POLY(A) SITE SELECTION IN THE YEAST TY RETROELEMENT REQUIRES AN UPSTREAM REGION AND SEQUENCE-SPECIFIC TITRATABLE FACTOR(S) IN-VITRO

In the Ty retrotransposon of Saccharomyces cerevisiae, as in most retr oelements, the polyadenylation site of the 5' long terminal repeat (LT R) is ignored and the one in the 3' LTR is efficiently used. We examin e here the contribution to this poly(A) site selection of the region t ermed 'U3', corresponding to the upstream nontranscribed portion of th e 5' LTR. Using an established assay in vitro, we find that 3' process ing is accurate and efficient with an RNA substrate corresponding to m ost of the LTR, whereas none is detectable with a shorter transcript l acking the U3 region, thus explaining why the 5' poly(A) site is ignor ed in genomic Ty mRNA. When HIS4 coding RNA, representing 'non-specifi c' sequence, replaces the U3 region, the Ty polyadenylation site is ac tivated to 50% of the wild-type level. Within one specific region (TS1 ) in U3, 90-95 nt upstream of the poly(A) site, the change of UAGUAU t o UCGCAU reduces processing efficiency by half, to the non-specific le vel provided by other sequences or by a deletion of the TS1 region. An other region (TS2) near the poly(A) site appears to be independently r esponsible for the remaining half of the processing activity. Alterati on of both TS1 and TS2 eliminates processing entirely. In competition assays, excess unlabeled U3, but not its mutated counterparts, reduces the processing of radiolabeled Ty mRNA, suggesting the involvement of some sequence-specific titratable factor(s) in the whole cell extract for U3-specific activation. Since we obtain similar reductions in com peting CYC1 mRNA processing with excess unlabeled Ty-derived RNAs, we conclude that polyadenylation of Ty element RNA, while possessing some unusual properties, also shares common components of the processing m achinery used with other yeast mRNA transcripts.