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J. Biol. Chem., Vol. 282, Issue 34, 24792-24805, August 24, 2007

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Metal Determines Efficiency and Substrate Specificity of the Nuclear NUDIX Decapping Proteins X29 and H29K (Nudt16)^*

From the Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211

The Xenopus X29 protein was identified by its high affinity binding to U8 small nucleolar RNA, a small nucleolar RNA required for ribosome biogenesis. X29 and its human homologue H29K (Nudt16) are nuclear nucleoside diphosphatase proteins localized within foci in the nucleolus and nucleoplasm. These proteins can remove m⁷G and m²²⁷G caps from RNAs, rendering them substrates for 5'-3' exonucleases for degradation in vivo. Here, a more complete characterization of these metal-dependent decapping proteins demonstrates that the metal identity determines both the efficiency of decapping and the RNA substrate specificity. In Mg⁺² the proteins hydrolyze the 5' cap from only one RNA substrate: U8 small nucleolar RNA. However, in the presence of Mn⁺² or Co⁺² all RNAs are substrates and the decapping efficiency is higher. The x-ray crystal structure of X29 facilitated structure-based mutagenesis. Mutation of single amino acids coordinating metal in the active site yielded mutant proteins confirming essential residues. In vitro assays with purified components are consistent with a lack of protein turnover, apparently due to an inability of the protein to release the decapped RNA, implicating critical in vivo interacting factors. Collectively, these studies indicate that the metal that binds the X29/H29K proteins in vivo may determine whether these decapping proteins function solely as a negative regulator of ribosome biogenesis or can decap a wider variety of nuclear-limited RNAs. With the potential broader RNA substrate specificity, X29/H29K may be the nuclear counterparts of the cytoplasmic decapping machinery, localized in specialized bodies involved in RNA decay.

Received for publication, May 21, 2007

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Current address: Biochemistry Section, Surgical Neurology Branch, NINDS, National Institutes of Health, Bethesda, MD 20892.

¹ To whom correspondence should be addressed: Dept. of Biochemistry, University of Missouri-Columbia, 117 Schweitzer Hall, Columbia, MO 65211. Tel.: 573-884-1424; Fax: 573-884-4812; E-mail: peculisb{at}missouri.edu.

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