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Eukaryotic Cell, June 2006, p. 905-915, Vol. 5, No. 6
1535-9778/06/$08.00+0     doi:10.1128/EC.00080-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Complete Cap 4 Formation Is Not Required for Viability in Trypanosoma brucei{dagger}

Jesse R. Zamudio,1 Bidyottam Mittra,1 Gusti M. Zeiner,1,{ddagger} Marcin Feder,2 Janusz M. Bujnicki,2 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 90095,1 Laboratory of Bioinformatics and Protein Engineering, International Institute of Molecular and Cell Biology, ul. ks. Trojdena 4, 02-109 Warsaw, Poland2

Received 20 March 2006/ Accepted 21 March 2006

In kinetoplastids spliced leader (SL) RNA is trans-spliced onto the 5' ends of all nuclear mRNAs, providing a universal exon with a unique cap. Mature SL contains an m7G cap, ribose 2'-O methylations on the first four nucleotides, and base methylations on nucleotides 1 and 4 (AACU). This structure is referred to as cap 4. Mutagenized SL RNAs that exhibit reduced cap 4 are trans-spliced, but these mRNAs do not associate with polysomes, suggesting a direct role in translation for cap 4, the primary SL sequence, or both. To separate SL RNA sequence alterations from cap 4 maturation, we have examined two ribose 2'-O-methyltransferases in Trypanosoma brucei. Both enzymes fall into the Rossmann fold class of methyltransferases and model into a conserved structure based on vaccinia virus homolog VP39. Knockdown of the methyltransferases individually or in combination did not affect growth rates and suggests a temporal placement in the cap 4 formation cascade: TbMT417 modifies A2 and is not required for subsequent steps; TbMT511 methylates C3, without which U4 methylations are reduced. Incomplete cap 4 maturation was reflected in substrate SL and mRNA populations. Recombinant methyltransferases bind to a methyl donor and show preference for m7G-capped RNAs in vitro. Both enzymes reside in the nucleoplasm. Based on the cap phenotype of substrate SL stranded in the cytosol, A2, C3, and U4 methylations are added after nuclear reimport of Sm protein-complexed substrate SL RNA. As mature cap 4 is dispensable for translation, cap 1 modifications and/or SL sequences are implicated in ribosomal interaction.

* Corresponding author. Mailing address: Department of Microbiology, Immunology and Molecular Genetics, 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.

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

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

Eukaryotic Cell, June 2006, p. 905-915, Vol. 5, No. 6
1535-9778/06/$08.00+0     doi:10.1128/EC.00080-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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