Two phases in the addition of a poly(A) tail.

The addition of a poly(A) tail has been examined in a HeLa cell nuclear extract using SV40 late RNAs that end at or near the natural poly(A) site. We find that the addition of a full-length, 200-nucleotide poly(A) tail occurs in two discrete phases. In the first phase, the addition of each adenosine is dependent on the highly conserved sequence AAUAAA. Mutations in that sequence result in an accumulation of products that contain 9 or fewer adenosine residues. In the second phase, poly(A) addition no longer requires AAUAAA but, instead, requires the oligo(A) primer synthesized during the first phase. Thus, RNAs carrying an AAUAAA mutation and a 3'-terminal oligo(A) segment are extended efficiently to full-length poly(A). The transition between phases occurs with the addition of the tenth adenosine residue. An activity exists that limits the length of poly(A) added in the extract to approximately 200 nucleotides. The two phases share at least one component and are likely to involve the same poly(A) polymerase.