A Targeted Deleterious Allele of the Splicing Factor SCNM1 in the Mouse

Abstract

The auxiliary spliceosomal protein SCNM1 contributes to recognition of nonconsensus splice donor sites. 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 SCNM1 R187X, which is defective in splicing the mutated donor site in the Scn8a medJ transcript. To further probe the in vivo function of SCNM1, we constructed a floxed allele and generated a mouse with constitutive deletion of exons 3-5. The SCNM1 D3-5 protein is produced and correctly localized to the nucleus, but is more functionally impaired than the C57BL/6J allele. Deficiency of SCNM1 did not significantly alter other brain transcripts. We characterized an ENU-induced allele of Scnm1 and evaluated the ability of wild-type SCNM1 to rescue lethal mutations of I-mfa and Brunol4. The phenotypes of the Scnm1 D3-5 mutant confirm the role of this splice factor in processing the Scn8a medJ transcript and provide a new allele of greater severity for future studies. SODIUM channel modifier 1 (Scnm1) is an auxiliary splice factor that was identified by its role in the strain-specific lethality of the sodium channel mutation Scn8a medJ (Buchner et al. 2003). A direct role for SCNM1 in splicing the Scn8a medJ transcript was subsequently demonstrated in a cell culture assay (Howell et al. 2007). The Scn8a medJ mutation is a 4-bp deletion in the splice donor site of exon 3, nucleotides 15 to 18, generating a weak site with a C nucleotide at the consensus 15G position (Kohrman et al. 1996). In the presence of a wild-type Scnm1 gene, only 10% of the Scn8a medJ transcripts are correctly spliced and encode an active channel, while $90% of the transcripts skip exon 2 and exon 3. In the presence of the C57BL/6J-specific Scnm1 R187X allele, the amount of full-length transcript is reduced to 5% and the Scn8a medJ mice do not survive (Buchner et al. 2003). It is not clear from the earlier studies whether Scnm1 R187X is a null allele or retains partial function.SCNM1 is a 229-amino-acid protein with a bipartite nuclear localization signal near the N terminus and one C2H2 zinc finger of the U1C type ( Figure 1A). It is an accessory component of the U1 splicesome protein complex and interacts with the spliceosomal Sm and U1-70K proteins (Howell et al. 2007). Along with other members of the U1C protein family, SCNM1 is thought to contribute to the recognition of different subsets of nonconsensus splice donor sites (Roca et al. 2005). By analogy with the effect of Scnm1 R187X on the splicing of Scn8a medJ

RBFOX1 Cooperates with MBNL1 to Control Splicing in Muscle, Including Events Altered in Myotonic Dystrophy Type 1

“…It is also possible that alternative splicing events in C57BL/6 mice are in general less sensitive to expression of CUG repeats. SCNM1 is a disease modifying allele that affects splicing, and that is most severely affected in C57BL/6 [58] , [59] . Such strain-specific differences may produce overlapping but globally distinct splicing signatures that may contribute to differences in the expression of the DM1 phenotype in FVB/n and C57BL/6 mice.…”

Theataxia3Mutation in the N-Terminal Cytoplasmic Domain of Sodium Channel Nav1.6 Disrupts Intracellular Trafficking

“…Mice with the genotype Scn8a medJ/medJ , Scnm1 R187X/R187X produce 5% of normal channel protein and do not survive beyond 3 weeks, but they do retain partial hind limb function like the ataxia3 mice (Kearney et al, 2002). Mice with 1.5% of normal levels of Na v 1.6 exhibit complete hind limb paralysis and lethality at 3 weeks (Howell et al, 2008) (Howell et al, 2008). The phenotype of the ataxia3 mice suggests that approximately 5% of Nav1.6-S21P reaches the cell surface in vivo .…”

Modifier Genes and the Plasticity of Genetic Networks in Mice

“…Positional cloning of the modifier identified a novel gene whose protein plays a direct and previously unsuspected role in pre-mRNA splicing [31] , [32] . The sensitizing modifier allele (C57BL/6) encodes a truncated protein (R187X), but is less severe than a subsequently generated null allele [33] . The interaction between Scnm1 and Scn8a med-J appears highly specific, as Scnm1 does not alter general RNA patterns in brain nor modify other tested mutations with similar defects [33] .…”