Abstract
RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, making Leishmania an exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine Methyl Transferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. In Leishmania major, PRMT7 is a cytoplasmic protein implicit in pathogenesis with unknown substrates. Using comparative methyl-SILAC proteomics for the first time in protozoa, we identified 40 putative targets, including 17 RBPs hypomethylated upon PRMT7 knockout. PRMT7 can modify Alba3 and RBP16 trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substrates in vitro. The absence of PRMT7 levels in vivo selectively reduces Alba3 mRNA-binding capacity to specific target transcripts and can impact the relative stability of RBP16 in the cytoplasm. RNA immunoprecipitation analyses demonstrate PRMT7-dependent methylation promotes Alba3 association with select target transcripts and thus indirectly stabilizes mRNA of a known virulence factor, δ-amastin surface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation of RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence in Leishmania. This work introduces Leishmania PRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.
Highlights
Protein arginine methyltransferases (PRMTs) are widely distributed across eukaryotes in nine different classes of enzymes (PRMT1–9) [1]
To evaluate the impact of PRMT7 loss upon the arginine monomethyl proteome of L. major, wild-type (WT) and prmt7 parasites were labeled with either L-methionine [33] or L-methionine-methyl-13CD3 and submitted for methyl-SILAC proteomic analysis (Figure 1A)
Expression of PRMT7 was similar between light and heavy methyllabeled parasites (Figure 1C) and no significant growth difference was observed between WT and Δprmt7 cells, light or heavy (Figure 1D)
Summary
Protein arginine methyltransferases (PRMTs) are widely distributed across eukaryotes in nine different classes of enzymes (PRMT1–9) [1]. They catalyze arginine methylation in multiple cellular processes including histone modification, transcriptional control, RNA processing, protein localization and cell signaling [1,2,3]. PRMT7 is a unique enzyme as it is the sole PRMT known to catalyze only monomethyl arginine (MMA; Type III PRMT), which may indicate a regulatory step that primes substrates prior to dimethylation [1,5]. The only unicellular eukaryotes known to carry a PRMT7 homolog are choanoflagellates and kinetoplastids, including Leishmania, where gene expression control is primarily post-transcriptional [4,7]. The heightened regulatory role of RBPs in Leishmania can lend clear functional insight not obscured by complex networks of transcriptional regulation [8]
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