Splicing Dysregulation of Non-Canonical GC-5' Splice Sites of Breast Cancer Susceptibility Genes ATM and PALB2

• Published in: Cancers 2024-10, Vol.16 (21), p.3562
• Main Authors: Llinares-Burguet, Inés, Sanoguera-Miralles, Lara, Valenzuela-Palomo, Alberto, García-Álvarez, Alicia, Bueno-Martínez, Elena, Velasco-Sampedro, Eladio A
• Format: Article
• Language: English
• Subjects: Amino acid sequence; Binding sites; Breast cancer; Cancer; Deep learning; Disease susceptibility; Exons; Gene mapping; Genes; Genetic aspects; Genetic engineering; Health aspects; Introns; Oncology, Experimental; Peptide mapping; Regulatory sequences; Splicing
• ISSN: 2072-6694; 2072-6694
• DOI: 10.3390/cancers16213562

: The non-canonical GC-5' splice sites (5'ss) are the most common exception (~1%) to the classical GT/AG splicing rule. They constitute weak 5'ss and can be regulated by splicing factors, so they are especially sensitive to genetic variations inducing the misrecognition of their respective exons. We aimed to investigate the GC-5'ss of the breast/ovarian cancer susceptibility genes, (exon 50), (exon 1), and (exon 12), and their dysregulation induced by DNA variants. : Splicing assays of the minigenes, mgATM_49-52, mgBRIP1_1-2, and mgPALB2_5-12, were conducted to study the regulation of the indicated GC-5'ss. : A functional map of the splicing regulatory elements (SRE) formed by overlapping exonic microdeletions revealed three essential intervals, c.7335_7344del, c.3229_3258del, and c.3293_3322del, which are likely targets for spliceogenic SRE-variants. We then selected 14 and 9 variants (Hexplorer score < -40) located at these intervals that were assayed in MCF-7 cells. Nine and three variants affected splicing, impairing the recognition of exons 50 and 12, respectively. Therefore, these variants likely disrupt the active SREs involved in the inclusion of both exons in the mature mRNA. DeepCLIP predictions suggested the participation of several splicing factors in exon recognition, including SRSF1, SRSF2, and SRSF7, involved in the recognition of other GC sites. The spliceogenic variants c.7336G>T (p.(Glu2446Ter)) and c.7340T>A (p.(Leu2447Ter)) produced significant amounts of full-length transcripts (55-59%), which include premature termination stop codons, so they would inactivate ATM through both splicing disruption and protein truncation mechanisms. : exon 50 and exon 12 require specific sequences for efficient recognition by the splicing machinery. The mapping of SRE-rich intervals in minigenes is a valuable approach for the identification of spliceogenic variants that outperforms any prediction software. Indeed, 12 spliceogenic SRE-variants were identified in the critical intervals.