TbPRMT6 Is a Type I Protein Arginine Methyltransferase That Contributes to Cytokinesis in Trypanosoma brucei

Abstract

Arginine methylation is a widespread posttranslational modification of proteins catalyzed by a family of protein arginine methyltransferases (PRMTs). In Saccharomyces cerevisiae and mammals, this modification affects multiple cellular processes, such as chromatin remodeling leading to transcriptional regulation, RNA processing, DNA repair, and cell signaling. The protozoan parasite Trypanosoma brucei possesses five putative PRMTs in its genome. This is a large number of PRMTs relative to other unicellular eukaryotes, suggesting an important role for arginine methylation in trypanosomes. Here, we present the in vitro and in vivo characterization of a T. brucei enzyme homologous to human PRMT6, which we term TbPRMT6. Like human PRMT6, TbPRMT6 is a type I PRMT, catalyzing the production of monomethylarginine and asymmetric dimethylarginine residues. In in vitro methylation assays, TbPRMT6 utilizes bovine histones as a substrate, but it does not methylate several T. brucei glycine/arginine-rich proteins. As such, it exhibits a relatively narrow substrate specificity compared to other T. brucei PRMTs. Knockdown of TbPRMT6 in both procyclic form and bloodstream form T. brucei leads to a modest but reproducible effect on parasite growth in culture. Moreover, upon TbPRMT6 depletion, both PF and BF exhibit aberrant morphologies indicating defects in cell division, and these defects differ in the two life cycle stages. Mass spectrometry of TbPRMT6-associated proteins reveals histones, components of the nuclear pore complex, and flagellar proteins that may represent TbPRMT6 substrates contributing to the observed growth and morphological defects.Posttranslational methylation of proteins on arginine residues has multiple roles in a wide array of cellular functions, such as chromatin remodeling leading to transcription activation or repression, RNA processing, DNA repair, and various forms of cell signaling (5,6,8,9,52,70,98). The process of arginine methylation involves the transfer of methyl groups from S-adenosyl-methionine (AdoMet) to arginine residues of proteins and is catalyzed by a group of enzymes known as protein arginine methyltransferases (PRMTs). PRMTs themselves are further divided into four classes, depending on the type of methylated arginine generated. The largest PRMT class comprises the type I enzymes, as characterized by the first discovered PRMT, PRMT1. Type I PRMTs initially catalyze the formation of monomethylated arginine (MMA) on the terminal -nitrogen, followed by addition of a second methyl group on the same -nitrogen, which yields asymmetric dimethylarginine (ADMA). The type II PRMTs are a smaller group, presently consisting only of PRMT5 and its homologues. These enzymes also catalyze the synthesis of MMA on the terminal -nitrogen, but in contrast to the type I enzymes, type II PRMTs add a second methyl group to the adjacent terminal -nitrogen, resulting in symmetric dimethylarginine (SDMA). Almost all known eukaryotic cells possess at least one type I PRMT and one type II PRMT in the form of PRM...

Distinct Roles of a Mitogen-Activated Protein Kinase in Cytokinesis between Different Life Cycle Forms of Trypanosoma brucei

“…It should be noted that none of the proteins are enriched at the anterior tip of the new FAZ or on the cleavage furrow, making them less likely to be the direct regulators of cytokinesis. These proteins are associated with basal bodies and/or the flagellum (39,41,47) or the microtubule cytoskeleton (53,54) or are localized predominantly in the cytosol (13,43,(48)(49)(50)(51)(52). Like many of the above-mentioned proteins, TbMAPK6 is also distributed throughout the cytosol in both life cycle forms (Fig.…”

“…However, the downstream factors of TbAUK1 and TbPLK in the cytokinesis regulatory pathway(s) have not been identified. In addition to TbAUK1 and TbPLK, a number of other proteins have also been identified as potential regulators of cytokinesis due to the fact that deficiency in these proteins leads to cytokinesis defects (13,39,41,43,(47)(48)(49)(50)(51)(52)(53)(54)(55). Nevertheless, precisely how these proteins are involved in controlling cytokinesis has not been well established.…”

Protein Arginine Methylation in Parasitic Protozoa

“…In T. brucei, the most profound effect on growth and morphology was observed upon depletion of TbPRMT6 (38). Both PF and BF cells ablated for TbPRMT6 grow slowly under normal culture conditions, and low serum stress in PF greatly exacerbates this effect.…”

“…We recently characterized the T. brucei homologues of HsPRMT6 and HsPRMT7 (37,38). Like HsPRMT6, TbPRMT6 is a type I enzyme with an apparently narrow substrate specificity, and it undergoes automethylation, although the consequence of this is currently unknown (38).…”

Protein-arginine Methyltransferase 1 (PRMT1) Methylates Ash2L, a Shared Component of Mammalian Histone H3K4 Methyltransferase Complexes

“…The radioactive fractions (solid trace) were analyzed for the presence of tritium and compared with the elution profiles of the known standards (dashed trace). Here, the tritiated methylated derivatives would be expected to elute slightly before the non-isotopically labeled derivatives (34,68). DMLys, dimethyllysine; ADMA, asymmetric dimethylarginine; SDMA, symmetric dimethylarginine; MMA, monomethylarginine; PHD, plant homeodomain finger; SPRY, SPIa and ryanodine receptor domain.…”