No abstract

Protein arginine methylation is a posttranslational modification that impacts cellular functions, such as RNA processing, transcription, DNA repair, and signal transduction. The majority of our knowledge regarding arginine methylation derives from studies of yeast and mammals. Here, we describe a protein arginine N-methyltransferase (PRMT), TbPRMT5, from the early-branching eukaryote Trypanosoma brucei. TbPRMT5 shares the greatest sequence similarity with PRMT5 and Skb1 type II enzymes from humans and Schizosaccharomyces pombe, respectively, although it is significantly divergent at the amino acid level from its mammalian and yeast counterparts. Recombinant TbPRMT5 displays broad substrate specificity in vitro, including methylation of a mitochondrial-gene-regulatory protein, RBP16. TbPRMT5 catalyzes the formation of -N Gmonomethylarginine and symmetric -N G ,N G -dimethylarginine and does not require trypanosome cofactors for 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 of the parasite and localizes to the cytoplasm. Genetic disruption via RNA interference in procyclic-form trypanosomes indicates that TbPRMT5 is not essential for growth in this life cycle stage. TbPRMT5-TAP ectopically expressed in procyclic-form trypanosomes is present in high-molecular-weight complexes and associates with an RG domain-containing DEAD box protein related to yeast Ded1 and two kinetoplastidspecific proteins. Thus, TbPRMT5 is likely to be involved in novel methylation-regulated functions in trypanosomes, some of which may include RNA processing and/or translation.Protein arginine methylation is an irreversible posttranslational modification catalyzed by protein arginine methyltransferases (PRMTs). PRMTs transfer a methyl group from Sadenosyl-L-methionine (AdoMet) to the guanidino nitrogen atoms of substrate arginine residues (34). Arginine residues found within RGG, RG, or RXR motifs are common, but not exclusive, sites of methylation (reviewed in reference 60). Interestingly, a large percentage of PRMT substrates are RNA binding proteins, especially those containing RG-rich motifs (9, 57). Other common substrates include histones and transcriptional coactivators (reviewed in references 7 and 95). Arginine methylation regulates protein function by modulating subcellular localization (44,65,82), protein-protein interactions (6, 31), and, less frequently, protein-RNA interactions (27,28,86). Through these mechanisms, protein arginine methylation exerts a range of effects on several cellular processes, 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 enzymes have been described. Type I PRMTs catalyze the formation of -N G

Sphingolipids are important constituents of cell membranes and also serve as mediators of cell signaling and cell recognition. Sphingolipid metabolites such as sphingosine-1-phosphate and ceramide regulate signaling cascades involved in cell proliferation and differentiation, autophagy, inflammation, and apoptosis. Little is known about how sphingolipids and their metabolites function in single-celled eukaryotes. In the present study, we investigated the role of sphingosine kinase (SPHK) in the biology of the protozoan parasite Trypanosoma brucei, the agent of African sleeping sickness. T. brucei SPHK (TbSPHK) is constitutively but differentially expressed during the life cycle of T. brucei. Depletion of TbSPHK in procyclic-form T. brucei causes impaired growth and attenuation in the G1/S phase of the cell cycle. TbSPHK-depleted cells also develop organelle positioning defects and an accumulation of tyrosinated α-tubulin at the elongated posterior end of the cell, known as the “nozzle” phenotype, caused by other molecular perturbations in this organism. Our studies indicate that TbSPHK is involved in G1-to-S cell cycle progression, organelle positioning, and maintenance of cell morphology. Cytotoxicity assays using TbSPHK inhibitors revealed a favorable therapeutic index between T. brucei and human cells, suggesting TbSPHK to be a novel drug target.