A combination of RNAse H and S1 nuclease circumvents an artefact inherent to conventional S1 analysis of RNA splicing

SI nucleate napping it commonly used to analyze transcription and processing of unlabellod RNAs. However, the SI protoool that appears best suited to demonstrate splioing of a particular RNA (using an intronless probe that is 3' end-labelled in the downstream exon) is not diagnostio as expeoted. Rather, both intron-oontaining RNA and intronless RNA confer protection of probe across the splice juncture. To unambiguously demonstrate correotly spliced RNAs that begin at a specific initiation site, we present a procedure in which unsplioed RNA moleoules are first cleaved by RNase B following annealing to an intronic DNA fragment and the regaining RNA is then subjected to SI analysis using an intronless probe present in vast excess. Only splioed, oorreotly initiated transcripts can protect the probe aoross the splioe junotion and up to residue +1. This RNase H/Sl Method provides a broadly applioable teohnique with which to demonstrate splicing and initiation of a variety of transcripts, especially ones fron transfeoted genes that out arise both from the normal and from activated oryptic initiation sites.