Regulation of poly(A) polymerase by 14‐3‐3ϵ

Poly(A) polymerase (PAP) is a key enzyme responsible for the addition of the poly(A) at the 3′ end of pre‐mRNA. The C‐terminal region of mammalian PAP carries target sites for protein–protein interaction with the 25 kDa subunit of cleavage factor I and with splicing factors U1A and U2AF65. We used a yeast two‐hybrid screen to identify 14‐3‐3ϵ as an additional protein binding to the C‐terminal region of PAP. Interaction between PAP and 14‐3‐3ϵ was confirmed by both in vitro and in vivo binding assays. This interaction is dependent on PAP phosphorylation. Deletion analysis of PAP suggests that PAP contains multiple binding sites for 14‐3‐3ϵ. The binding of 14‐3‐3ϵ to PAP inhibits the polyadenylation activity of PAP in vitro, and overexpression of 14‐3‐3ϵ leads to a shorter poly(A) mRNA tail in vivo. In addition, the interaction between PAP and 14‐3‐3ϵ redistributes PAP within the cell by increasing its cytoplasmic localization. These data suggest that 14‐3‐3ϵ is involved in regulating both the activity and the nuclear/ cytoplasmic partitioning of PAP through the phosphorylation‐dependent interaction.

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