The Interaction In Vitro of the Intermediate Filament Protein Vimentin with Synthetic Polyribo- and Polydeoxyribonucleotides

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Abstract
In a continuation of studies on the association of the intermediate filament protein vimentin with unfolded ribosomal subunits, rRNA and various naturally occurring RNA and DNA, the binding of vimentin to a variety of synthetic polyribo- and polydeoxyribonucleotides was investigated. The vimentin-binding potentials of the different nucleic acids were determined in competition with 28S rRNA from [mouse] Ehrlich ascites tumor cells. The reaction products were analyzed by sucrose gradient centrifugation at low ionic strength and in the presence of EDTA. Among the homopolynucleotides tested, poly(rG), poly(rX), poly(rI), poly(rs4U) [rs4U = 4-thiouridine] and poly(dG) were very good competitors, whereas poly(dC) was moderately so. Poly(rA), poly(rU), poly(rC), poly(dA), poly(dU), poly(dT) and poly(dI) were very weak competitors. Annealing of the active homopolymers to their complementary homopolymer strands resulted in a reduction of the vimentin-binding potential; however, the resulting duplexes still had substantial vimentin-binding capacity. A similar reduction was observed when the mononucleotides of the active homopolymers were randomly copolymerized with their complementary nucleotides or incorporated into strictly alternating, double-stranded DNA. DNA-type copolymers, duplexes and alternating copolymers containing the nuclear bases adenine, thymine and uracil were moderate competitors, but, in general, they were significantly more active than the corresponding homopolymers. This result, together with those of the analysis of a series of random heteropolyribonucleotides, indicates that there is some influence of the base sequence and/or secondary structure of the nucleic acids on their vimentin-binding capacities. Whereas the minimum length of oligo(dG) for efficient vimentin binding was between 11 and 18 nucleotides, (dT)18 was still incapable of interacting with vimentin. In order to obtain information on the salt stability of vimentin-nucleic acid adducts, affinity chromatographies on single-stranded calf thymus DNA-cellulose and on rRNA and homopolyribonucleotides covalently coupled to agarose were also performed. Although because of unspecific secondary interaction of vimentin with the matrix, these nucleic acid-substituted absorbents could not be used for this purpose under non-denaturing conditions, in the presence of 6M urea a relationship between the nucleic acid substitution and the salt stability of vimentin binding was observed. Finally, the nucleic acid-binding properties of vimentin are compared with those of cytosolic steroid hormone receptors, particularly with those of the androgen receptor.