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 as well as in malignant tumors. Conventional cloning and sequencing techniques have already confirmed that alternative splicing events within the survivin pre-mRNA result in five distinct alternative transcript variants of the gene. 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 novel findings of the current study 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.

含有 5 (BIRC5) 的人杆状病毒 IAP 重复序列，也称为生存素，是凋亡蛋白 (IAP) 家族的保守成员，通常在胚胎和胎儿发育过程中表达。尽管生存素在终末分化的细胞和/或组织中的表达水平较低，但在某些病理条件以及恶性肿瘤中可以发现它们的表达水平显着增加。传统的克隆和测序技术已经证实，存活素前 mRNA 内的可变剪接事件导致该基因的五种不同的替代转录变体。然而，在本研究中，我们实施了一种创新的、内部开发的、有针对性的 DNA-seq 分析来鉴定新的生存素高通量测序方法提供的具有更高深度和覆盖率的替代转录变体。衍生的 NGS 数据集的生物信息学分析揭示了survivin基因的注释外显子之间的几个新剪接点，以及存在一个 117 nt 的新外显子，跨越注释的外显子 3 和 3B。使用变体特异性引物通过基于 PCR 的分析验证 NGS 发现导致鉴定出 14 种新的存活蛋白可变剪接变体 ( BIRC5 v.4 - v.17)，这些变体在广泛的已建立小组中展示了广泛的表达谱人类细胞系。尽管当前研究的新发现提供了对生存素的清晰概述实际上从前 mRNA 产生的 mRNA，未来的研究应该集中在表征所有新的替代转录变体以及推定的蛋白质同种型的生物学功能的迫在眉睫的必要性。这些研究将进一步有助于我们了解生存素异构体之间的平衡如何调节恶性细胞增殖和凋亡，提供新的诊断、预后和预测生物标志物以及治疗靶点。