Unraveling novel survivin mRNA transcripts in cancer cells using an in-house developed targeted high-throughput sequencing approach

Abstract

The human baculoviral IAP repeat containing 5 (BIRC5), also known as survivin, is a conserved member of the inhibitor of apoptosis protein (IAPs) family, which is normally expressed during embryonic and fetal development. Although the expression levels of survivin are low in terminally differentiated cells and/or tissues, they can be found notably increased in certain pathological conditions including malignant tumors. Conventional cloning and sequencing techniques have already confirmed that alternative splicing events of the survivin pre-mRNA result in five distinct alternative transcript variants. In the present study, however, we implemented an innovative, in-house developed, targeted DNA-seq assay to identify novel survivin alternative transcript variants with increased depth and coverage that high-throughput sequencing approaches offer. Bioinformatics analysis of the derived NGS datasets unveiled several novel splice junctions between annotated exons of survivin gene as well as the existence of a novel exon of 117 nt, spanning between the annotated exons 3 and 3B. Validation of the NGS findings with PCR-based assays, using variant-specific primers, led to the identification of fourteen novel survivin alternative splice variants (BIRC5 v.4 - v.17), which demonstrate wide expression profiles in a broad established panel of human cell lines. Although the presented novel findings provide a crystal-clear overview of the survivin mRNAs that are actually generated from the pre-mRNA, future studies should focus on the impending necessity of characterizing the biological function of all novel alternative transcript variants as well as the putative protein isoforms. Such studies will further contribute to our understanding of how the balance between survivin isoforms regulate malignant cell proliferation and apoptosis, providing novel diagnostic, prognostic and predictive biomarkers as well as therapeutic targets.