Pre-mRNA Topology Is Important for 3′-End Formation in Saccharomyces cerevisiae and Mammals

Abstract

Various signal motifs that are required for efficient pre-mRNA 3′-end formation in the yeast Saccharomyces cerevisiae have been reported. None of these known signal sequences appears to be of the same general importance as is the mammalian AAUAAA motif. To establish the importance of yeast pre-mRNA termini in 3′-end formation, the ends of a pre-mRNA transcript synthesized in vitro were ligated before incubation in a yeast whole-cell extract. Such covalently closed circular RNAs were not cleaved at their poly(A) sites. Interestingly, pseudocircular RNAs with complementary 3′- and 5′-terminal sequences allowing the formation of panhandle structures were also resistant to cleavage. However, 3′-end processing was impeded neither by terminal hairpins at either or at both ends nor by RNA oligonucleotides complementary to either or both ends of a linear pre-mRNA. Intriguingly, mammalian pseudocircular pre-mRNAs also were not cleaved at their poly(A) sites when incubated in a HeLa cell nuclear extract. These results provide evidence for the general importance of RNA topology in the formation of an active 3′-end processing complex in S. cerevisiae and higher eukaryotes. The possibility of a torus-shaped factor involved in 3′-end formation is discussed.