Sequence requirements of the SV40 early polyadenylation signal

Abstract

Polyadenylation of eukaryotic messenger RNA molecules consists of a two step reaction; cleavage of the primary transcript and step-wise addition of adenylic acid residues to the newly generated 3' OH group. The complex which cleaves and polyadenylates RNA transcripts recognizes a specific signal element on the RNA which is comprised, in part, by the hexanucleotide AAUAAA. Additional sequence elements must be required for polyadenylation since the hexanucleotide is found at sites other than adjacent to the 3' end of many genes. The objective of the experiments performed in this study is to isolate the sequence which comprises the entire SV40 early orientation polyadenylation signal. In the first step toward accomplishing this objective, deletions were made in the DNA sequence upstream from the poly(A) addition site in a plasmid containing the SV40(E) polyadenylation signal. These deletion mutants were assayed for a functional polyadenylation signal in a transient expression assay system. Data from these experiments is consistent with the hypothesis that a hexanucleotide is essential for a functional polyadenylation signal and sequences upstream from that hexanucleotide are dispensable. The deletion mutants were subcloned into a vector containing an adenovirus L3 polyadenylation signal downstream from the SV40 3' end processing signal. The distribution of transcripts cleaved and polyadenylated at the experimental SV40 site versus the control adenovirus L3 site shows that the SV40 signal is unaffected until the deletion end-point extends past the distal hexanucleotide. The data described above indicate the upstream boundary of the poly(A) signal is the distal hexanucleotide. Previous studies indicate the downstream boundary is the 3' end of the downstream element. A polyadenylation signal with these boundaries was constructed using synthesized oligonucleotide DNA fragments. Messenger RNAs transcribed from plasmids containing the synthetic fragment are cleaved and polyadenylated at the wild-type SV40 poly(A) site. The distribution of cleavage at the poly(A) addition site in the synthetic signal versus at a downstream adenovirus L3 poly(A) site is the same as that observed with the wild-type SV40 sequence. These data indicate that the SV40(E) polyadenylation signal can be isolated to a 40 nucleotide region surrounding the poly(A) addition site.