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J. Biol. Chem., Vol. 280, Issue 37, 32101-32106, September 16, 2005

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Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)^*

From the Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021

Tgs1 is the enzyme responsible for converting 7-methylguanosine RNA caps to the 2,2,7-trimethylguanosine cap structures of small nuclear and small nucleolar RNAs. Whereas budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe encode a single Tgs1 protein, the primitive eukaryote Giardia lamblia encodes two paralogs, Tgs1 and Tgs2. Here we show that purified Tgs2 is a monomeric enzyme that catalyzes methyl transfer from AdoMet (K_m of 6 µM) to m⁷GDP (K_m of 65 µM; k_cat of 14 min^–1) to form m^2,7GDP. Tgs2 also methylates m⁷GTP (K_m of 30 µM; k_cat of 13 min^–1) and m⁷GpppA (K_m of 7 µM; k_cat) of 14 min^–1 but is unreactive with GDP, GTP, GpppA, ATP, CTP, or UTP. We find that the conserved residues Asp-68, Glu-91, and Trp-143 are essential for Tgs2 methyltransferase activity in vitro. The m^2,7GDP product formed by Tgs2 can be converted to m^2,2,7GDP by S. pombe Tgs1 in the presence of excess AdoMet. However, Giardia Tgs2 itself is apparently unable to add a second methyl group at guanine-N2. This result implies that 2,2,7-trimethylguanosine caps in Giardia are either synthesized by Tgs1 alone or by the sequential action of Tgs2 and Tgs1. The specificity of Tgs2 raises the prospect that some Giardia mRNAs might contain dimethylguanosine caps.

Received for publication, June 13, 2005 , and in revised form, July 22, 2005.

^* Supported by National Institutes of Health Grant GM52470. 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.

¹ An American Cancer Society Research Professor and to whom correspondence should be addressed. E-mail: s-shuman{at}ski.mskcc.org.

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This article has been cited by other articles:

				S. Hausmann, A. Ramirez, S. Schneider, B. Schwer, and S. Shuman Biochemical and genetic analysis of RNA cap guanine-N2 methyltransferases from Giardia lamblia and Schizosaccharomyces pombe Nucleic Acids Res., March 12, 2007; 35(5): 1411 - 1420. [Abstract] [Full Text] [PDF]