Investigating the mechanism of alternative splicing regulation of the RNA-binding proteins T-STAR and Sam68

Abstract

Alternative splicing is an important mechanism of pre-mRNA processing, regulated by many splicing factors. Some splicing factors as T-STAR, are poorly characterised due to absence of RNA targets identified. The first main aim of this study was to identify the principles for splicing regulation by T-STAR, by characterising its first identified physiological targets Neurexin-2 and Tomosyn-2. The second important aim was to compare the T-STAR and Sam68 splicing regulation on example of Neurexin-2 being a T-STAR specific target, and Tomosyn-2 being regulated by both T-STAR and Sam68. By EMSA analyses, I identified that Neurexin-2 is a direct target of both T-STAR and Sam68. By minigene assays and mutagenesis, I found that T-STAR response element in Neurexin-2 is composite. Individually each AU-rich region is redundant for splicing regulation, but loss of two key groups of AU-rich sequence elements inhibits splicing control by T-STAR. Several splicing regulator proteins have been shown to follow a pattern where binding upstream of a regulated exon leads to splicing repression and binding downstream leads to splicing activation. The T-STAR response element starts just 13 nucleotides downstream of the regulated Neurexin-2 exon, suggesting T-STAR protein binding might physically occlude the 5´splice site. Instead, I found this T-STAR response element can still potently repress splicing even from more distant downstream locations, and surprisingly even when placed upstream of the regulated exon. To find out how general this form of position-independent splicing regulation is I identified AU-rich downstream sequence elements that control splicing patterns of Tomosyn-2 in response to both T-STAR and Sam68. These Tomosyn-2 splicing response sequences similarly repressed splicing when moved upstream of the regulated exon. The T-STAR response element in Neurexin-2 predominantly contained UUAA sequences, while the T-STAR/Sam68 response element in Tomosyn-2 contained UAAA repeats. Conversion of the Neurexin response element to UAAA and UAAAA placed this exon under control of both Sam68 and T-STAR. Current data suggest that T-STAR and Sam68 proteins are unusual in that they repress splicing from either side of the exon. These proteins have subtly different target sequences that can enable them to control distinct patterns of target exons in the cell.