Different positioning elements select poly(A) sites at the 3 '-end of GCN4mRNA in the yeast Saccharomyces cerevisiae

Cleavage and polyadenylation of eukaryotic mRNA requires efficiency and pos itioning elements in the 3'-untranslated region (3'-UTR) of the mRNA. Speci fic point mutations were introduced into the yeast GCN4 3'-UTR to detect se quence motifs which are involved in the positioning of the poly(A) site. 3' -End processing activities of different GCN4 3'-UTR alleles were measured i n an in vivo test system. Point mutations in an AAGAA motif defocussed sele ction of the poly(A) sites of the GCN4 3'-UTR to various additional poly(A) sites instead of the single site of the wild-type GCN4 3'-UTR. A strain wi th an intact wild-type GCN4 3'-UTR but impaired in RNA15 encoding an RNA-bi nding processing factor showed a similar defocussed pattern of poly(A) site selection. Remarkably, two additional sequence motifs upstream of the AAGA A motif which resemble yeast efficiency motifs independently affected poly( A) site positioning but not efficiency of 3'-end processing. Mutations in o ne motif resulted in an additional upstream poly(A) site. Alterations of th e other motif shifted the poly(A) sites exclusively to two downstream poly( A) sites. These data suggest several contact points between the precursor m RNA and the polyadenylation machinery in yeast.