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RNA binding proteins (RBPs) are the primary gene regulators in kinetoplastids as transcriptional control is nearly absent, makingLeishmaniaan exceptional model for investigating methylation of non-histone substrates. Arginine methylation is an evolutionarily conserved protein modification catalyzed by Protein aRginine MethylTransferases (PRMTs). The chromatin modifier PRMT7 is the only Type III PRMT found in higher eukaryotes and a restricted number of unicellular eukaryotes. InLeishmania 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 RBP16trans-regulators (mammalian RPP25 and YBX2 homologs, respectively) as direct substratesin vitro. The absence of PRMT7 levelsin vivoselectively 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 stability ofδ-amastinsurface antigen. These results highlight a novel role for PRMT7-mediated arginine methylation upon RBP substrates, suggesting a regulatory pathway controlling gene expression and virulence inLeishmania. This work introducesLeishmaniaPRMTs as epigenetic regulators of mRNA metabolism with mechanistic insight into the functional manipulation of RBPs by methylation.

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the transregulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression, This work was supported by the Medical Research Council [grant number MR/L00092X/1 to PBW]. Funding for open access charge: Medical Research Council and University of York. LCMS within the York Centre of Excellence in Mass Spectrometry was founded through Science City York and Yorkshire Forward/Northern Way Initiative; and is supported by EPSRC [grant numbers EP/K039660/1, EP/M028127/1].

We demonstrate the use of two-color digital holographic microscopy (DHM) for imaging microbiological subjects. The use of two wavelengths significantly reduces artifacts present in the reconstructed data, allowing us to image weakly-scattering objects in close proximity to strongly-scattering objects. We demonstrate this by reconstructing the shape of the flagellum of a unicellular eukaryotic parasite Leishmania mexicana in close proximity to a more strongly-scattering cell body. Our approach also yields a reduction of approximately one third in the axial position uncertainty when tracking the motion of swimming cells at low magnification, which we demonstrate with a sample of Escherichia coli bacteria mixed with polystyrene beads. The two-wavelength system that we describe introduces minimal additional complexity into the optical system, and provides significant benefits.