The goal of this project is to identify mechanisms by which arginine (arg) methylation regulates gene expression in kinetoplastid parasites. Kinetoplastids, including Trypanosoma brucei, T. cruzi, and Leishmania spp., collectively infect approximately 20 million people and cause hundreds of thousands of deaths per year. There is an appalling lack of safe and effective drugs to combat these kinetoplastid-related diseases, and a deeper understanding of parasite biology is required to identify pathways that could act as novel drug targets. One unique aspect of kinetoplastid biology is the apparent absence of transcriptional control of RNA polymerase II. In the absence of transcriptional control, gene regulation relies largely on posttranscriptional processes such as RNA stability and translation, thereby making RNA binding proteins (RBPs) critical regulators of cellular function. In higher eukaryotes, RBPs are common targets of arg methylation, a modification that can dramatically affect the subcellular localization and/or macromolecular interactions of target RBPs. Together, these observations suggest that arg methylation of RBPs could play an especially important role in kinetoplastid gene regulation. We have characterized five protein arg methyltransferases (PRMTs) and identified over 800 arg methylproteins in T. brucei. In this application, we will examine the function of a novel arg methylated RBP, TbMiz1. In vitro, TbMiz1 binds to RNA with some sequence specificity, but it does not bind DNA. In vivo, TbMiz1 is methylated on at least three arg residues, and it is essential for growth of procyclic form T. brucei. The proposed studies will begin to assess TbMiz1 cellular functions and elucidate how these functions are impacted by arg methylation. TbMiz1 contains a C-terminal arg/glycine-rich (RG) domain, a domain commonly targeted for arg methylation. The three known methylargs are confined to this region. To understand the affects of arg methylation, we will create T. brucei cell lines conditionally expressing hypomethylated TbMiz1 mutants either lacking the entire RG domain or harboring lysine to arg substitutions at the three known methylargs.
In Aim 1, we will determine the ability of TbMiz1 to act as a substrate for each of the five TbPRMTs using both in vitro methylation assays and reciprocal in vivo co-immunoprecipitations (co-IPs).
In Aim 2, we will determine TbMiz1 subcellular localization by immunoblotting and immunofluorescence and compare localization of the hypomethylated mutant TbMiz1. We will identify protein binding partners of TbMiz1 by mass spectrometry of tandem affinity purified wild type TbMiz1 and determine if arg methylation impacts these interactions by mass spectrometry and co-IP of the hypomethylated mutants.
In Aim 3, we will identify in vivo RNA targets of TbMiz1 using RNA immunoprecipitation and deep sequencing (RIP-seq). RIP of the hypomethylated mutants followed by qRT-PCR of validated target RNAs will reveal whether methylation modulates specific TbMiz1-RNA interactions. Overall, the proposed experiments will provide insight into the functions of an essential RBP in T. brucei and elucidate how arg methylation can modulate these functions.
Kinetoplastid parasites are a group of important human pathogens including Trypanosoma brucei, T. cruzi, and Leishmania spp., which collectively cause high morbidity and mortality in tropical and subtropical regions of the world, and also threaten military and southern border U.S. populations. Kinetoplastids rely on RNA-binding proteins to regulate gene expression, thus RNA binding proteins are a good target candidate for urgently needed drug development. Here we propose the use of T. brucei as an experimental model system to study regulation of kinetoplastid RNA binding proteins.
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|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|
|Lott, Kaylen; Zhu, Lu; Fisk, John C et al. (2014) Functional interplay between protein arginine methyltransferases in Trypanosoma brucei. Microbiologyopen 3:595-609|
|Lott, Kaylen; Li, Jun; Fisk, John C et al. (2013) Global proteomic analysis in trypanosomes reveals unique proteins and conserved cellular processes impacted by arginine methylation. J Proteomics 91:210-25|