Abstract
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.
Highlights
Factor [8] and a protein kinase [9] in a manner not involving DNA turnover
Human topoisomerase I is involved in the activation of serine/arginine-rich cofactors of RNA splicing, either by an endogenous protein kinase activity [9] or by physical association with such an enzyme [21]
We report on a direct interaction of topoisomerase I and another RNA-splicing factor
Summary
Recombinant Proteins—Topoisomerase I was expressed in Saccharomyces cerevisiae and purified as described [15]. Precipitated PSF/ p54nrb heterodimer was renatured and purified by gel permeation chromatography in the presence of 1 M NaCl using a Superdex 200 HR30/10 column (Pharmacia Biotech). Native endogeneous topoisomerase I soluble in 1 M ammonium sulfate was adsorbed to phenyl-Sepharose (Pharmacia Biotech), eluted with 30 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM PMSF, 50% glycerol. Analytical gel permeation chromatography of coeluates of topoisomerase I, PSF, and p54nrb was carried out in the presence of 400 mM NaCl with or without 1 M urea using a Superdex 200 HR30/10 column. Concentrations of pure recombinant topoisomerase I and pure recombinant human PSF were determined according to Bradford and served as a standard for determining the respective protein concentrations in crude nuclear extracts, preparations of PSF/p54nrb heterodimers, and trimeric coeluates by comparative immunoblotting. Fluorescent images of cells are representative of the whole cell population inspected in at least 10 separate fields of view
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