The RNA-splicing factor PSF/p54 controls DNA-topoisomerase I activity by a direct interaction.
DNA-topoisomerase I has been implied in RNA splicing because it catalyzes RNA strand transfer and activates serine/arginine-rich RNA-splicing factors by phosphorylation. Here, we demonstrate a direct interaction between topoisomerase I and pyrimidine tract binding protein-associated splicing factor (PSF), a cofactor of RNA splicing, which forms heterodimers with its smaller homolog, the nuclear RNA-binding protein of 54 kDa (p54). Topoisomerase I, PSF, and p54 copurified in a 1:1:1 ratio from human A431 cell nuclear extracts. Specific binding of topoisomerase I to PSF (but not p54) was demonstrated by coimmunoprecipitation and by far Western blotting, in which renatured blots were probed with biotinylated topoisomerase I. Chemical cross-linking of pure topoisomerase I revealed monomeric, dimeric, and trimeric enzyme forms, whereas in the presence of PSF/p54 the enzyme was cross-linked into complexes larger than homotrimers. When topoisomerase I was complexed with PSF/p54 it was 16-fold more active than the pure enzyme, which could be stimulated 5- and 16-fold by the addition of recombinant PSF or native PSF/p54, respectively. A physiological role of this stimulatory mechanism seems feasible, because topoisomerase I and PSF showed a patched colocalization in A431 cell nuclei, which varied with cell cycle.
Straub
T
T
Medizinische Poliklinik, University of Wuerzburg, D-97070 Wuerzburg, Germany.
Grue
P
P
Uhse
A
A
Lisby
M
M
Knudsen
B R
BR
Tange
T O
TO
Westergaard
O
O
Boege
F
F
D002384:Catalysis; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004268:DNA-Binding Proteins; D004789:Enzyme Activation; D006801:Humans; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins / Q000378:metabolism*; D050810:Octamer Transcription Factors; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D004268:DNA-Binding Proteins; C083214:NONO protein, human; D034521:Nuclear Matrix-Associated Proteins; D009687:Nuclear Proteins; D050810:Octamer Transcription Factors; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D004247:DNA; D004264:DNA Topoisomerases, Type I