Poly(A) tail length is controlled by the nuclear poly(A)-binding protein regulating the interaction between poly(A) polymerase and the cleavage and polyadenylation specificity factor.

Poly(A) tails of mRNAs are synthesized in the cell nucleus with a defined length, approximately 250 nucleotides in mammalian cells. The same type of length control is seen in an in vitro polyadenylation system reconstituted from three proteins: poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF), and the nuclear poly(A)-binding protein (PABPN1). CPSF, binding the polyadenylation signal AAUAAA, and PABPN1, binding the growing poly(A) tail, cooperatively stimulate poly(A) polymerase such that a complete poly(A) tail is synthesized in one processive event, which terminates at a length of approximately 250 nucleotides. We report that PABPN1 is required to restrict CPSF binding to the AAUAAA sequence and to permit the stimulation of poly(A) polymerase by AAUAAA-bound CPSF to be maintained throughout the elongation reaction. The stimulation by CPSF is disrupted when the poly(A) tail has reached a length of approximately 250 nucleotides, and this terminates processive elongation. PABPN1 measures the length of the tail and is responsible for disrupting the CPSF-poly(A) polymerase interaction.

Stimulation of poly(A) polymerase through a direct interaction with the nuclear poly(A) binding protein allosterically regulated by RNA.

During polyadenylation of mRNA precursors in metazoan cells, poly(A) polymerase is stimulated by the nuclear poly(A) binding protein PABPN1. We report that stimulation depends on binding of PABPN1 to the substrate RNA directly adjacent to poly(A) polymerase and results in an approximately 80-fold increase in the apparent affinity of poly(A) polymerase for RNA without significant effect on catalytic efficiency. PABPN1 associates directly with poly(A) polymerase either upon allosteric activation by oligo(A) or, in the absence of RNA, upon deletion of its N-terminal domain. The N-terminal domain of PABPN1 may function to inhibit undesirable interactions of the protein; the inhibition is relieved upon RNA binding. Tethering of poly(A) polymerase is mediated largely by the C-terminal domain of PABPN1 and is necessary but not sufficient for stimulation of the enzyme; an additional interaction dependent on a coiled-coil structure located within the N-terminal domain of PABPN1 is required for a productive interaction.