Identification of a novel, non-snRNP protein complex containing U1A protein.
Mouse monoclonal antibodies (MAbs) were generated against Escherichia coli-produced U1snRNP-A (U1A) protein. U1A-specific MAbs as well as MAbs that reacted with both U1A and U2snRNP-B" (U2B") were isolated. MAb 12E12 was unique among the characterized MAbs because it failed to immunoprecipitate U1A protein produced by in vitro transcription and translation using rabbit reticulocyte lysates. However, when U1A protein was made using a wheat germ extract, MAb 12E12 could immunoprecipitate U1A quite readily, as did the other MAbs. These data suggest that the MAb 12E12 epitope is masked when U1A is prepared in reticulocyte lysate. Further studies showed that MAb 12E12 recognizes an epitope that is masked when U1A protein is bound to U1 RNA. The unique nature of MAb 12E12 was used to demonstrate that U1A could be immunoprecipitated from whole-cell extracts in a form that was free of U1 RNA and other snRNP components. However, this snRNP-free U1A (SF-A) was found to co-immunoprecipitate with a unique set of non-snRNP proteins. In order to confirm that U1A exists in at least two distinct complexes (snRNP bound and snRNP free), [35S]-labeled nucleoplasmic extracts were analyzed by sucrose density gradient fractionation and immunoprecipitation. MAb 12E12 specifically immunoprecipitated SF-A, which migrated in a novel non-snRNP complex. Specifically, proteins of approximately 58, 59, 63, 65, and 105 kDa co-sedimented and co-immunoprecipitated with SF-A. Our data show that a significant portion of the cellular U1A (at least 3% or approximately 30,000 molecules) exists in the nucleoplasm in one or more novel complexes. Our previous studies have demonstrated an effect of purified U1A on polyadenylation of pre-mRNAs and, consistent with this finding, purified antibodies to SF-A significantly diminish polyadenylation in vitro.
O'Connor
J P
JP
Department of Orthopaedic Surgery, University of Pennsylvania School of Medicine, Philadelphia 19104, USA.
Alwine
J C
JC
Lutz
C S
CS
D000818:Animals; D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus / Q000737:chemistry; D000939:Epitopes / Q000032:analysis; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D011233:Precipitin Tests; D012342:RNA, Small Nuclear / Q000032:analysis; D016601:RNA-Binding Proteins; D011817:Rabbits; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000032:analysis* / Q000502:physiology; D012261:Ribonucleoproteins / Q000737:chemistry*; D017411:Ribonucleoproteins, Small Nuclear / Q000032:analysis
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D000939:Epitopes; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear; C075339:U1A protein