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J. Biol. Chem., Vol. 282, Issue 22, 15995-16005, June 1, 2007

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Trypanosoma brucei Encodes a Bifunctional Capping Enzyme Essential for Cap 4 Formation on the Spliced Leader RNA^*

Yuko Takagi, Shalaka Sindkar, Dimitra Ekonomidis, Megan P. Hall¹, and C. Kiong Ho²

From the Departments of Biological Sciences and Microbiology and Immunology, State University of New York, Buffalo, New York 14260

The 5' end of kinetoplastid mRNA possesses a hypermethylated cap 4 structure, which is derived from standard m⁷GpppN (cap 0) with additional methylations at seven sites within the first four nucleosides on the spliced leader RNA. In addition to TbCe1 guanylyltransferase and TbCmt1 (guanine N-7) methyltransferase, Trypanosoma brucei encodes a second cap 0 forming enzyme. TbCgm1 (T. brucei cap guanylyltransferase-methyltransferase) is a novel bifunctional capping enzyme consisting of an amino-terminal guanylyltransferase domain and a carboxyl-terminal methyltransferase domain. Recombinant TbCgm1 transfers the GMP to spliced leader RNA (SL RNA) via a covalent enzyme-GMP intermediate, and methylates the guanine N-7 position of the GpppN-terminated RNA to form cap 0 structure. The two domains can function autonomously in vitro. TbCGM1 is essential for parasite growth. Silencing of TbCGM1 by RNA interference increased the abundance of uncapped SL RNA and lead to accumulation of hypomethylated SL RNA. In contrast, silencing of TbCE1 and TbCMT1 did not affect parasite growth or SL RNA capping. We conclude that TbCgm1 specifically cap SL RNA, and cap 0 is a prerequisite for subsequent methylation events leading to the formation of mature SL RNA.

Received for publication, February 22, 2007 , and in revised form, April 5, 2007.

^* This work was supported by American Heart Association Scientific Development Grant 0635082N (to C. K. H.) and by generous funding from the College of Arts and Sciences, State University of New York at Buffalo. 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 Present address: Dept. of Molecular Cell & Developmental Biology, University of California, Santa Cruz, CA 95064.

² To whom correspondence should be addressed. Tel.: 716-645-2363; Fax: 716-645-2975; E-mail: kiongho{at}buffalo.edu.

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