Uncoupling of mRNA 3' cleavage and polyadenylation by expression of a hammerhead ribozyme in yeast.

An artificial messenger RNA containing a derivative of a tobacco ringspot virus ribozyme was expressed in the bakers' yeast Saccharomyces cerevisiae. This mRNA was able to cleave itself efficiently in vivo. Using this system, the two steps of mRNA 3' processing, i.e. cleavage and the addition of a poly(A) tail, can be separated in yeast in vivo. The ribozyme-cleaved transcript was shown to be polyadenylated. The poly(A) tail length was similar to the poly(A) tail length of an endogenous yeast mRNA. Therefore, cleavage of the precursor RNA at the polyadenylation site and the addition of adenosine residues to the 5' product require independent cellular machineries in yeast and can be separately analyzed. This is in contrast to higher eukaryotes where both processes are coupled.