Tristetraprolin and Its Family Members Can Promote the Cell-Free Deadenylation of AU-Rich Element-Containing mRNAs by Poly(A) Ribonuclease

Abstract

Eukaryotic mRNA stability can be influenced by AU-rich elements (AREs) within mRNA primary sequences. Tristetraprolin (TTP) is a CCCH tandem zinc finger protein that binds to ARE-containing transcripts and destabilizes them, apparently by first promoting the removal of their poly(A) tails. We developed a cell-free system in which TTP and its related proteins stimulated the deadenylation of ARE-containing, polyadenylated transcripts. Transcript deadenylation was not stimulated when a mutant TTP protein was used that was incapable of RNA binding, nor when a mutant ARE was present that did not bind TTP. The ability of TTP to promote transcript deadenylation required Mg 2؉ , but not ATP or prior capping of the RNA substrate. Cotransfection and additivity studies with the poly(A) RNase (PARN) demonstrated that TTP promoted the ability of this enzyme to deadenylate ARE-containing, polyadenylated transcripts, while having no effect on transcripts lacking an ARE. There was no effect of TTP to act synergistically with enzymatically inactive PARN mutants. We conclude that TTP can promote the deadenylation of ARE-containing, polyadenylated substrates by PARN. This interaction may be responsible for the ability of TTP and its family members to promote the deadenylation of such transcripts in intact cells.Steady-state levels of cellular mRNAs are determined by the balance between their biosynthesis and turnover. Different mRNAs can exhibit marked differences in turnover rates within the same cell, and the turnover rates of individual mRNAs can also vary significantly in response to changes in the cellular environment. In mammalian cells, the earliest step in mRNA turnover is thought to be removal of the poly(A) tail, or deadenylation, and this process in turn largely determines the overall decay rate of the mRNA (15,16,31,36).It has been appreciated for many years that cis-acting AUrich elements (AREs), often within the 3Ј-untranslated regions (3Ј-UTR) of the mRNA, can confer decreased stability on the mRNAs that contain them (32).

Expression, Purification, and Biochemical Characterization of the Antiinflammatory Tristetraprolin: A Zinc-Dependent mRNA Binding Protein Affected by Posttranslational Modifications,

“…The formation of a large oligomer of TTP was also supported by GMSA, in which active TTP-ARE complexes were found in the wells of the autoradiograph at high TTP concentrations. Immunoblotting showed that some TTP was in a size of dimer even under SDS denaturation ( Figure 5), in a manner similar to those following cross-linking with disuccinimidyl suberate (42) and the E. coli glycerol facilitator on SDS-PAGE (43). The major form of active TTP from transfected mammalian cells being a tetramer is in agreement with previous results using the MBP-hTTP fusion protein purified from overexpressed E. coli cells (9).…”

p38 Mitogen-activated Protein Kinase Stabilizes mRNAs That Contain Cyclooxygenase-2 and Tumor Necrosis Factor AU-rich Elements by Inhibiting Deadenylation

“…Immunodepletion of the Xenopus orthologue of HuR from oocyte extracts had no effect on the ARE-directed deadenylation of an exogenous RNA in vitro (46). Recently, TTP has been shown to promote ARE-directed deadenylation in a cell-free system (29). However, in cells lacking TTP, such as the HeLa cells used in this study, it is unknown which factor is responsible for targeting ARE-containing mRNAs for rapid deadenylation or for inhibition of deadenylation by p38 MAPK.…”

“…Despite the identification of numerous ARE-binding proteins (ARE-BPs), it is unclear which (if any) provides a link between p38 MAPK and the ARE. One ARE-BP, tristetraprolin (TTP), is destabilizing and directs deadenylation (29). It is an in vitro substrate of MAPKAPK-2 (30); however, TTP is not necessarily involved in the basic stabilization mechanism because it is not expressed in HeLa cells in which regulation of reporter mRNAs by p38 MAPK has been studied.…”

Destabilization of vascular endothelial growth factor mRNA by the zinc-finger protein TIS11b

“…TIS11b belongs to a family of RNA-binding proteins (comprising also TIS11/TTP/tristetraprolin and TIS11d) that share characteristic tandem CCCH-type zinc-finger domains. The canonical family member, TTP, has been shown to bind to AREs in the 3 0 -UTR of tumour necrosis factor a (TNFa) mRNA through these zinc-finger domains, and to decrease TNFa mRNA stability by promoting its deadenylation and degradation (Carballo et al, 1998;Lai et al, 2003). All members of the family are involved in regulation of several short-lived cytokine mRNAs including granulocyte macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (Carballo et al, 2000;Stoecklin et al, 2000) and appear to share similar ARE-binding and mRNA-destabilizing activities.…”