RNA binding properties of the AU-rich element-binding recombinant Nup475/TIS11/tristetraprolin protein.
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 Cys(3)His 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-gamma, and interferon-beta 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.
D001483:Base Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D015347:RNA Probes; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D051816:Tristetraprolin
D017931:DNA Primers; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D015347:RNA Probes; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D051816:Tristetraprolin; D012313:RNA