Characterization of regulatory mechanisms for BIM exon 3 alternative splicing
Date of Issue2017-02-07
School of Biological Sciences
BCL-2-interacting mediator of cell death (BIM) is a proapoptotic member of the BCL-2 protein family. BIM upregulation is required for tyrosine kinase inhibitor (TKI)-mediated cell death in many kinase-driven cancers including chronic myeloid leukemia (CML) and epidermal growth factor receptor mutated non-small cell lung cancer (EGFR NSCLC). Previously, a 2.9 kb deletion polymorphism in BIM intron 2 was found to contribute to TKI resistance in CML and EGFR NSCLC patients. The deletion allele switched splicing from BIM exon 4 (E4) to exon 3 (E3) in a mutually exclusive manner, generating isoforms lacking the proapoptotic BCL2-homology domain 3 (BH3) encoded by E4. This suggests that modulating BIM splicing to exclude E3 might resensitize the polymorphism containing cancer cells to TKI. In this project, the mechanisms of BIM E3 alternative splicing regulation were studied. To identify the cis-acting splicing elements regulating BIM E3, a series of sequential 10 nucleotide deletions throughout E3 and upstream intronic region were generated in two BIM minigenes with and without the polymorphic fragment. By comparing the E3/E4 ratio of the deletion mutants to that of the full-length minigenes by quantitative real-time PCR, putative splicing enhancers and silencers regulating BIM exon 3 were defined. The exonic enhancers were subsequently verified by point mutations and heterologous minigene analysis. The length of the BIM E3 polypyrimidine tract, which is an essential sequence within 3’ splice sites was varied, and it was found that an unusually long minimum of 16 uridines was required for maximal E3 inclusion. The splicing factor, SRSF1 was confirmed to promote BIM E3 inclusion by knockdown/overexpression study. In addition minor alleles for four annotated single nucleotide polymorphisms (SNPs) were found to potently alter E3 splicing in BIM minigenes and the allele combinations showed additive effects. These annotated SNPs will be subsequently tested for their effects on TKI responses. The splicing regulatory elements elucidated in our study may reveal therapeutic targets for sensitization of TKI responses in cancers dependent on BIM expression for drug sensitivity.