MKP-1 mRNA stabilization and translational control by RNA-binding proteins HuR and NF90

Area of Science:

• Cellular Biology
• Molecular Biology
• Biochemistry

Background:

• Mitogen-activated protein kinase phosphatase 1 (MKP-1) is crucial for dephosphorylating and inactivating key stress-activated kinases like JNK and p38.
• Oxidative stress, such as that induced by hydrogen peroxide (H2O2), is known to upregulate MKP-1.
• The posttranscriptional mechanisms governing this upregulation remain incompletely understood.

Purpose of the Study:

• To investigate the posttranscriptional mechanisms responsible for the robust induction of MKP-1 by oxidants in HeLa cells.
• To identify RNA-binding proteins (RNA-BPs) involved in regulating MKP-1 mRNA stability and translation under oxidative stress.
• To elucidate the role of specific RNA-BPs in mediating the cellular response to oxidative stress via MKP-1.

Main Methods:

• Treatment of HeLa cells with hydrogen peroxide (H2O2) to induce oxidative stress.
• Analysis of MKP-1 mRNA stability and association with the translation machinery.
• Identification of RNA-BPs binding to the MKP-1 3' untranslated region using biotinylated transcripts.
• Ribonucleoprotein immunoprecipitation (RIP) assays to assess in vivo interactions between MKP-1 mRNA and RNA-BPs.
• RNA interference (RNAi) to silence specific RNA-BPs (HuR, NF90, TIAR, TIA-1) and assess their impact on MKP-1 expression and kinase activity.

Main Results:

• H2O2 treatment significantly stabilized MKP-1 mRNA and increased its association with translational machinery.
• Four RNA-BPs (HuR, NF90, TIAR, TIA-1) were identified as binding to the MKP-1 3' UTR.
• H2O2 increased the association of MKP-1 mRNA with HuR and NF90, while decreasing association with TIAR and TIA-1.
• Silencing of HuR or NF90 reduced H2O2-induced MKP-1 mRNA stability.
• HuR silencing markedly decreased MKP-1 translation.
• Reduced MKP-1 expression in HuR-silenced cells led to elevated JNK and p38 phosphorylation upon H2O2 treatment.

Conclusions:

• Oxidative stress-induced upregulation of MKP-1 is significantly influenced by enhanced mRNA stability and translation.
• The RNA-binding proteins HuR and NF90 play critical roles in mediating MKP-1 mRNA stabilization and translation under oxidative stress.
• These findings highlight a posttranscriptional regulatory network involving RNA-BPs that controls MKP-1 expression and cellular response to oxidative stress.