Orientation-dependent function of a short CYC1 DNA fragment in directing mRNA 3' end formation in yeast.

Abstract

We have cloned an 82-base-pair region spanning the site of normal 3' end formation of Saccharomyces cerevisiae CYC1 mRNA into an integrative vector carrying the 5' end of the actin gene (including its intron) fused in frame to HIS4ABC sequences. This vector can confer the ability to grow on histidinol if HIS4C (encoding histidinol dehydrogenase) is sufficiently expressed. With the CYC1 fragment cloned in its wild-type (forward) orientation within the actin intron, transformants cannot grow on histidinol, whereas cells transformed with the vector carrying the reverse orientation of this fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate less than 40% of the control level of full-length mRNA and reveal the presence of a short, stable (approximately equal to 300 nucleotides) poly(A) RNA that represents 60-70% of the transcripts originating from the same promoter. The reverse orientation of the insert allows near-normal levels of full-length mRNA. Mapping of the 3' end of the truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYC1 transcript. Dominant and recessive suppressor mutations permit growth on histidinol despite the inserted fragment. Genetic analyses indicate that most of the dominant mutants are cis-acting and that the recessive mutants define a minimum of three complementation groups, indicating that defects in several different genes can restore higher levels of HIS4C expression.