Abstract
Cleavage and polyadenylation of eukaryotic mRNA requires efficiency and positioning elements in the 3'-untranslated region (3'-UTR) of the mRNA. Specific point mutations were introduced into the yeast GCN4 3'-UTR to detect sequence motifs which are involved in the positioning of the poly(A) site. 3'-End proces-sing activities of different GCN4 3'-UTR alleles were measured in an in vivo test system. Point mutations in an AAGAA motif defocussed selection 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 with an intact wild-type GCN4 3'-UTR but impaired in RNA15 encoding an RNA-binding processing factor showed a similar defocussed pattern of poly(A) site selection. Remarkably, two additional sequence motifs upstream of the AAGAA motif which resemble yeast efficiency motifs independently affected poly(A) site positioning but not efficiency of 3'-end processing. Mutations in one motif resulted in an additional upstream poly(A) site. Alterations of the other motif shifted the poly(A) sites exclusively to two downstream poly(A) sites. These data suggest several contact points between the precursor mRNA and the polyadenylation machinery in yeast.
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