Regulation of retention of FosB intron 4 by PTB.
One effect of stressors such as chronic drug administration is that sequence within the terminal exon of the transcription factor FosB is recognized as intronic and removed by alternative splicing. This results in an open-reading-frame shift that produces a translation stop codon and ultimately a truncated protein, termed DeltaFosB. In vitro splicing assays with control and mutated transcripts generated from a fosB mini-gene construct indicated a CU-rich sequence at the 3' end of intron 4 (I4) plays an important role in regulating fosB pre-mRNA splicing due to its binding of polypyrimidine tract binding protein (PTB). PTB binding to this sequence is dependent upon phosphorylation by protein kinase A and is blocked if the CU-rich sequence is mutated to a U-rich region. When this mutated fosB minigene is expressed in HeLa cells, the splicing efficiency of its product is increased compared to wild type. Moreover, transient transfection of PTB-1 in HeLa cells decreased the splicing efficiency of a wild type fosB minigene transcript. Depletion of PTB from nuclear extracts facilitated U2AF65 binding to wild type sequence in vitro, suggesting these proteins function in a dynamic equilibrium to modulate fosB pre-mRNA alternative splicing. These results demonstrate for the first time that phosphorylated PTB promotes intron retention and thereby silences the splicing of fosB I4.
Marinescu
Victor
V
Department of Cellular and Molecular Pharmacology, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, Illinois, United States of America.
Loomis
Patricia A
PA
Ehmann
Svetlana
S
Beales
Mitchell
M
Potashkin
Judith A
JA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D007438:Introns; D010766:Phosphorylation; D038941:Polypyrimidine Tract-Binding Protein / Q000502:physiology*; D016760:Proto-Oncogene Proteins c-fos / Q000235:genetics*; D012326:RNA Splicing; D012689:Sequence Homology, Nucleic Acid