G-quadruplex/m⁶A-regulated BCL2 pre-mRNA alternative splicing and comparative characterization of its two isoforms

Alternative splicing of the BCL2 precursor mRNA (pre-mRNA) generate two protein-coding transcript isoforms, BCL2α and BCL2β. Numerous reports indicate that BCL2, namely BCL2α, contributes to cancer development through promoting cancer cell survival, while the mechanism underlying BCL2 pre-mRNA alternative splicing and the functional role of the BCL2β in oncogenesis remain unknown. Here, we discovered an RNA G-quadruplex (rG4) motif and three m⁶A RNA methylation motifs downstream rG4's proximity at the 5'-end of BCL2 pre-mRNA exon 3. In vitro and in cellulo assays verified rG4 structure formation in this region. The methylation status at these m⁶A motifs was also demonstrated in breast cancer cells. rG4 structure favored BCL2β splicing, but the underlying mechanisms in triple negative breast cancer (TNBC) and non-TNBC cells were different. In TNBC cells, rG4 stabilization promoted m⁶A modification that decreased both overall BCL2 mRNA levels and BCL2α splicing, but enhanced BCL2β production; conversely, in non-TNBC cells, rG4 structure reduced m⁶A modification that increased both overall BCL2 mRNA and BCL2α isoform levels, but reduced BCL2β splicing. When exploring splicing factors involved in BCL2 pre-mRNA splicing, we identified SRSF1 as a novel rG4 binding protein that resolved rG4 structure, subsequently promoting BCL2α splicing and reducing BCL2β isoform. Further, BCL2α exhibited oncogenic activities as previously reported, but BCL2β reduced breast cancer cell survivability and migration, and sensitized cells to chemotherapeutics-induced apoptosis. Overall, in this study, we revealed the mechanisms regulating BCL2 pre-mRNA alternative splicing through rG4 structure, m⁶A modification and SRSF1 binding, and discovered antiproliferative and proapoptotic activity of BCL2β.