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.

Poly(A) site choice during mRNA 3′-end formation in the Schizosaccharomyces pombe wos2 gene

“…4), of a similar structure to that required for polyadenylation in the human U1A pre-mRNA (Gunderson et al 1994). In some instances, RNA topology is known to play an important role in 3¢-end formation, both in yeasts and in mammals (Irniger et al 1991;Stumpf et al 1996). Therefore, we suggest that polyadenylation sites in wos2 transcripts may be determined by RNA topology.…”

“…Some sequence motifs have been proposed to play an important role in cleavage and poly(A) site recognition (Stumpf et al 1996), but none of them seems to be of general significance for 3¢-end formation in yeast transcripts (Dichtl and Keller 2001) -perhaps because RNA signals are frequently composed of a combination of sequence and structural motifs. This seems to be the case in wos2.…”

Effects of mutations in the Saccharomyces cerevisiae RNA14 gene on the abundance and polyadenylation of its transcripts

“…3). It has already been reported that pre-mRNA topology is important for 3¢ end formation and poly(A) site recognition (van Gelder et al 1993;Graveley et al 1996;Stumpf et al 1996). With this in mind, it is striking that the only two long, hypothetical, stem-loop structures (with a stem of 15 nucleotides) found in the RNA14 sequence (including the 5¢ and 3¢ untranslated sequences) are located at or near the cleavage sites of the short and middle transcripts.…”

Formation of mRNA 3′ Ends in Eukaryotes: Mechanism, Regulation, and Interrelationships with Other Steps in mRNA Synthesis

“…Pol II does not appear to be involved in the poly(A) addition step. The interaction of the CTD with CPSF and CstF (299) may stabilize the cleavage complex or, as proposed by Hirose and Manley (205), have allosteric effects, such as those found for CTD and the capping enzyme (443). This involvement of Pol II in cleavage is consistent with earlier studies showing that protein-encoding mRNAs transcribed by Pol I and Pol III were for the most part not polyadenylated (257,429,432) and with the recent demonstration of the colocalization of CstF and phosphorylated pol II in vivo (439).…”

“…The presence of mRNA secondary structure around signal sequences may be important in some yeast genes (222,401), and a long-range interaction of 5Ј and 3Ј untranslated regions has been demonstrated for MFA2 mRNA (130). While the contribution of RNA conformation has not been rigorously investigated for any site, circular RNA substrates are not cleaved in vitro (443).…”