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In Vitro Studies of the Prp9·Prp11·Prp21 Complex Indicate a Pathway for U2 Small Nuclear Ribonucleoprotein Activation

Wiest, Debra K. and O'Day, Christine L. and Abelson, John (1996) In Vitro Studies of the Prp9·Prp11·Prp21 Complex Indicate a Pathway for U2 Small Nuclear Ribonucleoprotein Activation. Journal of Biological Chemistry, 271 (52). pp. 33268-33276. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:WIEjbc96

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Abstract

Pre-mRNA splicing takes place on a large ribonucleoprotein particle, the spliceosome which contains the five small nuclear ribonucleoproteins (snRNPs), U1, U2, U4, U5, and U6. In Saccharomyces cerevisiae the mRNA splicing factors, Prp9, Prp11, and Prp21, are necessary for addition of the U2 snRNP to the pre-mRNA in an early step of spliceosome assembly. This paper describes a study of interactions between these proteins and their role in spliceosome assembly. The proteins were expressed in Escherichia coli. Prp9 and Prp11 were purified by metal affinity chromatography. Prp21 was purified using a solubilization/renaturation protocol. We have combined these separately purified proteins and present direct evidence of a Prp9·Prp11·Prp21 protein complex that is functional in in vitro splicing assays. Characteristics of this Prp9·Prp11·Prp21 complex were further investigated using proteins synthesized in vitro. In addition, we found that Prp9, Prp11, and Prp21 influence the structure of the U2 snRNP in a manner that alters the accessibility of the branch point pairing region of the U2 snRNA to oligonucleotide-directed RNaseH cleavage. We present a model, based on the data presented here and in the accompanying paper, for a combined role of Prp9, Prp11, Prp21, and Prp5 in activating the U2 snRNP for assembly into the pre-spliceosome.


Item Type:Article
Additional Information:©1996 by The American Society for Biochemistry and Molecular Biology, Inc. (Received for publication, May 1, 1996, and in revised form, August 12, 1996) We thank Jaime Arenas for providing the Prp21 expression plasmids. The research was supported by Grant GM32637 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. [D.K.W. was] [s]upported by a grant from the Jane Coffin Childs Memorial Fund for Medical Research.
Record Number:CaltechAUTHORS:WIEjbc96
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:WIEjbc96
Alternative URL:http://www.jbc.org/cgi/content/abstract/271/52/33268
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:6455
Collection:CaltechAUTHORS
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Deposited On:05 Jan 2007
Last Modified:26 Dec 2012 09:21

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