Functional study ofLeishmania braziliensisprotein arginine methyltransferases (PRMTs) reveals that PRMT1 and PRMT5 are required for macrophage infection

Abstract

In trypanosomatids, regulation of gene expression occurs mainly at the posttranscriptional level, and RNA-binding proteins (RBPs) are key players in determining the fates of transcripts. RBPs are major targets of protein arginine methyltransferases (PRMTs), which posttranslationally regulate the RNA-binding capacity and other macromolecular interactions of RBPs by transferring methyl groups to protein arginine residues. Herein, we present the results of a study that functionally characterized the five predicted PRMTs in Leishmania braziliensis by gene knockout and endogenous protein HA tagging using CRISPR/Cas9 gene editing. We report that arginine methylation profiles vary among Leishmania species and that target protein methylation changes across different L. braziliensis life cycle stages, with higher PRMT expression in the promastigote stages than in the axenic amastigote stage. Knockout of some of the L. braziliensis PRMTs led to significant changes in global arginine methylation patterns without affecting promastigote axenic growth. Deletion of either PRMT1 or PRMT3 disrupted most type I PRMT activity, resulting in a global increase in monomethyl arginine (MMA) levels, which is mainly catalyzed by PRMT7. Putative targets and/or PRMT-interacting proteins were identified by coimmunoprecipitation using HA-tagged PRMTs, revealing a network of target RBPs and suggesting functional interactions between them and a relevant participation in epigenetic control of gene expression. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection in vitro, and that in the absence of PRMT1 and PRMT5, axenic amastigote proliferation is impaired. The results indicate that arginine methylation is modulated across life cycle stages in L. braziliensis and show possible functional overlap and cooperation among the different PRMTs in targeting proteins. Overall, our data suggest important regulatory roles of these proteins throughout the L. braziliensis life cycle, showing that arginine methylation is important for parasite-host cell interactions.

LeishmaniaRibosomal Protein (RP) paralogous genes compensate each other’s expression maintaining protein native levels

“…To obtain metacyclic promastigotes, procyclic forms were cultivated for 5 days in M199 and metacyclics were enriched from stationary phase cultures using a Ficoll ® gradient (23). Transfections were performed using the AMAXA Nucleofector equipment, as already published by our group (24). Briefly, 1•10 7 promastigotes were resuspended in 100 µL Tb-BSF buffer and added to 60 µL of DNA generated by PCR (for Δ or tagging).…”

“…Proteins interacting with the control RNA sequence were unconsidered and the results were based on the proteins specifically interacting with the 3'UTR sequences in triplicate. Detailed information on Mass Spectrometry, Database searching and criteria for protein identification have been described elsewhere (24).…”

Virtual Special Issue: Epigenetics 2022

“…14−17 Also notable is the increase of interest in bromodomains in protozoa; their potential as novel targets against protozoans-mediated diseases is discussed in a Viewpoint 18 and two articles. 19,20 Finally, the characterization of acetyl-CoA synthetase as an interesting target for the development of antiplasmodial drugs, 21 the profiling of epigenetic modulators against hemoprotozoan parasites, 22 and two publications focused on the role of the host 23,24 complete the contributions from ACS Infectious Diseases to this virtual special issue. We thank Drs.…”

Virtual Special Issue: Epigenetics 2022

“…14−17 Also notable is the increase of interest in bromodomains in protozoa; their potential as novel targets against protozoans-mediated diseases is discussed in a Viewpoint 18 and two articles. 19,20 Finally, the characterization of acetyl-CoA synthetase as an interesting target for the development of antiplasmodial drugs, 21 the profiling of epigenetic modulators against hemoprotozoan parasites, 22 and two publications focused on the role of the host 23,24 complete the contributions from ACS Infectious Diseases to this virtual special issue. We thank Drs.…”