Proteins C1 and C2 of heterogeneous nuclear ribonucleoprotein complexes bind RNA in a highly cooperative fashion: support for their contiguous deposition on pre-mRNA during transcription.

Abstract

Proteins C1 and C2 together comprise about one-third the protein mass of mammalian core 40S heterogeneous nuclear ribonucleoprotein particles (40S hnRNP) and exist as heterotetramers of (C1)3C2. On the basis of nonequilibrium binding studies, it has been suggested that the C proteins specifically bind oligo(U)- and poly(U)-rich sequences, and preferentially associate with uridine-rich regions near the 3' termini of many introns. We describe here a more quantitative characterization of the equilibrium binding properties of native and recombinant C protein to homoribopolymers using fluorescence spectroscopy. Like C protein from HeLa cells, the recombinant proteins spontaneously oligomerize to form tetramers with the same hydrodynamic properties as native protein. Near-stoichiometric binding titrations of the fluorescent homoribopolymer polyethenoadenosine (poly[r(epsilon A)]) with recombinant (C1)4 and (C2)4 homotetramers along with competition binding assays with poly(A) and poly(C) indicate that the binding site size (n) is between 150 and 230 nucleotides. This site size range is in close agreement with that previously determined for native C protein through hydrodynamic and ultrastructural studies (approximately 230 nucleotides). (C1)4 and (C2)4 bind poly(G) with intrinsic affinities (Ki) of 10(9) M-1, which are a hundredfold higher than their affinities for poly(U). In opposition to reports that C protein does not bind poly(A) and poly(C), we find that the C proteins bind these substrates with moderate Ki, but with high cooperativity (omega). The overall affinity (K omega) for the binding of both proteins to poly(A) and poly(C) is 10-fold higher (> 10(8) but < 10(9) M-1) than their affinities for poly(U). The highly cooperative binding of C protein to these substrates provides a mechanistic basis for the distribution of C protein along the length of nucleic acid substrates.