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Nucleic Acids Research, 1988, Vol. 16, No. 13 5999-6014
© 1988

Articles

Use of RNase H and primer extension to analyze RNA splicing

Susan H. Erster, Linda A. Finn, David A. Frendewey and David M. Helfman

Cold Spring Harbor Laboratory Cold Spring Harbor, NY 11724, USA

Received February 17, 1988. Revised June 6, 1988. Accepted June 6, 1988.

A new method for the characterization of pre-mRNA splicing products is presented. In this method RNA molecules are hybridized to an oligodeoxynucleotide complementary to exon sequences upstream of a given 5' splice site, and the RNA strands of the resulting RNA:DNA hybrids are cleaved by RNase H. The cleaved RNAs are then subjected to primer extension using a 32P-labelled primer complementary to exon sequences downstream of an appropriate 3' splice site. Since the primer extension products all terminate at the site of RNase H cleavage, their lengths are indicative of the splice sites utilized. The method simplifies the study of the processing of complex pre-mRNAs by allowing the splicing events between any two exons to be analyzed. We have used this approach to characterize the RNAs generated by expression of the rat tropomyosin 1 (Tm 1) gene in various rat tissues and in cultured cells after transient transfection. The results demonstrate that this method is suitable for the analysis of alternative RNA processing in vivo.


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