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J. Biol. Chem., Vol. 277, Issue 50, 48558-48564, December 13, 2002
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From the Digestive Health Center of Excellence, University of
Virginia Health Sciences Center, Charlottesville, Virginia 22908
Regulation of messenger RNA stability by AU-rich
elements is an important means of regulating genes induced by growth
factors and cytokines. Nup475 (also known as tristetraprolin, or TIS11) is the prototype for a family of zinc-binding Cys3His
motif proteins required for proper regulation of tumor necrosis factor
mRNA stability in macrophages. We developed an Escherichia
coli expression system to produce soluble Nup475 protein in
quantity to study its RNA binding properties. Nup475 protein bound a
tumor necrosis factor AU-rich element over a broad range of pH and salt
concentrations by RNA gel shift. This binding was inhibited by excess
zinc metal, providing a potential mechanism for previous reports of
zinc stabilization of AU-rich element (ARE) containing messenger RNAs.
Immobilized Nup475 protein was used to select its optimal binding site
by RNA SELEX and revealed a strong preference for the extended sequence UUAUUUAUU, rather than a simple AUUUA motif. These findings were confirmed by site-directed mutagenesis of the tumor necrosis factor ARE
and RNA gel shifts on c-fos, interferon- This manuscript is dedicated to the memory of Daniel Nathans.
RNA Binding Properties of the AU-rich Element-binding Recombinant
Nup475/TIS11/Tristetraprolin Protein*
,
, and
interferon-
ARE fragments. A weaker binding activity toward
adenine-rich sites, such as a poly(A) tail RNA fragment, can partially
disrupt the Nup475-tumor necrosis factor AU-rich element complex.
*
This work was supported by National Institutes of Health
Grants DK02501 and DK60720 and an American Digestive Health
Foundation Industry Research Scholar Award (to M. T. W.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: MR-4 Bldg., Rm. 1036, Lane Rd., University of Virginia Health Sciences Center, Charlottesville, VA 22908. Tel.: 434-243-4831; Fax: 434-243-6169; E-mail: mtw3p@virginia.edu.
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