CHARACTERIZATION OF 2 MAJOR CELLULAR POLY(RC)-BINDING HUMAN PROTEINS, EACH CONTAINING 3 K-HOMOLOGOUS (KH) DOMAINS

We have revealed and characterised two nucleic-acid-binding proteins; termed PCBP-1 (M(r) 37 525, pI 7.07) and PCBP-2 (M(r) 38579, pI 6.76), that together with heterogeneous ribonucleoparticle (hnRNP)-K correspond to the major cellular poly(rC)-binding proteins. mRNA for both PCBPs were detected in all the human tissues analysed. Both proteins contain three K-homologous (KH) domains which share similarity with other KH domain proteins, including the fragile-X protein FMR1, and which are positioned as in hnRNP-K and nova, i.e. with two closely spaced domains at the N-terminus and one at the C-terminus. PCBPs do not contain RGG boxes or any other known nucleic-acid-binding motifs. Expression in the vaccinia virus system showed that both proteins are post-translationally modified in vivo, a fact that was confirmed by [P-32]orthophosphate labelling. Northwestern-blot analysis showed that the nonphosphorylated forms bind tenaciously to poly(rC) in vitro, while significantly less binding was observed for the phosphorylated variants. Escherichia coli expressed proteins also bound poly(rG), albeit at a lower level. In addition, PCBP-2 bound poly(rU), whereas very little binding to poly(rA) was observed for both proteins.