Mechanism Of The Aberrant Splicing Of DVL2 Induced By Cancer-associated SF3B1 Mutation

Backgroud:RNA splicing is a critical step in the process of gene expressing in eukaryotic cell.Splicing factor SF3B1 is a component of U2 sn RNP complex,and is frequently mutated in cancers.The cancer-associated SF3B1 mutation causes alternative RNA splicing,especially at 3′ splice sites.RNA sequencing analysis reveals that CLL samples with SF3B1 mutation showed alternative 3′ splicing between exons 10 and 11 of DVL2,leading to an in-frame 24-amino-acid deletion.The truncated form of DVL2 protein leads to enhanced Notch signaling,which promotes tumorigenesis.However,the molecular mechanism of SF3B1 mutation which causes the alternative splicing of DVL2 has not been elucidated.Objective:This study aims to investigate the molecular mechanism of SF3B1 mutation that causes alternative splicing of DVL2,to identify the branchpoint(BP)for the canonical and aberrant splicing of DVL2,to analyze the affinity between BPS and U2 sn RNA for the canonical and aberrant splicing of DVL2,and to investigate whether the canonical 3′ss and Py tract are required for the aberrant splicing of DVL.Method:Since,the highest frequency of cancer-associated SF3B1 mutation is K700 E mutation.K700 E mutated SF3B1 expression construct was generated to explore the molecular mechanism of alternative splicing of DVL2 induced by SF3B1 mutation.SVM-BPfinder was used to identify the branchpoint for the canonical and aberrant splicing of DVL2,and we mutated these adenosines to guanosines in DVL2 minigene to identify which adenosines were branchpoint for the canonical and aberrant splicing of DVL2.To confirm the preference of the mutated SF3B1 on the alternative BPS,we swapped the position of the canonical BPS with the position of the alternative BPS in DVL2 minigene.In addition,to investigate whether the canonical 3′ss and Py tract were required for the aberrant splicing of DVL2,the nucleotide upstream of the AG,a nucleotide essential for the function of 3′ss,was mutated and the canonical and the alternative Py tract was deleted in the DVL2 minigene.Result:In this study,we confirmed that the-24 A and-34 A were the canonical branchpoint(BP)and the-16 A was the alternative branchpoint(BP).U2 sn RNP with mutated SF3B1 preferred to use BPS with high affinity to U2 sn RNA for mediating splicing at 3′ss.BPS1 for the canonical splicing of DVL2 minigene played a dominant role in mediating the canonical DVL2 splicing.Both the binding affinity to U2 sn RNA and the distance to 3′ss were important for the selection of BPS to mediate RNA splicing.Conclusion:The aberrant splicing of DVL2 is not dependent on the canonical Py tract and 3′ss,which reveals a novel mechanism of aberrant splicing induced by SF3B1 mutations.