Regulation of mammalian poly(A) polymerases by protein-protein interactions

Abstract

Mammalian poly(A) polymerase (PAP), a key enzyme in the pre-mRNA 3`-end processing reaction, carries the catalytic domain in the N-terminal region, an RNA binding domain, two nuclear localization signals, and a serine/threonine-rich regulatory domain in the C-terminal region. Since the poly(A) tail is implicated in stability, nuclear export, and translation efficiency of mRNA, the control of the poly(A) tail addition by PAP could be an important mechanism that regulates the level of the gene expression. The PAP activity can be regulated through interaction between PAP and regulatory proteins. In order to determine whether these regulatory proteins exist and how they regulate the PAP activity, partner proteins that would interact with the C-terminal region of PAP were searched using yeast two-hybrid screenings. The 25-kD subunit of cleavage factor I (CFI-25), 14-3-3ε, and cyclophilin were identified as candidate proteins that bind to the C-terminal region of PAP: 1) The glutathione S-transferase-pull down assay and the immunoprecipitation experiment revealed that PAP directly interacts with CFI-25 and that the C-terminal 69 residues of PAP and the N-terminal 60 residues of CFI-25 are sufficient for the interaction between CFI-25 and PAP. Since CFI is known to function in the assembly of the pre-mRNA 3`-processing complex, this interaction may play an important role in the assembly of the processing complex and/or in the regulation of PAP activity within the complex. 2) 14-3-3ε, a signaling protein identified as a discrete phosphoserine/threonine binding module, inhibited PAP by interacting with the C-terminal region of PAP. This interaction was dependent on PAP-phosphorylation. Overexpression of 14-3-3ε also led to the shorter length of poly(A) tails of mRNA in vivo. PAP and 14-3-3ε was co-localized in cytoplasm of cotransfected cells. These data suggested a potential role for 14-3-3ε in modulating PAP activity through a phosphorylation-dependent interaction. T...