Characterization of the Molecular Mechanism of the Tissue-Specific and Developmentally Regulated Alternative Splicing of SCN9A Exon 5

Chiavelli, Chiara (2015). Characterization of the Molecular Mechanism of the Tissue-Specific and Developmentally Regulated Alternative Splicing of SCN9A Exon 5. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000ef98

Abstract

The voltage-gated sodium channel Nav 1.7, encoded by SCN9A gene, is expressed in dorsal root ganglia (DRG) and in sympathetic ganglia and it is considered to be one of the major player in the regulation of pain perception. The linkage to several human pain disorders confirms its pivotal role in pain pathway. The gene contains an exon 5 pair, 5N and 5A, that are mutually exclusive spliced. These exons encode part of the voltage sensor of the channel; although the alternative splice variants differ by only two amino acids, they differentially affect the amplitude of the currents generated and the excitability of nociceptive neurons.

In this study I mapped some of the splicing regulatory elements that determine SCN9A exon 5 developmentally regulated mutually exclusive splicing. In the first stages of development, characterized by a major inclusion of the neonatal form, ETR3 plays an essential role acting on both sides, favouring 5N and inhibiting 5A inclusion. For the switching towards a major inclusion of exon 5A some elements have been identified. Fox1 helps the recognition of the weak 5A 5' splice site, interacting with four cis-acting elements downstream of exon 5A. Furthermore an exonic splicing enhancer (ESE), bound by SRSF1 and SRSF6, improves the usage of 5A weak 3' splice site. At the same time 5N inclusion is inhibited by the presence of two silencers, one exonic recognized by PTB and one intronic close to 5N 5' splice site.

The duplication of this exon is well conserved in six out of nine voltage-gated sodium channel (VGSC) genes and some elements mapped for SCN9A gene display a great homology in other family members. The investigation of possible common factors reveals some interesting results: for example the ESE in the adult form is present in at least other three VGSCs.