Conserved sequence elements in the final exon of MDM2-eight-exon skipping event reveal a ‘cassette regulon’ model of alternative splicing controlled by a distal regulatory element

Modern sequencing technologies have revealed that cancers exhibit widespread dysregulation of alternative splicing, which plays a significant role in driving tumor hallmarks. Alternative splicing enables a single gene to generate multiple mRNA variants, a fundamental mechanism that shapes the distinctive characteristics of normal cells as well as cancer cells. These splicing events are governed by complex interactions between <i>cis</i> - and <i>trans</i> -acting factors and understanding these mechanisms is crucial for effectively targeting cancer cells. This study highlights a unique splicing event in which genotoxic stress induces the skipping of eight-exons in the proto-oncogene <i>MDM2</i> , resulting in the formation of <i>MDM2-ALT1</i> , an isoform that is overexpressed in various cancers. To effectively target this splicing event, it is important to unfold the regulatory mechanism behind it. We hypothesize that the event occurs either by the autonomous regulation of individual exons ( <i>Exon autonomous model</i> ), where each exon is regulated independently or by the coordinated exclusion of an eight-exon-regulon unit ( <i>Exon Regulon model</i> ). Utilizing <i>in-silico</i> tools, a comprehensive modular minigene system, CRISPR-mutated cell line, and murine models, we demonstrate that this complex <i>MDM2</i> splicing event is regulated by sequences within a distal terminal exon, which emerge as critical regulators, orchestrating the alternative splicing of <i>MDM2</i> . Constitutive expression of the <i>Mdm2-MS2</i> isoform (the mouse ortholog of the human <i>MDM2-ALT1</i> splice variant), achieved by mutating the <i>Srsf2</i> binding-site on exon 11, modulates proliferation and apoptosis dynamics in NIH3T3 cells. Moreover, in a p53-wildtype <i>in-vivo</i> setting, this isoform confers a protective effect against age-induced neoplasia. Our findings support the <i>Exon Regulon model</i> of <i>MDM2</i> splicing, regulated by distal elements analogous to distal enhancer elements that control transcription. These finding sheds light on intricacies in the splicing code that could have significant implications for developing splice variant targeting cancer therapies.