Altered serine/arginine-rich protein phosphorylation and exonic enhancer-dependent splicing in Mammalian cells lacking topoisomerase I
Abstract
DNA topoisomerase I (Topo I) specifically phosphorylates arginine-serine-rich (SR proteins) splicing factors and is potentially involved in pre-mRNA-splicing regulation. Using a Topo I-deficient murine B lymphoma-derived subclone (P388-45/C) selected for its resistance to high dosage of the antitumor drug camptothecin, we show that Topo I depletion results in the hypophosphorylation of SR proteins and impairs exonic splicing enhancer (ESE)-dependent but not constitutive splicing. The Affymetrix GeneChip system analysis revealed that several alternatively spliced genes, characterized by small exons and large introns, are down-regulated in Topo I-deficient cells. Given that ectopic expression of green fluorescent protein-Topo I fusion in Topo I-deficient cells restores both wild-type phosphorylation of SR proteins and ESE-dependent splicing, we conclude that Topo I-mediated phosphorylation plays a specific role in ESE-regulated splicing.
Keywords
Alternative Splicing Animals Antineoplastic Agents/pharmacology Camptothecin/pharmacology DNA Topoisomerases
Leukemic Green Fluorescent Proteins Leukemia P388/enzymology/genetics Luminescent Proteins/biosynthesis/genetics Mice Nuclear Proteins/*metabolism Phosphoproteins/*metabolism Phosphorylation RNA Splicing/*physiology RNA-Binding Proteins Recombinant Fusion Proteins/biosynthesis/genetics/metabolism Trans-Activation (Genetics)
Neoplasm Exons/physiology Gene Expression Regulation
Type I/*deficiency/genetics Down-Regulation Drug Resistance