Nucleic Acids Research, Vol 25, Issue 2 354-361, Copyright © 1997 by Oxford University Press
W Hong, M Bennett, Y Xiao, R Feld Kramer, C Wang and R Reed
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.
ARTICLES
Association of U2 snRNP with the spliceosomal complex E
Membrane Biology Laboratory, Institute of Molecular and Cell Biology, National University of Singapore, 10 Kent Ridge Crescent, Singapore 0511, Singapore.
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