The upstream fragment generated by 3' cleavage of the pre-mRNA receives a poly(A) tail of approximately 250 AMP residues in a reaction depending on the AAUAAA sequence 10 to 30 nucleotides upstream of the 3' end. Polyadenylation is carried out by three proteins: Poly(A) polymerase carries the catalytic activity. The enzyme has no specificity for any particular RNA sequence, and it also has a very low affinity for the RNA.
Under physiological conditions, the activity of poly(A) polymerase thus depends on two auxiliary factors, both of which bind to specific RNA sequences and recruit the enzyme by a direct contact. One of these proteins is the heterotetrameric CPSF, which binds the AAUAAA sequence and is also essential for 3' cleavage. The second is the nuclear poly(A) binding protein (PABPN1), which binds the growing poly(A) tails once this has reached a length of about ten nucleotides. Stimulation of poly(A) polymerase by both proteins is synergistic and results in processive elongation of the RNA, i.e. the polymerase adds AMP residues without dissociating from the RNA. The processive reaction is terminated when the tail has reached a length of about 250 nucleotides.