Fractionation and Characterization of a Yeast mRNA Splicing Extract
- Creators
- Cheng, Soo-Chen
- Abelson, John
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.
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.Attached Files
Published - CHEpnas86.pdf
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Additional details
- PMCID
- PMC323302
- Eprint ID
- 10893
- Resolver ID
- CaltechAUTHORS:CHEpnas86
- Created
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2008-06-15Created from EPrint's datestamp field
- Updated
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2019-10-03Created from EPrint's last_modified field