Rbfox proteins regulate alternative splicing of neuronal sodium channel SCN8A

Abstract

The SCN8A gene encodes the voltage-gated sodium channel Na(v)1.6, a major channel in neurons of the CNS and PNS. SCN8A contains two alternative exons,18N and 18A, that exhibit tissue specific splicing. In brain, the major SCN8A transcript contains exon 18A and encodes the full-length sodium channel. In other tissues, the major transcript contains exon 18N and encodes a truncated protein, due to the presence of an in-frame stop codon. Selection of exon 18A is therefore essential for generation of a functional channel protein, but the proteins involved in this selection have not been identified. Using a 2.6 kb Scn8a minigene containing exons 18N and 18A, we demonstrate that co-transfection with Fox-1 or Fox-2 initiates inclusion of exon 18A. This effect is dependent on the consensus Fox binding site located 28 bp downstream of exon 18A. We examined the alternative splicing of human SCN8A and found that the postnatal switch to exon 18A is completed later than 10 months of age. In purified cell populations, transcripts containing exon 18A predominate in neurons but are not present in oligodendrocytes or astrocytes. Transcripts containing exon 18N appear to be degraded by nonsense-mediated decay in HEK cells. Our data indicate that RBFOX proteins contribute to the cell-specific expression of Na(v)1.6 channels in mature neurons.

Fig. 1

Alternative splicing of SCN8A. (A) Structure of the full length, four domain, 1980 amino acid residue Nav1.6 channel protein. The transmembrane segments encoded by exon 18 are shown in black. (B) Genomic structure of exons 18A and 18N. SCN8A contains 26 protein coding exons; exons 17 to 19 are expanded. Exon 18N contains an in-frame stop codon. (C) A consensus Fox binding site is located downstream of exon 18A in SCN8A genes from human (H), mouse (M) and fugu (F).

Fig. 2

Splicing of an Scn8a minigene. (A) A. Structure of the 2.6 kb minigene containing exon 18N, exon 18A, and the Fox binding site TGCATG (asterisk). The CMV promoter and β-globin exons 1 and 2 are indicated; intron sizes in kb; exons not to scale. (B) RT-PCR products amplified from RNA isolated after transfection of HEK cells with the minigene alone (none) or co-transfection with Fox-1 or Fox-2. (C) RT-PCR products from HEK cells transfected with a minigene carrying a 1 bp mutation in the Fox binding site (from TGCATG to TGCgTG). w, wildtype minigene; m1, mutant minigene. RT-PCR primers 15 and 16 in β-globin exons 1 and 2, respectively (Table S1). Predicted lengths in bp: 8N+A=352, 18A=284, 18N=229, Δ18=161. The identity of all RT-PCR products in this and other figures was confirmed by sequencing.

Fig. 3

Splicing of endogenous SCN8A trancripts in HEK cells. (A) RT-PCR products amplified from RNA from untransfected HEK cells with a forward primer in exon 17 and reverse primer in exon 19. PCR product lengths: 18N=216 bp; Δ18=146 bp. The product containing 18A (269 bp) is not present. (B) Transcripts containing exon 18A alone can be amplified only after transfection of the Fox-1 cDNA. forward primer in exon 17, reverse primer in exon 18A. M, markers: 100-bp ladder.

Fig. 4

Treatment with cycloheximide increases the abundance of SCN8A transcripts containing exon 18N. (A) HEK cells were incubated with cycloheximide (CHX, 500 ug/ml) for 5 hrs. RT-PCR was carried out with primers 11–12 (Table S1). Product sizes: 18N=216 bp, Δ18=146 bp. (B) RT-PCR products from transcripts of ATP7A and FIG4 that do not contain in-frame stop codons. Control, no CHX.

Fig. 5

SCN8A expression in purified neurons and human brain. (A) RT-PCR of RNA from rat brain at ages P1, P12 and adult demonstrates the postnatal switch to transcripts containing exon 18A. Purified retinal ganglia neurons (RGC) (P6–7) express predominantly exon 18A. Purified astrocytes (P1) and oligodendrocytes (P12) do not contain transcripts with exon 18A. M, molecular weight markers in bp. (B) Time course of developmental switch to exon 18A in human brain. The pattern is delayed in comparison with rodent brain; the human pattern at 10 months postnatal resembles rodent brain at postnatal day 1 (P1). fibr, human fibroblast control; E18w, 18 weeks of gestation. Forward primer in exon 17, reverse primer in exon 19; predicted product sizes, 18A=286, 18N=231, Δ18=163; identity of RT-PCR products was confirmed by sequencing. M, molecular weight markers in bp

Fig. 6

Model for restriction of active Na_v1.6 channels to neurons. Splice factors Fox-1 and Fox-2 in neurons generate transcripts containing exon 18A that encode the active sodium channel. In the absence of Fox-1 and Fox-2 in non-neuronal cells, transcripts containing exon 18N are produced and degraded by nonsense-mediated decay. Translation of remaining 18N transcripts generates a truncated protein lacking channel activity (Plummer et al., 1997). The much greater total abundance of Na_v1.6 transcripts in neurons compared with other cells is also a consequence of regulation at the level of transcription.