4.6 Article

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

期刊

出版社

FRONTIERS MEDIA SA
DOI: 10.3389/fmolb.2022.943105

关键词

alternative splicing; G-quadruplex; ellipticine; apoptosis; circular dichroism; NMR

资金

  1. Biotechnology and Biological Sciences Research Council (BBSRC) grant [BB/R006555/1]
  2. University of Leicester, College of Life Sciences PhD scholarship

向作者/读者索取更多资源

The BCL2L1 gene expresses two isoforms of Bcl-x protein, with different actions, through alternative splicing. Over-expression of one isoform is associated with cancer cell survival and growth. The study shows that a G-quadruplex structure forms downstream of one isoform and is stabilized by the compound GQC-05. GQC-05 selectively binds to the Bcl-x G-quadruplex and shifts the balance between G-quadruplex and duplex structures, increasing accessibility of a specific splice site.
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.

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