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EUKARYOTIC CELL, Sept. 2007, p. 1665–1681 Vol. 6, No. 91535-9778/07/$08.00⫹0 doi:10.1128/EC.00133-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.Evolutionarily Divergent Type II Protein Arginine Methyltransferasein Trypanosoma brucei䌤Deborah A. Pasternack,1Joyce Sayegh,2Steven Clarke,2and Laurie K. Read1*Department of Microbiology and Immunology and Witebsky Center for Microbial Pathogenesis and Immunology,State University of New York School of Medicine, Buffalo, New York 14214,1and Department of Chemistry andBiochemistry and Molecular Biology Institute, University of California at Los Angeles,Los Angeles, California 90095-15692Received 20 April 2007/Accepted 22 June 2007Protein arginine methylation is a posttranslational modification that impacts cellular functions, such asRNA processing, transcription, DNA repair, and signal transduction. The majority of our knowledge regardingarginine methylation derives from studies of yeast and mammals. Here, we describe a protein arginineN-methyltransferase (PRMT), TbPRMT5, from the early-branching eukaryote Trypanosoma brucei. TbPRMT5shares the greatest sequence similarity with PRMT5 and Skb1 type II enzymes from humans and Schizosac-charomyces pombe, respectively, although it is significantly divergent at the amino acid level from its mamma-lian and yeast counterparts. Recombinant TbPRMT5 displays broad substrate specificity in vitro, includingmethylation of a mitochondrial-gene-regulatory protein, RBP16. TbPRMT5 catalyzes the formation of ␻-NG-monomethylarginine and symmetric ␻-NG,NGⴕ-dimethylarginine and does not require trypanosome cofactorsfor this activity. These data establish that type II PRMTs evolved early in the eukaryotic lineage. In vivo,TbPRMT5 is constitutively expressed in the bloodstream form and procyclic-form (insect host) life stages ofthe parasite and localizes to the cytoplasm. Genetic disruption via RNA interference in procyclic-form try-panosomes indicates that TbPRMT5 is not essential for growth in this life cycle stage. TbPRMT5-TAPectopically expressed in procyclic-form trypanosomes is present in high-molecular-weight complexes andassociates with an RG domain-containing DEAD box protein related to yeast Ded1 and two kinetoplastid-specific proteins. Thus, TbPRMT5 is likely to be involved in novel methylation-regulated functions in trypano-somes, some of which may include RNA processing and/or translation.Protein arginine methylation is an irreversible posttransla-tional modification catalyzed by protein arginine methyltrans-ferases (PRMTs). PRMTs transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the guanidino nitrogenatoms of substrate arginine residues (34). Arginine residuesfound within RGG, RG, or RXR motifs are common, but notexclusive, sites of methylation (reviewed in reference 60). In-terestingly, a large percentage of PRMT substrates are RNAbinding proteins, especially those containing RG-rich motifs(9, 57). Other common substrates include histones and tran-scriptional coactivators (reviewed in references 7 and 95). Ar-ginine methylation regulates protein function by modulatingsubcellular localization (44, 65, 82), protein-protein interac-tions (6, 31), and, less frequently, protein-RNA interactions(27, 28, 86). Through these mechanisms, protein argininemethylation exerts a range of effects on several cellular pro-cesses, including transcription (18, 96), signal transduction (1,22), DNA repair (10), and RNA processing (12, 32, 82).Arginine methyltransferases have been identified in animals,fungi, plants, and protozoa, and in total, four classes of en-zymes have been described. Type I PRMTs catalyze the for-mation of ␻-NG-monomethylarginine (MMA) and asymmetric␻-NG,NG-dimethylarginine (aDMA). Type II enzymes catalyzethe formation of MMA and symmetric ␻-NG,NG⬘-dimethyl-arginine (sDMA). Type III and type IV PRMTs catalyze onlyMMA or ␦-NG-monomethylarginine formation, respectively.Most of our knowledge about PRMTs derives from analysis ofmammals and yeasts. Of the 11 putative PRMTs identified inthe genome of Homo sapiens, five display clear type I activity(PRMT1, PRMT3, PRMT4/CARM1, PRMT6, and PRMT8),one displays clear type II activity (PRMT5), and work is inprogress on the others (reviewed in reference 50). In contrastto humans, only three PRMTs have been described in Saccha-romyces cerevisiae. They are PRMT1 and PRMT5 homologs(called HMT1p and HSL7p), as well as a novel type IV PRMTcalled RMT2p, which has been described only in buddingyeast, although homologs are present in a variety of fungi andplants (20, 99). Genes homologous to PRMT1 and PRMT5have also been described in the yeast Schizosaccharomycespombe, as well as an additional type I PRMT that is a homologof PRMT3 (4, 38, 75). Although genes encoding putativePRMTs are present in the genomes of various plants andprotozoa, analysis of arginine methylation in these groups hasbeen limited. Type II PRMT activity mediated by SKB1 spe-cific for histone H4 is reported to control flowering time inArabidopsis thaliana (92), whereas type I PRMT homologshave been characterized from the parasitic protozoa Trypano-soma brucei (TbPRMT1 [72]) and Toxoplasma gondii (TgCARM1and TgPRMT1 [81]). Phylogenetic analysis suggests thatPRMTs originated early in the eukaryotic lineage, since nohomologs have been identified in bacteria, archaea, or thebasal eukaryote Giardia lamblia (50).* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, SUNY Buffalo School of Medicine, 138 FarberHall, Buffalo, NY 14214. Phone: (716) 829-3307. Fax: (716) 829-2158.E-mail: [email protected]䌤Published ahead of print on 29 June 2007.1665 at UCLA BIOMEDICAL LIB/SERIALS on January 24, 2008 ec.asm.orgDownloaded fromT. brucei, the causative agent of African trypanosomiasis, isan early-branching eukaryote that lacks transcriptional controland displays complex mechanisms of gene expression. Tran-scription of protein-coding genes is polycistronic, yet the dif-ferential expression of steady-state RNA has been observedbetween bloodstream form (BF) and procyclic-form (PF) lifestages of the parasite (37). Trypanosomes exhibit unique biol-ogy, in which gene expression is coordinated posttranscription-ally through mechanisms that include trans splicing, RNA sta-bility, RNA editing, and translation (23, 80, 89). A vast numberof RNA binding proteins are presumably required to coordi-nate these


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