Mutually exclusive splicing regulates the Nav 1.6 sodium channel function through a combinatorial mechanism that involves three distinct splicing regulatory elements and their ligands

Lorena Zubovic 0 Marco Baralle 0 Francisco E. Baralle 0 0 International Centre for Genetic Engineering and Biotechnology (ICGEB) 34012 , Trieste, Italy Mutually exclusive splicing is a form of alternative pre-mRNA processing that consists in the use of only one of a set of two or more exons. We have investigated the mechanisms involved in this process for exon 18 of the Nav 1.6 sodium channel transcript and its significance regarding geneexpression regulation. The 18N exon (neonatal form) has a stop codon in phase and although the mRNA can be detected by amplification methods, the truncated protein has not been observed. The switch from 18N to 18A (adult form) occurs only in a restricted set of neural tissues producing the functional channel while other tissues display the mRNA with the 18N exon also in adulthood. We demonstrate that the mRNA species carrying the stop codon is subjected to Nonsense-Mediated Decay, providing a control mechanism of channel expression. We also map a string of cis-elements within the mutually exclusive exons and in the flanking introns responsible for their strict tissue and temporal specificity. These elements bind a series of positive (RbFox-1, SRSF1, SRSF2) and negative (hnRNPA1, PTB, hnRNPA2/B1, hnRNPD-like JKTBP) splicing regulatory proteins. These splicing factors, with the exception of RbFox-1, are ubiquitous but their levels vary during development and differentiation, ensuing unique sets of tissue and temporal levels of splicing factors. The combinatorial nature of these elements is highlighted by the dominance of the elements that bind the ubiquitous factors over the tissue specific RbFox-1. - Mutually exclusive splicing (ME) distinguishes itself from classical cassette exon splicing in that ME exons are never observed together in a given cell/tissue. Therefore, contrary to alternative splicing where a ratio of transcripts containing the alternative exons can arise, ME splicing results in one specific isoform delineating the need for a strict restriction on the isoform(s) present (1). This form of regulation is particularly common in the mammalian nervous system where a large percent (4060%) of neuronal pre-mRNA undergo alternative splicing in a tissue and developmental regulated manner (2). From a mechanistic perspective the intriguing question regards the mechanisms involved in preventing ME exons from being spliced to each other, considering that the introns between them contain functional 50- and 30-splice sites. Several models have being established to be responsible for ME splicing, among these are steric interference between splice sites (35) and incompatible splice sites flanking pairs of ME exons (1,6). However, the majority of ME exon pairs do not have a mechanism that absolutely forbids their splicing together and the mechanism(s) that determine the splicing choices are not well known. In these cases, the regulated selection of the individual exons must be sufficiently coordinated to minimize inappropriate splicing without the need for an absolute physical impediment to double-exon inclusion (7). To accomplish this it is evident that the splicing machinery must make use of the specialized regulatory proteins that bind cis-elements in the pre-mRNA in either exonic or intronic regions, and alter the splice site recognition functioning as repressors of one ME exon and activators of its partner (8). Elucidating how these cis-elements determine which ME exon is chosen is important in understanding the basic mechanism of pre-mRNA splicing and in particular of neural pre-mRNA splicing (4,911). ME splicing occurs extensively in a tissue and developmentally regulated manner within a family of proteins responsible for the rising phase of action potentials in electrically excitable cells, namely the voltage-gated sodium channels (1214). This modulation of splicing may result in transcripts that at one extreme have the insertion of an in frame stop codon while on the other alter only a few amino acids, resulting in biochemical and pharmacologically distinct sodium channel isoforms (1,4). It was of interest to see if there could be a common mechanism behind the ME splicing in this family and as a first step of an extensive study we have investigated the ME splicing of exon 18 in the Nav 1.6 (SCN8A) sodium channel transcript. The voltage gated sodium channel a subunit SCN8A is one of the most abundant sodium channels in neurons. The gene encoding the protein has been shown to undergo ME splicing of exon 18 that encodes transmembrane segments S3 and S4 in domain III of the protein. Prior studies of SCN8A isoforms demonstrated that fetal neurons and non-neuronal cells produce two variant transcripts, one predominant transcript containing the alternative exon 18N (neonatal) and one that skips exon 18. As exon 18N includes a stop codon, unless degraded by NMD, it is predicted that fetal neurons and non-neuronal cells would express a truncated non functional variant of Nav 1.6 sodium channel (15). The proportion of transcripts containing exon 18N is highest in mouse fetal brain between E12.5 and P1.5, while at later stages the predominant transcripts contain exon 18A (adult), the major transcript in adult brain and spinal cord that results in a functional channel (15). The understanding of the ME mechanism of these exons aside the mechanistic aspect regarding ME choice may also have a therapeutic value as Nav 1.6 plays an important role in normal axonal conduction and may significantly contribute to the pathophysiology of the injured nervous system (16,17). A greater understanding of how different cell types modulate the ME splicing abolishing channel function may therefore provide clues for possible therapeutic intervention. Our results support a model in which the ME splicing regulates channel expression through nonsense-mediated mRNA decay of the transcript containing exon 18N. The exclusion of exon 18A in non-neuronal tissue is regulated primarily by the interaction of hnRNP proteins with an exonic splicing silencer (ESS) that we have identified in this exon, while exon 18N inclusion is due to SR proteins that function through an exonic splicing enhancer (ESE) mapped within this sequence. In neuronal cells the ratio of these protein levels differs and in addition a neuron specific factor (RbFox-1) is present. This combination results in exon 18A inclusion in the final transcript and exon 18N exclusion. MATERIALS AND METHODS Construction of minigene and expression plasmids cDNA for muscle-specific RbFox-1 (NM_145891.2), brain-specific RbFox-1 (NM_001142334.1), RbFox-2 (NP_001026865.1) and RbFox-3 (NP_001076044.1) were synthesized by Genescript and subsequently cloned into pFlag CMV-4 Expression vector (Sigma-Aldrich). Hybrid minigenes E18A and E18N were made as previously described (18), using a DNA fragments extending 150-bp upstream and 100-bp downstream of the exon 18A or 18N. SCN8A WT was made by via amplifying three fragme (...truncated)