Research Council of Australia for theaward of CJ Martin Research Fellowship 358774.

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Communicating editor: K. V. Anderson

New Allele of Splicing Factor Scnm1 1427