Pharmacological Depletion of Polyamines with DFMO in Neuroblastoma cell line SKNBE2

Metabolites are central to cellular homeostasis. Whereas much emphasis has been placed on their relevance to meet energetic and biosynthetic demands, metabolic intermediates also function as signaling molecules. Here we show that polyamines, small polycations that are essential for cell biology, regulate the process of alternative pre-mRNA splicing. We find that the inhibition of polyamine synthesis increases the phosphorylation of spliceosomal proteins, concomitant with remarkable perturbation of alternative splicing in cells and tissues. Mechanistically, molecular modeling together with biochemical assays revealed that polyamines bind to acidic phosphorylatable motifs in splicing factors of the U2 snRNP SF3 subcomplex, thus preventing the action of kinases. The molecular process through which polyamines regulate protein phosphorylation is a phenomenon that we define as “metabolic shielding”. Overall design: The neuroblastoma cell line SKNBE2 was obtained from the Children's Oncology Group Cell Culture Repository and cultured in RPMI (Gibco). To inhibit polyamine biosynthesis cells were treated for 4 days with 250 µM of DFMO obtained from Pat Woster (Medical University of South Carolina). At sub-confluency cells were washed with ice-cold PBS and RNA was isolated from cell extracts by direct addition of 3-volumes of QIAzol® (Qiagen, Cat. No. 79306). After thorough mixing, RNA was purified using the Direct-Zol RNA Mini Prep Plus kit. Stranded library preparation was performed at the Genomic Platform (UNIGE) following standard protocols. Libraries were sequenced on an Illumina NovaSeq 6000, SR 100 bp.