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

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thesis
posted on 14.01.2022, 13:13 by Mohammed A. H. Bhogadia
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-XL and Bcl-XS, which are anti and pro-apoptotic respectively. In many cancers, activation of oncogenic pathways leads to overexpression of the Bcl-XL isoform, resulting in cancer cell survival and growth (Boucher et al., 2000). Increasing the amount of the XS 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 XS/XL 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 XS 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.

History

Supervisor(s)

Cyril Dominguez; Ian Eperon

Date of award

20/10/2021

Author affiliation

Department of Molecular and Cell Biology

Awarding institution

University of Leicester

Qualification level

Doctoral

Qualification name

PhD

Language

en