Biophysical characterisation of a G-quadruplex in the Bcl-x pre-mRNA and the binding specificity of the ligand GQC-05

Bcl-x is a member of the B-cell lymphoma 2 (Bcl-2) protein family. This protein family consists of both pro and anti-apoptotic proteins that regulate mitochondrial membrane permeability in cells. The two alternative 5’ splice sites (5’ss) in exon 2 of Bcl-x gives rise to two antagonistic splice variants Bcl-X_L and Bcl-X_S, which are anti and pro-apoptotic respectively. In many cancers, activation of oncogenic pathways leads to overexpression of the Bcl-X_L isoform, resulting in cancer cell survival and growth (Boucher et al., 2000). Increasing the amount of the X_S isoform, thereby promoting apoptosis, is a novel way to kill cancer cells. Our recent studies have identified an RNA secondary structure known as a G-quadruplex (G4) that has the potential to form near both splice sites and alter the splicing pattern of Bcl-x (Weldon et al., 2017, 2018). The ellipticine derivative GQC-05, a previously identified DNA G4 specific ligand (Brown et al., 2011), has been shown to bind proximal to both 5’ss resulting in a 8-fold increase in X_S/X_L ratio, analogues of which showed considerably less extensive effects (Weldon et al., 2018). Using various biophysical techniques, we have characterised the RNA secondary structure element near the X_S 5’ss and have postulated the possible effects of GQC-05 on these structural features and its role in altering splice site selection. Our results show that a stable G4 exists downstream of the Xs 5’ss and this structure is stabilised in the presence of GQC-05. We also show that GQC-05 displays less G4 binding specificity in buffer, but shows greater G4 selectivity in the presence of a nuclear extract. Therefore, we aim to understand how these secondary structure elements stabilised by GQC-05 lead to splice site bias, enabling us to design more potent molecules as novel anti-cancer compounds.