3′ Untranslated Regions Are Modular Entities That Determine Polyadenylation Profiles

The 3′ ends of eukaryotic mRNAs are generated by cleavage of nascent transcripts followed by polyadenylation, which occurs at numerous sites within 3′ untranslated regions (3′ UTRs) but rarely within coding regions. An individual gene can yield many 3′-mRNA isoforms with distinct half-lives. We dissect the relative contributions of protein-coding sequences (open reading frames [ORFs]) and 3′ UTRs to polyadenylation profiles in yeast. ORF-deleted derivatives often display strongly decreased mRNA levels, indicating that ORFs contribute to overall mRNA stability. Poly(A) profiles, and hence relative isoform half-lives, of most (9 of 10) ORF-deleted derivatives are very similar to their wild-type counterparts. Similarly, in-frame insertion of a large protein-coding fragment between the ORF and 3′ UTR has minimal effect on the poly(A) profile in all 15 cases tested. Last, reciprocal ORF/3′-UTR chimeric genes indicate that the poly(A) profile is determined by the 3′ UTR. Thus, 3′ UTRs are self-contained modular entities sufficient to determine poly(A) profiles and relative 3′-isoform half-lives. In the one atypical instance, ORF deletion causes an upstream shift of poly(A) sites, likely because juxtaposition of an unusually high AT-rich stretch directs polyadenylation closely downstream. This suggests that long AT-rich stretches, which are not encountered until after coding regions, are important for restricting polyadenylation to 3′ UTRs.