Polymerase chain reaction mapping of yeast GAL7 mRNA polyadenylation sites demonstrates that 3' end processing in vitro faithfully reproduces the 3' ends observed in vivo

Abstract

In general, synthetic RNA transcripts corresponding to the 3' ends of Saccharomyces cerevisiae genes appear to be accurately cleaved and polyadenylated in vitro under appropriate conditions in yeast cell extracts. Initially, however, the endpoints observed in vitro for the GAL7 gene failed to correlate adequately with those reported in vivo as derived from traditional S1 nuclease protection analyses. This led us to apply an independent method for analyzing mRNA 3' ends, using the polymerase chain reaction, with a first strand primer that incorporated a BamHI restriction site sequence near its 5' end, followed by (dT)17. This proved to be a sensitive and accurate means for determining precisely the major and minor polyadenylation sites of the GAL7 mRNA. Moreover, there was complete agreement between the sites identified with this technique when applied to cellular RNA and those generated in vitro by our 3' end mRNA processing reaction. This provides further support for the likelihood that processing in vitro faithfully reflects the endonucleolytic cleavage and polyadenylation events that occur within the living cell.