Protein arginine (arg) methylation is a common posttranslational modification of RNA binding proteins (RNA BPs) in yeast and mammals. In Trypanosoma brucei, gene regulation is largely effected posttranscriptionally, and thus likely involves a plethora of RNA BPs. For these reasons, we hypothesize that RNA BP arg methylation plays an especially important role trypanosome gene regulation. Previously, we showed that the mitochondrial RNA BP, RBP16, is an in vivo substrate of the protein arg methyltransferase (PRMT), TbPRMT1. Arg methylation increases the functional diversity of RBP16 and modulates its macromolecular interactions. Additionally, depletion of either TbPRMT1 or TbPRMT5 results in distinct defects in mitochondrial gene expression. We identified several additional RNA BPs and RNA metabolic enzymes that associate with T. brucei PRMTs. One of these is TbDed1, a predicted RNA helicase that is methylated by TbPRMT5, exhibits ATPase activity, and is essential for optimal procyclic form (PF) growth. Ongoing in vivo and in vitro analyses of the five TbPRMTs revealed that TbPRMT6 is essential for optimal PF growth, and TbPRMT7 is a novel kinetoplastid-specific type III PRMT. Our long-term goal is to understand the roles of protein arg methylation in T. brucei gene regulation, and here we propose four specific aims.
In Aim 1, we will use in vitro methylation assays and RNAi to complete the comprehensive assessment of T. brucei's capacity for protein arg methylation and evaluate the consequences of PRMT depletion for trypanosome growth and morphology. We will also address redundancy and cooperation of selected TbPRMT pairs by dual RNAi.
In Aim 2, a battery of novel methylproteins from mitochondria, nuclei, and cytosol will be identified by anti-methylarg immunoprecipitation/mass spectrometry.
In Aim 3, we will analyze the mechanisms by which arg methylation impacts mitochondrial gene expression. Using cells expressing distinct hypomethylated versions of the model methylprotein, RBP16, we will elucidate the roles of specific methylargs in mRNA stability and editing, higher order complex formation, and mRNA association. Proteins that read RBP16 methylmarks will be identified.
In Aim 4, we will determine the role of arg methylation in creating functional diversity in the DEAD box protein, TbDed1. Cells either depleted of TbDed1 or expressing predominantly hypomethylated TbDed1 will be assayed for gene regulatory functions including trans splicing, translation initiation, and the formation of cytoplasmic RNP granules implicated in RNA turnover and translational control. We will also determine the impact of methylation on TbDed1 ATPase, RNA helicase, and RNA annealing activities to address the molecular bases of cellular methylarg function. Collectively, the proposed studies will significantly increase our understanding of gene regulation in a medically and economically important parasite. They will also provide insight into novel functions of a common posttranslational modification in higher eukaryotes.
The long-term goal of this project is to elucidate the roles protein arginine methylation in Trypanosoma brucei gene expression, particularly at the RNA level. These studies will greatly increase our understanding of gene regulation in a medically and economically important parasite. In addition, because the effects of arginine methylation on RNA metabolism are not well understood in general, they will also provide insight into a common posttranslational modification in higher eukaryotes.
|Kafková, Lucie; Tu, Chengjian; Pazzo, Kyle L et al. (2018) Trypanosoma brucei PRMT1 Is a Nucleic Acid Binding Protein with a Role in Energy Metabolism and the Starvation Stress Response. MBio 9:|
|Kafková, Lucie; Debler, Erik W; Fisk, John C et al. (2017) The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex. J Biol Chem 292:2089-2100|
|An, Bo; Zhang, Ming; Johnson, Robert W et al. (2015) Surfactant-aided precipitation/on-pellet-digestion (SOD) procedure provides robust and rapid sample preparation for reproducible, accurate and sensitive LC/MS quantification of therapeutic protein in plasma and tissues. Anal Chem 87:4023-9|
|Lott, Kaylen; Mukhopadhyay, Shreya; Li, Jun et al. (2015) Arginine methylation of DRBD18 differentially impacts its opposing effects on the trypanosome transcriptome. Nucleic Acids Res 43:5501-23|
|Tu, Chengjian; Beharry, Kay D; Shen, Xiaomeng et al. (2015) Proteomic profiling of the retinas in a neonatal rat model of oxygen-induced retinopathy with a reproducible ion-current-based MS1 approach. J Proteome Res 14:2109-2120|
|Kamisoglu, Kubra; Sukumaran, Siddharth; Nouri-Nigjeh, Eslam et al. (2015) Tandem analysis of transcriptome and proteome changes after a single dose of corticosteroid: a systems approach to liver function in pharmacogenomics. OMICS 19:80-91|
|An, Bo; Zhang, Ming; Qu, Jun (2014) Toward sensitive and accurate analysis of antibody biotherapeutics by liquid chromatography coupled with mass spectrometry. Drug Metab Dispos 42:1858-66|
|Gui, Shanying; Gathiaka, Symon; Li, Jun et al. (2014) A remodeled protein arginine methyltransferase 1 (PRMT1) generates symmetric dimethylarginine. J Biol Chem 289:9320-7|
|Tu, Chengjian; Sheng, Quanhu; Li, Jun et al. (2014) ICan: an optimized ion-current-based quantification procedure with enhanced quantitative accuracy and sensitivity in biomarker discovery. J Proteome Res 13:5888-97|
|Nouri-Nigjeh, Eslam; Sukumaran, Siddharth; Tu, Chengjian et al. (2014) Highly multiplexed and reproducible ion-current-based strategy for large-scale quantitative proteomics and the application to protein expression dynamics induced by methylprednisolone in 60 rats. Anal Chem 86:8149-57|
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