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J. Biol. Chem., Vol. 283, Issue 6, 3161-3172, February 8, 2008

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The TbMTr1 Spliced Leader RNA Cap 1 2 '-O-Ribose Methyltransferase from Trypanosoma brucei Acts with Substrate Specificity^*

Bidyottam Mittra, Jesse R. Zamudio, Janusz M. Bujnicki^¶, Janusz Stepinski^||, Edward Darzynkiewicz^||, David A. Campbell¹, and Nancy R. Sturm

From the Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, California 90095, the Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, ul. ks. Trojdena 4, 02-109 Warsaw, Poland, the ^¶Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland, and the ^||Department of Biophysics, Institute of Experimental Physics, Warsaw University, 93 Zwirki and Wigury St., 02-089 Warsaw, Poland

In metazoa cap 1 (m⁷GpppNmp-RNA) is linked to higher levels of translation; however, the enzyme responsible remains unidentified. We have validated the first eukaryotic encoded cap 1 2 '-O-ribose methyltransferase, TbMTr1, a member of a conserved family that modifies the first transcribed nucleotide of spliced leader and U1 small nuclear RNAs in the kinetoplastid protozoan Trypanosoma brucei. In addition to cap 0 (m⁷GpppNp-RNA), mRNA in these parasites has ribose methylations on the first four nucleotides with base methylations on the first and fourth (m⁷Gpppm^6,6AmpAmpCmpm³Ump-SL RNA) conveyed via trans-splicing of a universal spliced leader. The function of this cap 4 is unclear. Spliced leader is the majority RNA polymerase II transcript; the RNA polymerase III-transcribed U1 small nuclear RNA has the same first four nucleotides as spliced leader, but it receives an m^2,2,7G cap with hypermethylation at position one only (m^2,2,7Gpppm^6,6AmpApCpUp-U1 snRNA). Here we examine the biochemical properties of recombinant TbMTr1. Active over a pH range of 6.0 to 9.5, TbMTr1 is sensitive to Mg²⁺. Positions Lys⁹⁵-Asp²⁰⁴-Lys²⁵⁹-Glu²⁸⁵ constitute the conserved catalytic core. A guanosine cap on RNA independent of its N⁷ methylation status is required for substrate recognition, but an m^2,2,7G-cap is not recognized. TbMTr1 favors the spliced leader 5' sequence, as reflected by a preference for A at position 1 and modulation of activity for substrates with base changes at positions 2 and 3. With similarities to human cap 1 methyltransferase activity, TbMTr1 is an excellent model for higher eukaryotic cap 1 methyltransferases and the consequences of cap 1 modification.

Received for publication, September 4, 2007 , and in revised form, November 9, 2007.

^* This work was supported by National Institutes of Health Grant AI056034 (to D. A. C. and N. R. S.), Polish Ministry of Science and Higher Education Grant 2P04A00628, Howard Hughes Medical Institute Grant 55005604 (to E. D.), Polish Ministry of Science and Higher Education Grant N301 2396 33 (to J. M. B.), National Science Foundation Louis Stokes Alliance for Minority Participation Award HRD-0115115:3, and United States Public Health Service National Research Service Award GM07104 (to J. R. Z.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1 and Figs. S1–S3.

¹ To whom correspondence should be addressed: 609 Charles E. Young Dr., University of California, Los Angeles, CA 90095-1489. Tel.: 310-206-5556; Fax: 310-206-5231; E-mail: dc{at}ucla.edu.

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