Biophysical characterisation of the Bcl-x pre-mRNA and binding specificity of the ellipticine derivative GQC-05: Implication for alternative splicing regulation.
Biophysical characterisation of the Bcl-x pre-mRNA and binding specificity of the ellipticine derivative GQC-05: Implication for alternative splicing regulation.
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DOI：
10.3389/fmolb.2022.943105
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发表时间：
2022
影响因子：
5
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中科院分区：
文献类型：
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BCL 2L 1基因通过使用外显子2中的两个选择性5′剪接位点（5′ss）中的任一个表达Bcl-x蛋白的两种同种型。这些蛋白质具有拮抗作用，Bcl-XL是抗凋亡的，Bcl-XS是促凋亡的。在许多癌症中，Bcl-XL同种型过度表达，导致癌细胞存活和生长，因此将剪接转换为Xs同种型可能具有治疗益处。我们先前提出了一个假定的G-四链体（G4）存在于XS 5′ss的下游，并表明椭圆藤碱衍生物GQC-05，一种先前鉴定的DNA G4特异性配体，在体外和细胞中诱导XS/XL比率的增加。在这里，我们证明了这种G4在体外形成，并且在GQC-05的存在下结构得到稳定。我们还表明，GQC-05结合RNA非特异性在缓冲液条件下，但选择性的Bcl-x G4在核提取物的存在下，突出了生物物理测量功能环境之外的局限性。我们还证明，GQC-05能够将竞争的G4和双链体结构之间的平衡向G4构象转移，导致XS 5′ss的可及性增加，支持我们先前关于GQC-05作用机制的模型。
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-XL being anti-apoptotic and Bcl-XS pro-apoptotic. In a number of cancers the Bcl-XL isoform is over-expressed, resulting in cancer cell survival and growth, so switching splicing to the Xs isoform could have therapeutic benefits. We have previously proposed that a putative G-quadruplex (G4) exists downstream of the XS 5′ss and shown that the ellipticine derivative GQC-05, a previously identified DNA G4-specific ligand, induces an increase in the XS/XL 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 XS 5′ss, supporting our previous model on the mechanism of action of GQC-05.
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