COLOCALIZATION OF A HIGH-MOLECULAR-MASS PHOSPHOPROTEIN OF THE NUCLEARMATRIX (P255) WITH SPLICEOSOMES

It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear prote in (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of splice osome components as revealed by antibodies to the m(3)G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immu noblot studies indicated that p255 is resistant to extraction with non -ionic detergents, nucleases and high ionic strength buffers and may t hus be defined biochemically as a nuclear matrix phosphoprotein. To de termine the nature of the association of p255 with the nuclear structu re, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We f ound that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase t ransition, where it colocalized with SC-35. Nuclear matrix preparation s that were digested with DNases and RNases showed that interphasic p2 55 still localized to nuclear speckles even though snRNA and snRNP ant igens were removed. Heat-shocked cells labelled with monoclonal antibo dy CC-3 exhibited more rounded and less interconnected speckles, ident ical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuc lear structures, to which they are more tightly bound than the snRNP a ntigens. They further suggest that both proteins are implicated in spl iceosome assembly or attachment.