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Nucleic Acids Research, 1992, Vol. 20, No. 8 1949-1957
© 1992

MOLECULAR BIOLOGY

Phosphorothioate substitution identifies phosphate groups important for pre-mRNA splicing

Kathryn L. Maschhoff and Richard A. Padgett*

Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas 5323 Harry Hines Boulevard, Dallas, TX 75235, USA

*To whom correspondence should be addressed

Received January 9, 1992. Revised March 20, 1992. Accepted March 20, 1992.

Substitution of pre-mRNA In vitro splicing substrates with {alpha}-phosphorothioate ribonucleotide analogs has multiple effects on the processes of spllceosome formation and splicing. A major effect of substitution is on the splicing cleavage/ligation reactions. Substitution at the 5' splice junction blocks the first cleavage/ligation reaction while substitution at the 3' splice junction blocks the second cleavage/ligation reaction. A second effect of phosphorothioate substitution is the inhibition of spliceosome formation. A substitution/interference assay was used to determine positions where substitution inhibits spliceosome formation or splicing. Substitution in the 3' splice site polypyrlmidine tract was found to Inhibit spliceosome formation and splicing. This effect was enhanced with multiple substitutions in the region. No sites of substitution within the exons were found which affected spliceosome formation or splicing.


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