The nuclear matrix phosphoprotein p255 associates with splicing complexes as part of the [U4/U6.U5] tri-snRNP particle.
The monoclonal antibody CC3 recognizes a phosphorylated epitope present on an interphase protein of 255 kDa. Previous work has shown that p255 is localized mainly to nuclear speckles and remains associated with the nuclear matrix scaffold following extraction with non-ionic detergents, nucleases and high salt. The association of p255 with splicing complexes is suggested by the finding that mAb CC3 can inhibit in vitro splicing and immunoprecipitate pre-messenger RNA and splicing products. Small nuclear RNA immunoprecipitation assays show that p255 is a component of the U5 small nuclear ribonucleoprotein (snRNP) and the [U4/U6.U5] tri-snRNP complex. In RNase protection assays, mAb CC3 immunoprecipitates fragments containing branch site and 3' splice site sequences. As predicted for a [U4/U6.U5]-associated component, the recovery of the branch site-protected fragment requires binding of U2 snRNP and is inhibited by EDTA. p255 may correspond to the previously identified p220 protein, the mammalian analogue of the yeast PRP8 protein. Our results suggest that changes in the phosphorylation of p255 may be part of control mechanisms that interface splicing activity with nuclear organization.
Chabot
B
B
Département de Microbiologie, Faculté de Médecine, Université de Sherbrooke, Québec, Canada.
Bisotto
S
S
Vincent
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D015530:Nuclear Matrix / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D010766:Phosphorylation; D012326:RNA Splicing; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism
D000911:Antibodies, Monoclonal; D034961:Antigens, Nuclear; D009687:Nuclear Proteins; D010750:Phosphoproteins; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear