Decoding the concealed transcriptional signature of the apoptosis-related BCL2 antagonist/killer 1 (BAK1) gene in human malignancies

BCL2 antagonist/killer (BAK) is a multidomain pro-apoptotic effector protein, encoded by the human BAK1 gene, which has emerged as a key checkpoint in the apoptotic machinery. Disassembly of BAK's tertiary structure, such as the truncation of the α1 helix, leads to deregulation of the pro-apoptotic functions and reduction of the protein's stability, thus being implicated in human malignancies. Although many studies have already clarified the vital role of BAK in cellular mechanisms, its pre-mRNA maturation process under cancerous and physiological human cells is neglected. In the present work, we developed and employed a custom multiplexed nanopore sequencing approach that enabled the identification and structural characterization of previously undescribed BAK1 mRNA transcripts (BAK1 v.2-v.11). The described novel mRNAs are derived from multiple types of alternative splicing events, including exon skipping and intron retentions. The implemented multiplexed long-read sequencing approach provided the detailed expression profile of the novel mRNAs in a wide panel of human malignancies and at the same time allowed their relative quantification as compared to the annotated BAK1 v.1. The validation of each novel transcript was carried out with qPCR-based assays. Our results strongly support that most of the novel BAK1 mRNAs harbor open reading frames with conserved BH domains that provide new insights into the correlated mechanisms of apoptosis suppression and cancer. The current study highlights for the first time the hidden aspects of BAK1's transcriptional landscape in both physiological and cancerous human cells and distinguishes the amino acid sequence of the putative BAK isoforms that may possess key apoptosis-related functions not only in diseases, but also under normal cellular conditions.