Association of U2 snRNP with the spliceosomal complex E.
In metazoans, the E complex is operationally defined as an ATP-independent spliceosomal complex that elutes as a single peak on a gel filtration column and can be chased into spliced products in the presence of an excess of competitor pre-mRNA. The A complex is the first ATP-dependent functional spliceosomal complex. U1 snRNP first binds tightly to the 5'splice site in the E complex and U2 snRNP first binds tightly to the branch site in the A complex. In this study, we have generated and characterized a monoclonal antibody (mAb 4G8) directed against SAP 62, a component of U2 snRNP and a subunit of the essential mammalian splicing factor SF3a. We show that this antibody is highly specific for SAP 62, detecting only SAP 62 on Western blots and immunoprecipitating only SAP 62 from nuclear extracts. The anti-SAP 62 antibody also immunoprecipitates U2 snRNP and the A complex. Significantly, however, we find that the E complex is also efficiently immunoprecipitated by the anti-SAP 62 antibody. This antibody does not cross-react with any E complex-specific components, indicating that SAP 62 itself is associated with the E complex. To determine whether other U2 snRNP components are associated with the E complex, we used antibodies to the U2 snRNP proteins B"and SAP 155. These antibodies also specifically immunoprecipitate the E complex. These observations indicate that U2 snRNP is associated with the E complex. However, we find that U2 snRNP is not as tightly bound in the E complex as it is in the A complex. The possible significance of the weak association of U2 snRNP with the E complex is discussed.
Hong
W
W
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore.
Bennett
M
M
Xiao
Y
Y
Feld Kramer
R
R
Wang
C
C
Reed
R
R
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002352:Carrier Proteins / Q000276:immunology / Q000378:metabolism; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D009687:Nuclear Proteins / Q000276:immunology / Q000378:metabolism; D009698:Nucleoproteins / Q000276:immunology / Q000378:metabolism; D011233:Precipitin Tests; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017381:Spliceosomes / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D009687:Nuclear Proteins; D009698:Nucleoproteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; C083433:SF3A2 protein, human