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Molecular and Cellular Biology, September 2007, p. 6084-6092, Vol. 27, No. 17
0270-7306/07/$08.00+0     doi:10.1128/MCB.00647-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The 2'-O-Ribose Methyltransferase for Cap 1 of Spliced Leader RNA and U1 Small Nuclear RNA in Trypanosoma brucei{triangledown} ,{dagger}

Jesse R. Zamudio,1 Bidyottam Mittra,1 Silvie Foldynová-Trantírková,1,2 Gusti M. Zeiner,1,{ddagger} Julius Lukes,2 Janusz M. Bujnicki,3 Nancy R. Sturm,1* and David A. Campbell1

Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California 90095-1489,1 Biology Centre, Czech Academy of Sciences, and Faculty of Biology, University of South Bohemia, 37005 Ceské Budejovice, Czech Republic,2 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, Warsaw, and Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Poznan, Poland3

Received 12 April 2007/ Accepted 18 June 2007

mRNA cap 1 2'-O-ribose methylation is a widespread modification that is implicated in processing, trafficking, and translational control in eukaryotic systems. The eukaryotic enzyme has yet to be identified. In kinetoplastid flagellates trans-splicing of spliced leader (SL) to polycistronic precursors conveys a hypermethylated cap 4, including a cap 0 m7G and seven additional methylations on the first 4 nucleotides, to all nuclear mRNAs. We report the first eukaryotic cap 1 2'-O-ribose methyltransferase, TbMTr1, a member of a conserved family of viral and eukaryotic enzymes. Recombinant TbMTr1 methylates the ribose of the first nucleotide of an m7G-capped substrate. Knockdowns and null mutants of TbMTr1 in Trypanosoma brucei grow normally, with loss of 2'-O-ribose methylation at cap 1 on substrate SL RNA and U1 small nuclear RNA. TbMTr1-null cells have an accumulation of cap 0 substrate without further methylation, while spliced mRNA is modified efficiently at position 4 in the absence of 2'-O-ribose methylation at position 1; downstream cap 4 methylations are independent of cap 1. Based on TbMTr1-green fluorescent protein localization, 2'-O-ribose methylation at position 1 occurs in the nucleus. Accumulation of 3'-extended SL RNA substrate indicates a delay in processing and suggests a synergistic role for cap 1 in maturation.

* Corresponding author. Mailing address: Department of Microbiology, Immunology and Molecular Genetics, David Geffen School of Medicine, 609 Charles E. Young Drive East, University of California at Los Angeles, Los Angeles, CA 90095-1489. Phone: (310) 206-5556. Fax: (310) 206-5231. E-mail: nsturm{at}ucla.edu

{triangledown} Published ahead of print on 2 July 2007.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

{ddagger} Present address: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA 94305.

Molecular and Cellular Biology, September 2007, p. 6084-6092, Vol. 27, No. 17
0270-7306/07/$08.00+0     doi:10.1128/MCB.00647-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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