Investigation of Mcl-1 alternative splicing regulation by GQC-05, a putative G–quadruplex ligand

The Bcl-2 family of proteins are regulators of apoptosis. One of the Bcl-2 genes, Mcl-1, produces two antagonistic isoforms by alternative splicing. The larger and more abundant protein isoform, Mcl-1L inhibits apoptosis, whereas the smaller protein isoform Mcl-1S favours apoptosis. RNA G-quadruplex (rG4s) have been shown to regulate various RNA processing events, including splicing, so modulating rG4 formation by interactions with small molecules is a possible route to modulate alternative splicing. A small molecule, GQC-05, was previously shown to stabilise rG4 formation in another Bcl-2 family member, Bcl-x. Like Bcl-x, Mcl-1 pre-mRNA sequence contain repeat of G nucleotides at the splice sites, which might have the potential to form rG4s. Previous studies in the lab showed that GQC-05 shifted splicing of Mcl-1 towards the pro-apoptotic isoform in HeLa cells more than those in Bcl-x pre-mRNA. The aim of this study is to determine firstly whether rG4s are exist in Mcl-1 pre-mRNA, and if they do, secondly how GQC-05 affects these rG4s in the regulation of alternative splicing in Mcl-1.The computational RNA G4 prediction tool, QGRS Mapper, has shown very similar G-scores between Bcl-x and Mcl-1 RNA. In vitro splicing assays of Mcl-1 has shown intron 1 regulates the splicing efficiency. Structural mapping results from WT and 7-deaza-G substituted Mcl-1 in both E- and A-complex conditions showed structural rearrangements, particularly at the 5’ ss, 3’ ss and the polypyrimidine-tract. The addition of GQC-05 displayed variations in the RNase H cleavage suggesting that GQC-05 either remodel the pre-mRNA structure or competes with splicing factors to bind the RNA. Pulldown assays show that GQC-05 is not specific to rG4s but could bind other secondary structures. Mutations of the G-runs in Mcl-1 modify the Mcl-1S/Mcl-1L splicing ratio, and generally increasing Mcl-1 exon 2 skipping in the presence of GQC-05.