Interaction of the human autoantigen p150 with splicing snRNPs.
An important goal of studies on pre-mRNA splicing is to identify factors that mediate the snRNP-snRNP and snRNP-pre-mRNA interactions that take place in the spliceosome. The U4/U6 snRNP is one of the four snRNPs that are subunits of spliceosomes. A rare patient autoimmune serum (MaS serum) has recently been identified that specifically immunoprecipitates U4/U6 snRNP from HeLa cell extracts through recognition of a 150 kDa autoantigen (p150) (Okano and Medsger, Journal of Immunology, 146, 535-542, 1991). Here we show that in addition to U4/U6 snRNP, p150 can also be detected associated with 20 S U5, U4/U6.U5 and 17 S U2 snRNPs, but not with U1 snRNP. In each particle p150 is present in sub-stoichiometric levels relative to the major snRNP proteins. We show that MaS serum selectively immunoprecipitates a sub-population of U4/U6 snRNPs in which the m3G-cap structure is masked and that p150 is preferentially associated with U6 snRNA in the U4/U6 particle. Anti-p150 antibodies show widespread nucleoplasmic staining, excluding nucleoli, with an elevated concentration in coiled bodies. This changes to a discrete punctate pattern when cells are treated with alpha-amanitin. Both the cytological and biochemical data indicate that the p150 autoantigen is a snRNP-associated factor in vivo. We also present biochemical evidence confirming that assembly of U4/U6 and U5 snRNPs into a U4/U6.U5 tri-snRNP particle is an integral step in the spliceosome assembly pathway. Addition of the purified U4/U6.U5 tri-snRNP restores splicing activity to inactivated HeLa nuclear extracts in which splicing had been inhibited by specific depletion of either the U4/U6 or U5 snRNPs.
Blencowe
B J
BJ
European Molecular Biology Laboratory, Heidelberg, Germany.
Carmo-Fonseca
M
M
Behrens
S E
SE
Lührmann
R
R
Lamond
A I
AI
D001323:Autoantibodies; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012326:RNA Splicing; D017413:Ribonucleoprotein, U2 Small Nuclear / Q000378:metabolism; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism; D014158:Transcription, Genetic
D001323:Autoantibodies; D001324:Autoantigens; D017413:Ribonucleoprotein, U2 Small Nuclear; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear