Regulation of Bcl-X splicing by G-quadruplexes enables regulation by G-quadruplex ligands to favour apoptosis

• Main Author: Weldon, Carika Elshae
• Other Authors: Dominguez, Cyril; Eperon, Ian
• Published: University of Leicester 2015
• Subjects: 572
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.674538

The Bcl-2 family of proteins are the primary regulators of the intrinsic apoptotic pathway, with anti-apoptotic members up regulated in cancer. Bcl-X is of particular interest, as it can be alternatively spliced to yield antagonistic proteins, the proapoptotic Bcl-XS and the anti-apoptotic Bcl-XL. Heterogeneous ribonuclearprotein (hnRNP) F/H, which binds to G-tract RNA, was shown to shift splicing in favour of the XS 5’ss (Garneau et al., 2005). G-tracts can form G-quadruplexes (G4s), which have been implicated to effect various stages of RNA processing, including splicing. Structural studies of hnRNP F/H revealed that it prefers to bind single stranded G-tract RNA (Dominguez et al., 2010) and competes with G4 formation (Samatanga et al., 2013). It was proposed that G4s may also form within the Bcl-X pre-mRNA that will alter 5’ss selection. In this study, two main questions were asked: (1) Is the Bcl-X pre-mRNA capable of forming any G4s (2) if so, do these G4s alter the 5’ss selection. Once a better understanding of 5’ss selection in Bcl-X was attained, a novel non-intrusive method was developed that identified putative G4s regions in the presence of competing secondary structures. Three main regions were highlighted for further investigation. By screening over 30 G4 ligands for their effect on 5’ss selection in Bcl-X, it was revealed that G4 ligands not only cause a shift to favour the pro-apoptotic site, but that such an effect is restricted to certain structural classes of G4 ligands. By investigating the mechanism of action of the best G4 ligand from the screen, GQC-05, two independent non-canonical G4s were found to be stabilized by GQC-05 owing to its dual effect on 5’ss selection. This not only expands the range of potential G4 forming sequences in RNA but also offers a new therapeutic strategy for alternative splicing regulation of apoptosis.