Characterisation of 3′ end formation of the yeast HIS3 mRNA

Abstract

The nucleotide (nt) sequence of the 3′ end of the yeast HIS3 mRNA was determined by PCR amplification of the 3′ end. Analysis of 28 individual clones revealed that at least 13 distinct polyadenylation sites are present. The sites of polyadenylation are extremely heterogeneous and do not show any obvious similarity other than that they occur after pyrimidine residues in most cases. Most mutants carrying internal deletions of the 3′ untranslated region (3′ UTR) did not abolish 3' end formation and showed polyadenylation at normal sites. Deletion of a 90-nt region that contains an A + T-rich sequence close to the 3' end of the HIS3 coding sequence and a subset of processing sites resulted in a drastic reduction in the levels of full-length HIS3 mRNA and concomitant transcription past the normal HIS3 3′ end. The 90-nt region appears to be sufficient to direct the formation of at least a subset of the HIS3 3′ ends since mutants that carry deletions of flanking regions of this sequence show detectable levels of HIS3 mRNA. Spacing between the upstream A-T sequence and the site of processing is variable. In the light of the extreme heterogeneity of the sites, a possible mechanism for 3′ processing is discussed.