Sequences on the 3' side of hexanucleotide AAUAAA affect efficiency of cleavage at the polyadenylation site

The hexanucleotide AAUAAA has been demonstrated to be part of the signal for cleavage and polyadenylation at appropriate sites on eucaryotic mRNA precursors. Since this sequence is not unique to polyadenylation sites, it cannot be the entire signal for the cleavage event. We have extended the definition of the polyadenylation cleavage signal by examining the cleavage event at the site of polyadenylation for the simian virus 40 late mRNAs. Using viable mutants, we have determined that deletion of sequences between 3 and 60 nucleotides on the 3' side of the AAUAAA decreases the efficiency of utilization of the normal polyadenylation site. These data strongly indicate a second major element of the polyadenylation signal. The phenotype of these deletion mutants is an enrichment of viral late transcripts longer than the normally polyadenylated RNA in infected cells. These extended transcripts appear to have an increased half-life due to the less efficient cleavage at the normal polyadenylation site. The enriched levels of extended transcripts in cells infected with the deletion mutants allowed us to examine regions of the late transcript which normally are difficult to study. The extended transcripts have several discrete 3' ends which we have analyzed in relation to polyadenylation and other RNA processing events. Two of these ends map to nucleotides 2794 and 2848, which lie within a region of extensive secondary structure which marks the putative processing signal for the formation of the simian virus 40-associated small RNA. A third specific 3' end reveals a cryptic polyadenylation site at approximately nucleotides 2980 to 2985, more than 300 nucleotides beyond the normal polyadenylation site. This site appears to be utilized only in mutants with debilitated normal sites. The significance of sequences on the 3' side of an AAUAAA for efficient polyadenylation at a specific site is discussed.