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Fractionation and Characterization of a Yeast mRNA Splicing Extract

Cheng, Soo-Chen and Abelson, John (1986) Fractionation and Characterization of a Yeast mRNA Splicing Extract. Proceedings of the National Academy of Sciences of the United States of America, 83 (8). pp. 2387-2391. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:CHEpnas86

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Abstract

We have fractionated a yeast whole cell extract that can accurately splice synthetic actin and CYH2 pre-mRNAs. Three fractions, designated I, II, and III, have been separated by use of ammonium sulfate fractionation and chromatography on heparin agarose. Each fraction alone has no splicing activity. Fractions I and II allow the first step of the splicing reaction to proceed, giving rise to the splicing intermediates, free exon 1, and intron-exon 2. Addition of fraction III completes the reaction. Micrococcal nuclease treatment of the whole cell extract or of either fraction I or II abolished splicing activity, indicating that fractions I and II have RNA moieties that are required in the splicing reaction. The nature of the RNAs was examined using antibodies directed against the trimethylated cap structure unique to small nuclear RNAs. Preincubation of the whole cell extract with protein A-Sepharose coupled to trimethylated cap antibody abolished splicing activity. This indicates that at least one essential RNA component contains a trimethyl cap. Thus, in yeast as in mammalian systems, small nuclear RNAs are involved in mRNA splicing.


Item Type:Article
Additional Information:Copyright © 1986 by the National Academy of Sciences. Contributed by John Abelson, December 18, 1985. We thank Douglas Black and Reinhard Luhrmann for providing us with anti-(m3G)cap antibodies, and Andrew Newman and Joan Steitz for their valuable suggestions and encouragements. We are also indebted to Benoit Chabot, Douglas Black, Joan Steitz, and Christine Guthrie for a critical reading of the manuscript. The research was supported by Grant GM 30356 from the National Institutes of Health. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Record Number:CaltechAUTHORS:CHEpnas86
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:CHEpnas86
Alternative URL:http://www.pnas.org/cgi/content/abstract/83/8/2387
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:10893
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:15 Jun 2008
Last Modified:14 Nov 2014 19:20

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