Biophysical characterisation of the Bcl-x pre-mRNA and binding specificity of the ellipticine derivative GQC-05: Implication for alternative splicing regulation.

ABSTRACT: The BCL2L1 gene expresses two isoforms of Bcl-x protein via the use of either of two alternative 5' splice sites (5'ss) in exon 2. These proteins have antagonistic actions, Bcl-X_L being anti-apoptotic and Bcl-X_S pro-apoptotic. In a number of cancers the Bcl-X_L isoform is over-expressed, resulting in cancer cell survival and growth, so switching splicing to the X_s isoform could have therapeutic benefits. We have previously proposed that a putative G-quadruplex (G4) exists downstream of the X_S 5'ss and shown that the ellipticine derivative GQC-05, a previously identified DNA G4-specific ligand, induces an increase in the X_S/X_L ratio both in vitro and in cells. Here, we demonstrate that this G4 forms in vitro and that the structure is stabilised in the presence of GQC-05. We also show that GQC-05 binds RNA non-specifically in buffer conditions, but selectively to the Bcl-x G4 in the presence of nuclear extract, highlighting the limitations of biophysical measurements taken outside of a functional environment. We also demonstrate that GQC-05 is able to shift the equilibrium between competing G4 and duplex structures towards the G4 conformation, leading to an increase in accessibility of the X_S 5'ss, supporting our previous model on the mechanism of action of GQC-05.

SUBMITTER: Bhogadia M 

PROVIDER: S-EPMC9428248 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature