In vitro trans-splicing in Saccharomyces cerevisiae

Creators: Ghetti, Andrea; Abelson, John N.

Abstract

The interactions established at the 5'-splice site during spliceosome assembly are likely to be important for both precise recognition of the upstream intron boundary and for positioning this site in the active center of the spliceosome. Definition of the RNA-RNA and the RNA-protein interactions at the 5' splice site would be facilitated by the use of a small substrate amenable to modification during chemical synthesis. We describe a trans-splicing reaction performed in Saccharomyces cerevisiae extracts in which the 5' splice site and the 3' splice site are on separate molecules. The RNA contributing the 5' splice site is only 20 nucleotides long and was synthesized chemically. The trans-splicing reaction is accurate and has the same sequence, ATP, and Mg^2+ requirements as cis-splicing. We also report how deoxy substitutions around the 5'-splice site affect trans-splicing efficiency.

Additional Information

© 1995 National Academy of Sciences. Contributed by John N. Abelson, August 30, 1995. We are grateful to Maria Konarska and Robin Reed for sharing their trans-splicing results prior to publication. We also thank James their trans-splicing results prior to publication. We also thank James Bruzik, Christine Guthrie, David McPheeters, Anna Marie Pyle, Christian Siebel, and John Wagner for comments on the manuscript. 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.

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