Physical and functional characterization of a type II protein arginine methyltransferase in Trypanosoma brucei
Pasternack, Deborah A.
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The protozoan parasite and causative agent of human and animal African trypanosomiasis, Trypanosoma brucei , is a leading cause of morbidity and mortality among epidemic rural regions of sub-Saharan Africa. T. brucei is transmitted to the bloodstream of the mammalian host by the tsetse fly, where it resides extracellularly and evades immune detection by a mechanism called antigenic variation. Due to the antigenic nature of the parasite, the prospect for vaccine development is grim. Instead, disease treatment relies solely on chemotherapeutic strategies that target the unique and exploitable biology of the trypanosome. Such unique aspects of T. brucei biology include polycistronic transcription, pre-mRNA trans -splicing, and kinetoplastid RNA editing. The developmental regulation of trypanosome gene expression relies on coordinated post-transcriptional events in which RNA binding proteins play a leading role. With this in mind, our focus is directed towards a family of protein arginine methyltransferases (PRMTs), which are implicated in a variety of post-transcriptional events and hypothesized to play a regulatory role in trypanosome gene expression. Protein arginine methylation is a post-translational modification that modulates the function of a variety nucleic acid binding proteins, impacting transcriptional and post-transcriptional gene expression. These modifications are catalyzed by a family of PRMTs described in mammals and yeast for which five homologs have been identified in the T. brucei genome. The initial characterization of TbPRMT1 and TbPRMT5 has been explored, the latter of which is described in Chapters II and III of this thesis. TbPRMT1 is a type I PRMT and catalyzes asymmetric dimethylarginine modifications, while TbPRMT5 is a type II PRMT and catalyzes symmetric dimethylarginine modifications. Both TbPRMT1 and TbPRMT5 are constitutively expressed in bloodstream and procyclic form trypanosomes, localize to the cytoplasm, and are not essential for growth. Both enzymes methylate a variety of substrates in vitro , including the mitochondrial regulatory protein, RBP16. In addition, TbPRMT5 was shown to associate in vivo with a kinetoplastid-specific nucleotidyltransferase and an ATP-dependent RNA helicase, the latter of which is an in vitro TbPRMT5 substrate. In vivo analysis of higher-order TbPRMT5-TAP-containing complexes indicates that TbPRMT5 associates with two predominant protein complexes with molecular weights of approximately 250 and 700 kDa. The latter of these complexes is unstable and does not withstand glycerol gradient fractionation. While TbPRMT1 and TbPRMT5 do not play a global role in trans -splicing or decay of nuclear encoded RNAs, they both play a role in mitochondrial gene expression. In Chapter III of this dissertation, I show that disruption of TbPRMT5 in procyclic form trypanosomes by RNA interference results in the destabilization of never edited COI and ND4 and both edited and unedited apocytochrome b (CYb) and COII mitochondrial RNAs (Chapter III). In addition, TbPRMT5 disruption resulted in a modest but significant increase in the steady-abundance of mitochondrial-encoded guide RNAs (gRNAs), suggesting that TbPRMT5 plays a role in gRNA turnover. Furthermore, I demonstrate that TbPRMT5 potentially modulates the function of at least four mitochondrial methylproteins. Several in vivo hypomethylation defects were observed in mitochondrial lysates of TbPRMT5-disrupted cells, and TbPRMT5 appears to affect the expression and/or mitochondrial localization of one putative in vivo substrate. The characterization of TbPRMT5 substrates, as well as the remaining T. brucei PRMTs, is currently being explored. In summary, I provide evidence that TbPRMT5-catalyzed arginine methylation plays important regulatory roles in T. brucei mitochondrial gene expression, and its association with RNA modifying enzymes suggests that it likely impacts additional specific RNA metabolic events in the nucleus and/or cytoplasm.