Functional Study of <i>Leishmania braziliensis</i> Protein Arginine Methyltransferases (PRMTs) Reveals That PRMT1 and PRMT5 Are Required for Macrophage Infection

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
targets of protein arginine methyltransferases (PRMTs), which posttranslationally
regulate the RNA-binding capacity and other RBP interactions by transferring
methyl groups to arginine residues (R-methylation). Herein, we 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 R-methylation profiles vary among Leishmania species and across L. braziliensis lifecycle stages, with the peak PRMT expression occurring in promastigotes.
A list of PRMT-interacting proteins was obtained in a single coimmunoprecipitation
assay using HA-tagged PRMTs, suggesting a network of putative targets
of PRMTs and cooperation between the R-methylation writers. Knockout
of each L. braziliensis PRMT led to significant
changes in global arginine methylation patterns without affecting
cell viability. Deletion of either PRMT1 or PRMT3 disrupted most type
I PRMT activity, resulting in a global increase in monomethyl arginine
levels. Finally, we demonstrate that L. braziliensis PRMT1 and PRMT5 are required for efficient macrophage infection
in vitro, and for axenic amastigote proliferation. The results indicate
that R-methylation is modulated across lifecycle 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.