Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes.
It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear protein (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of spliceosome components as revealed by antibodies to the m3G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immunoblot studies indicated that p255 is resistant to extraction with non-ionic detergents, nucleases and high ionic strength buffers and may thus be defined biochemically as a nuclear matrix phosphoprotein. To determine the nature of the association of p255 with the nuclear structure, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We found that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase transition, where it colocalized with SC-35. Nuclear matrix preparations that were digested with DNases and RNases showed that interphasic p255 still localized to nuclear speckles even though snRNA and snRNP antigens were removed. Heat-shocked cells labelled with monoclonal antibody CC-3 exhibited more rounded and less interconnected speckles, identical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuclear structures, to which they are more tightly bound than the snRNP antigens. They further suggest that both proteins are implicated in spliceosome assembly or attachment.
Bisotto
S
S
CHUL Research Center, Laval University, Ste-Foy, Québec, Canada.
Lauriault
P
P
Duval
M
M
Vincent
M
M
D016475:3T3 Cells; D000230:Adenocarcinoma / Q000473:pathology; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001943:Breast Neoplasms / Q000473:pathology; D016466:CHO Cells; D002461:Cell Line, Transformed; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts; D005455:Fluorescent Antibody Technique; D006358:Hot Temperature; D015151:Immunoblotting; D051379:Mice; D008970:Molecular Weight; D015530:Nuclear Matrix / Q000737:chemistry*; D009687:Nuclear Proteins / Q000032:analysis* / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification*; D012326:RNA Splicing; D012260:Ribonucleases / Q000494:pharmacology; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear / Q000032:analysis / Q000276:immunology; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C499372:SRSF2 protein, mouse; D000068103:Serine-Arginine Splicing Factors; D012260:Ribonucleases