P38 Mitogen-activated Protein Kinase Stabilizes mRNAs That Contain Cyclooxygenase-2 and Tumor Necrosis Factor AU-rich Elements by Inhibiting Deadenylation

Jonathan L.E Dean,Simon J Sarsfield,Elizabeth Tsounakou,Jeremy Saklatvala

doi:10.1074/jbc.m306345200

Abstract

AU-rich elements (AREs) in 3'-untranslated regions of mRNAs confer instability. They target mRNAs for rapid deadenylation and degradation and may enhance decapping. The p38 MAPK pathway stabilizes many otherwise unstable ARE-containing mRNAs encoding proteins involved in inflammation; however, the mRNA decay step(s) regulated by the signaling pathway are unknown. To investigate whether it regulates deadenylation or the decay of the mRNA body, we used a tetracycline-regulated beta-globin mRNA reporter system to transcribe pulses of mRNA of uniform length. We measured on Northern gels the migration of reporter mRNAs isolated from cells transfected only with reporter plasmid or co-transfected with an active mutant of MAPK kinase-6, and treated either with or without the p38 MAPK inhibitor SB 203580. Differences in migration were shown by RNase H mapping with oligo(dT) to be due to poly(A) shortening. Insertion of an ARE into the beta-globin reporter mRNA promoted rapid deadenylation and decay of hypo-adenylated reporter mRNA. p38 MAPK activation inhibited the deadenylation of reporter mRNAs containing either the cyclooxygenase-2 or tumor necrosis factor AREs. The regulation of deadenylation by p38 MAPK was found to be specific because deadenylation of the beta-globin reporter mRNA either lacking an ARE or containing the c-Myc 3'-untranslated region (which is not p38 MAPK-responsive) was unaffected by p38 MAPK. It was concluded that the p38 MAPK pathway predominantly regulates deadenylation, rather than decay of the mRNA body, and this provides an explanation for why p38 MAPK regulates mRNA stability in some situations and translation in others.

Highlights

The control of mRNA stability is fundamental in regulating gene expression
To check that the stability of mRNA produced using this pulse-chase approach was regulated by p38 mitogen-activated protein kinase (MAPK), HeLa tetoff cells were transiently transfected with pTet-BBB-COX-2 AU-rich elements (AREs) reporter plasmid together with or without a plasmid expressing MKK6E, an active mutant of the p38 MAPK upstream activator (MAPK kinase-6)
We found that p38 MAPK stabilizes reporter mRNAs containing either the COX-2 or tumor necrosis factor (TNF) AREs by inhibiting deadenylation

Summary

Introduction

The control of mRNA stability is fundamental in regulating gene expression. This is reinforced by the fact that mRNA stability can be modulated by cell signaling events [1,2,3]. One ARE-BP, tristetraprolin (TTP), is destabilizing and directs deadenylation [29] It is an in vitro substrate of MAPKAPK-2 [30]; 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.. In order to identify the link between mRNA stability and p38 MAPK, it is necessary to ascertain which specific mRNA decay steps are regulated by the signaling pathway. To do this we transcribed pulses of reporter mRNA in cells containing active or inactive p38 MAPK and followed poly(A) tail length and decay of the mRNA by Northern blotting. We show that p38 MAPK stabilizes mRNA by blocking ARE-directed deadenylation

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