The control of mRNA deadenylation by MAPKAP kinase 2 and tristetraprolin

Abstract

The abundance of a given mRNA is a function of its rates of synthesis and degradation. The regulation of mRNA stability is therefore an important means of modulating gene expression. Control of mRNA stability is mediated by cisacting sequences within 5’ or 3’ untranslated regions (UTR), such as adenosine/uridine-rich elements (ARE) contained in the 3’ UTRs of many inflammatory mediator mRNAs, and by the action of trans-acting RNA-binding factors that interact with these cis-acting elements. Tristetraprolin (TTP) is an ARE-binding protein that destabilises inflammatory mRNAs by promoting their deadenylation. TTP is phosphorylated and inactivated in the cell by MAPKactivated protein kinase (MK)-2, a kinase downstream of p38, which is activated by pro-inflammatory stimuli. I have successfully set up an in vitro assay to investigate TTP-directed deadenylation of ARE-containing mRNAs (IVDA, in vitro deadenylation assay) and this assay was used to investigate different aspects of the deadenylation mechanism. I show that a TTP-directed deadenylase activity is present in HeLa and RAW 264.7 cells and that the mechanism whereby TTP promotes deadenylation of ARE-containing RNA is regulated by MK2 in vitro. In fact, phosphorylation of Ser-52 and Ser-178 of TTP by MK2 resulted in inhibition of TTP-directed deadenylation of AREcontaining RNA. The use of 14-3-3 protein antagonists showed that regulation of TTP-directed deadenylation by MK2 is independent of 14-3-3 binding to TTP. To investigate the mechanism whereby TTP promotes deadenylation, it was necessary to identify the deadenylases involved. The carbon catabolite repressor protein (CCR)4-CCR4-associated factor (CAF)1 complex was identified as the major source of deadenylase activity in HeLa cells responsible for TTP-directed deadenylation. CAF1a and CAF1b were found to interact with TTP in an RNA-independent fashion. MK2 phosphorylation was found to reduce the ability of TTP to promote deadenylation by inhibiting the recruitment of CAF1 deadenylase in a mechanism that does not involve sequestration of TTP by 14-3-3.