Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)

doi:10.1074/jbc.M506438200

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Papers In Press, published online ahead of print July 25, 2005
J. Biol. Chem, 10.1074/jbc.M506438200
Submitted on June 13, 2005
Revised on July 22, 2005
Accepted on July 25, 2005

Giardia lamblia RNA Cap Guanine-N2 Methyltransferase (Tgs2)

Stéphane Hausmann and Stewart Shuman

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021

Corresponding Author: s-shuman{at}ski.mskcc.org

Tgs1 is the enzyme responsible for converting 7-methylguanosine RNA caps to the 2,2,7-trimethylguanosine (TMG) 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 (Km 6 µM) to m7GDP (Km 65 µM, kcat 14 min-1) to form m2,7GDP. Tgs2 also methylates m7GTP (Km 30 µM, kcat 13 min-1) and m7GpppA (Km 7 µM, kcat 14 min-1), but is unreactive with GDP, GTP, GpppA, ATP, CTP or UTP. We find that conserved residues Asp68, Glu91, and Trp143 are essential for Tgs2 methyltransferase activity in vitro. The m2,7GDP product formed by Tgs2 can be converted to m2,2,7GDP 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 TMG 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.

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