Tristetraprolin inhibits poly(A)-tail synthesis in nuclear mRNA that contains AU-rich elements by interacting with poly(A)-binding protein nuclear 1.

backgroundTristetraprolin binds mRNA AU-rich elements and thereby facilitates the destabilization of mature mRNA in the cytosol. METHODOLOGY/PRINCIPAL

findingsTo understand how tristetraprolin mechanistically functions, we biopanned with a phage-display library for proteins that interact with tristetraprolin and retrieved, among others, a fragment of poly(A)-binding protein nuclear 1, which assists in the 3'-polyadenylation of mRNA by binding to immature poly(A) tails and thereby increases the activity of poly(A) polymerase, which is directly responsible for polyadenylation. The tristetraprolin/poly(A)-binding protein nuclear 1 interaction was characterized using tristetraprolin and poly(A)-binding protein nuclear 1 deletion mutants in pull-down and co-immunoprecipitation assays. Tristetraprolin interacted with the carboxyl-terminal region of poly(A)-binding protein nuclear 1 via its tandem zinc finger domain and another region. Although tristetraprolin and poly(A)-binding protein nuclear 1 are located in both the cytoplasm and the nucleus, they interacted in vivo in only the nucleus. In vitro, tristetraprolin bound both poly(A)-binding protein nuclear 1 and poly(A) polymerase and thereby inhibited polyadenylation of AU-rich element-containing mRNAs encoding tumor necrosis factor α, GM-CSF, and interleukin-10. A tandem zinc finger domain-deleted tristetraprolin mutant was a less effective inhibitor. Expression of a tristetraprolin mutant restricted to the nucleus resulted in downregulation of an AU-rich element-containing tumor necrosis factor α/luciferase mRNA construct. CONCLUSION/SIGNIFICANCE: In addition to its known cytosolic mRNA-degrading function, tristetraprolin inhibits poly(A) tail synthesis by interacting with poly(A)-binding protein nuclear 1 in the nucleus to regulate expression of AU-rich element-containing mRNA.