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

Abstract

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~80-fold increase in the apparent af®nity of poly(A) polymerase for RNA without signi®cant effect on catalytic ef®ciency. 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 suf®cient 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.

Regulation of the Nuclear Poly(A)-binding Protein by Arginine Methylation in Fission Yeast

“…In vitro data also indicate that mammalian PABP2 stimulates poly(A) synthesis by recruiting the poly(A) polymerase to the RNA substrate (17). However, detection of hyperadenylated RNAs in pab2-null cells indicates that factors other than Pab2 can stimulate tethering of PAP to the 3Ј-end of nascent transcripts as well as PAP processivity in vivo.…”

“…The affinity of PAPB2 to synthetic poly(A) tails is in the nanomolar range and requires both the RRM and the C-terminal arginine-rich domain for optimal and specific interaction with poly(A) (15,16). Experiments using in vitro polyadenylation assays led to a model in which PABP2 stimulates processive poly(A) synthesis by direct and simultaneous interactions with the growing poly(A) tail and the poly(A) polymerase (17). Although in vitro experiments have provided information about the biochemical properties of PABP2, little is known about the mechanism by which it regulates poly(A) tail synthesis in vivo.…”

The Drosophila melanogaster Gene cg4930 Encodes a High Affinity Inhibitor for Endonuclease G

“…L3pre-based RNAs were synthesized with [␣-32 P]UTP as described (33). Size-fractionated poly(A) was 5Ј-labeled with [␥-32 P]ATP and T4 polynucleotide kinase under standard conditions.…”

Protein factors in pre-mRNA 3′-end processing

“…PABP binds directly to nascent stretches of 11-14 adenylate nucleotides as they become available [196], and this binding continues until the proper poly(A) tail length is reached (~200 to 300 bases in mammals) [197]. In addition, direct binding of PABP to premRNA, adjacent to PAP, increases the efficiency of polyadenylation 80-fold [198]. Pab1p binds directly to Rna15p, which possibly recruits Pab1p to the polyadenylation site [192].…”