Members of the Tristetraprolin Family of Tandem CCCH Zinc Finger Proteins Exhibit CRM1-dependent Nucleocytoplasmic Shuttling

Ruth S Phillips,Silvia B.V Ramos,Perry J Blackshear

doi:10.1074/jbc.m111457200

Abstract

Members of the tristetraprolin (TTP) family of CCCH tandem zinc finger proteins can bind directly to certain types of AU-rich elements (AREs) in mRNA. Experiments in TTP-deficient mice have shown that TTP is involved in the physiological destabilization of at least two cytokine mRNAs, those encoding tumor necrosis factor alpha and granulocyte-macrophage colony-stimulating factor. The two other known mammalian members of the TTP family, CMG1 and TIS11D, also contain ARE-binding CCCH tandem zinc finger domains and can also destabilize ARE-containing mRNAs. To investigate the effects of primary sequence on the subcellular localization of these proteins, we constructed green fluorescent protein fusions with TTP, CMG1, and TIS11D; these were predominantly cytoplasmic when expressed in 293 or HeLa cells. Deletion and mutation analyses revealed functional nuclear export signals in the amino terminus of TTP and in the carboxyl termini of CMG1 and TIS11D. This type of leucine-rich nuclear export signal interacts with the nuclear export receptor CRM1; abrogation of CRM1 activity resulted in nuclear accumulation of TTP, CMG1, and TIS11D. These proteins are thus nucleocytoplasmic shuttling proteins and rely on CRM1 for their export from the nucleus. Although TTP, CMG1, and TIS11D lack known nuclear import sequences, mapping experiments revealed that their nuclear accumulation required an intact tandem zinc finger domain but did not require RNA binding ability. These findings suggest possible roles for nuclear import and export in the regulation of cellular TTP, CMG1, and TIS11D activity.

Highlights

The tristetraprolin (TTP)1 family of CCCH tandem zinc finger (TZF) proteins consists of three known members in mammals, with a fourth member identified in other vertebrates ʈ To whom correspondence should be addressed: A2-05 NIEHS, National Institutes of Health, 111 Alexander Dr, Research Triangle Park, NC 27709
Transfected TTP, CMG1, and TIS11D Fusion Proteins with GFP Are Predominantly Cytoplasmic at Steady State in 293 Cells—To examine the subcellular localization of wild-type TTP, CMG1, and TIS11D, 293 cells were transfected with plasmids encoding GFP fusion proteins, fixed, and examined by confocal microscopy
To show that fusion to GFP had no effect on protein localization, 293 cells were transfected with epitope-tagged versions of TTP and CMG1 and examined by immunocytochemistry

Summary

Introduction

The tristetraprolin (TTP) family of CCCH tandem zinc finger (TZF) proteins consists of three known members in mammals, with a fourth member identified in other vertebrates ʈ To whom correspondence should be addressed: A2-05 NIEHS, National Institutes of Health, 111 Alexander Dr, Research Triangle Park, NC 27709. TTP is able to regulate the stability of interleukin-3 (IL-3) mRNA in cell transfection studies, this has not yet been shown to be a physiological role (1, 11, 12). Decay of these cytokine mRNAs is normally regulated in part by the presence of AU-rich elements (ARE) located in their 3Ј-untranslated regions (13). Two additional mammalian members of the TTP family have been identified to date They are referred to here as CMG1 (15) ( known as TIS11B (16), ERF1 (17), BRF1 (18), or Berg (19)) and TIS11D (20) ( known as ERF2 (20) or BRF2 (21)). We present evidence that TTP, CMG1, and TIS11D can shuttle between the

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Results

Conclusion