Use of RNase H and primer extension to analyze RNA splicing.
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.
Erster
S H
SH
Cold Spring Harbor Laboratory, NY 11724.
Finn
L A
LA
Frendewey
D A
DA
Helfman
D M
DM
D000818:Animals; D001483:Base Sequence; D004722:Endoribonucleases / Q000378:metabolism*; D005091:Exons; D012322:RNA Precursors / Q000032:analysis; D012326:RNA Splicing; D016914:Ribonuclease H; D014335:Tropomyosin / Q000235:genetics
D012322:RNA Precursors; D014335:Tropomyosin; D004722:Endoribonucleases; D016914:Ribonuclease H