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 CYCI 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 CYCI 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 ofthis fragment are able to grow well. RNA transfers demonstrate that transformants containing the forward orientation accumulate <40% of the control level of full-length mRNA and reveal the presence ofa short, stable (=300 nucleotides) poly(A) RNA that represents 60-70% ofthe transcripts originating from the same promoter. The reverse orientation of the insert allows nearnormal levels offull-length mRNA. Mapping ofthe 3' end ofthe truncated RNA indicates that poly(A) addition is variable in length but occurs at the same location as in the normal CYCI 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.Sherman (3) when they found that the cycl-512 mutation [a 38-base-pair (bp) deletion spanning the wild-type CYCI mRNA 3' end point] resulted in a 90%o reduction in the overall accumulation oftranscripts encoding the CYCI product; many of the mRNAs were longer, and all were polyadenylylated. Several other yeast genes share limited homology to a subset of the sequences deleted by the cycl-512 mutation. Local sequence changes in these regions can restore partial function (4-6); however, no definitive sequence has yet been proved to specify 3' end formation. Analysis of cycl-512 revertants demonstrated that mutations in trans-acting factors can also act to restore the efficiency of 3' end formation and accumulation of stable mRNAs (4, 7).In this report we describe a combined genetic and biochemical approach to understanding 3' end formation and polyadenylylation of yeast mRNA. We have used an in-frame fusion of the actin and HIS4 genes (8) (8); genetic markers include ura3-52 for selection of URA3 transformants, his4-401 [a 2-kilobase (kb) deletion encompassing the promoter and most of the coding sequences], and the dominant HOLI mutation to permit uptake of the histidine precursor, histidinol. In addition, either ade2 or ade5 markers were introduced into the strains to permit selection of diploids used in complementation tests. Yeast were transformed after spheroplasting (9), and other standard genetic techniques and growth media were as described by Sherman et al. (10). In SC + Hol medium, 0.064% histidinol was included in s...

Ssu72 Protein Mediates Both Poly(A)-Coupled and Poly(A)-Independent Termination of RNA Polymerase II Transcription

“…4). Consistent with previous studies demonstrating that this minimal CYC1 element can direct cleavage and polyadenylation when located in the intron of a similar reporter gene construct (43), the CYCpAmin insertion resulted in sensitivity to a copper concentration of 0.25 mM. No increase in copper-resistant growth was observed with this construct in ssu72 or nrd1 mutant strains incubated for 3 days at 30°C.…”

“…The SNR13 125-182, 150-232, and 150-182 fragments were likewise introduced into the XhoI site after PCR amplification with primers that introduced XhoI sites flanking the SNR13 sequences. An XhoI-SalI fragment containing the minimal 83-bp CYC1 3Ј-processing element (43) was similarly cloned into the XhoI site of pGAC24 in both orientations, creating pGAC24-CYC83F and pGAC24-CYC83R.…”

Point mutations upstream of the yeast ADH2 poly(A) site significantly reduce the efficiency of 3'-end formation.

“…In this case, there was 35% readthrough of transcription, resulting in intermediate levels of ,B-galactosidase activity. This is the same relative amount of activity measured by Ruohola et al (37). In their study, the CYCJ 3'-end sequence was inserted into the actin gene intron, which was fused in frame to the HIS4 gene, and readthrough of transcription was measured by quantitation of the resulting RNAs.…”

“…Instead, we saw only the short transcript correctly terminated at the ADH2 site. Other studies also indicate that small insertions upstream of, but not adjacent to, the UACUAAC site of yeast introns have little effect on splicing (21,27,37).…”

Unusual aspects of in vitro RNA processing in the 3' regions of the GAL1, GAL7, and GAL10 genes in Saccharomyces cerevisiae.

“…Detailed analyses of cis-acting sequences involved in 3'-end formation of CYCl (14,(18)(19)(20) (9) propose that there are different classes of polyadenylation sites in S. cerevisiae, a lack of consensus elements would not be surprising unless the genes being compared were in the same class.…”