Altered expression of an RBP-associated arginine methyltransferase 7 in Leishmania major affects parasite infection.

Protein arginine methylation is a widely conserved post-translational modification performed by arginine methyltransferases (PRMTs). However, its functional role in parasitic protozoa is still under-explored. The Leishmania major genome encodes five PRMT homologs, including PRMT7. Here we show that LmjPRMT7 expression and arginine monomethylation are tightly regulated in a lifecycle stage-dependent manner. LmjPRMT7 levels are higher during the early promastigote logarithmic phase, negligible at stationary and late-stationary phases and rise once more post-differentiation to intracellular amastigotes. Immunofluorescence and co-immunoprecipitation studies demonstrate that LmjPRMT7 is a cytosolic protein associated with several RNA-binding proteins (RBPs) from which Alba20 is monomethylated only in LmjPRMT7-expressing promastigote stages. In addition, Alba20 protein levels are significantly altered in stationary promastigotes of the LmjPRMT7 knockout mutant. Considering RBPs are well-known mammalian PRMT substrates, our data suggest that arginine methylation via LmjPRMT7 may modulate RBP function during Leishmania spp. lifecycle progression. Importantly, genomic deletion of the LmjPRMT7 gene leads to an increase in parasite infectivity both in vitro and in vivo, while lesion progression is significantly reduced in LmjPRMT7-overexpressing parasites. This study is the first to describe a role of Leishmania protein arginine methylation in host-parasite interactions.

Leishmania Ribosomal Protein (RP) paralogous genes compensate each other's expression maintaining protein native levels.

In the protozoan parasite Leishmania, most genes encoding for ribosomal proteins (RPs) are present as two or more copies in the genome. However, their untranslated regions (UTRs) are predominantly divergent and might be associated with a distinct regulation of the expression of paralogous genes. Herein, we investigated the expression profiles of two RPs (S16 and L13a) encoded by duplicated genes in Leishmania major. The genes encoding for the S16 protein possess identical coding sequences (CDSs) and divergent UTRs, whereas the CDSs of L13a diverge by two amino acids and by their UTRs. Using CRISPR/Cas9 genome editing, we generated knockout (Δ) and endogenously tagged transfectants for each paralog of L13a and S16 genes. Combining tagged and Δ cell lines we found evidence of differential expression of both RPS16 and RPL13a isoforms throughout parasite development, with one isoform consistently more abundant than its respective copy. In addition, compensatory expression was observed for each paralog upon deletion of the corresponding isoform, suggesting functional conservation between these proteins. This differential expression pattern relates to post-translational processes, given compensation occurs at the level of the protein, with no alterations detected at transcript level. Ribosomal profiles for RPL13a indicate a standard behavior for these paralogues suggestive of interaction with heavy RNA-protein complexes, as already reported for other RPs in trypanosomatids. We identified paralog-specific bound to their 3'UTRs which may be influential in regulating paralog expression. In support, we identified conserved cis-elements within the 3'UTRs of RPS16 and RPL13a; cis-elements exclusive to the UTR of the more abundant paralog or to the less abundant ones were identified.

Cell homeostasis in a Leishmania major mutant overexpressing the spliced leader RNA is maintained by an increased proteolytic activity.

Although several stage-specific genes have been identified in Leishmania, the molecular mechanisms governing developmental gene regulation in this organism are still not well understood. We have previously reported an attenuation of virulence in Leishmania major and L. braziliensis carrying extra-copies of the spliced leader RNA gene. Here, we surveyed the major differences in proteome and transcript expression profiles between the spliced leader RNA overexpressor and control lines using two-dimensional gel electrophoresis and differential display reverse transcription PCR, respectively. Thirty-nine genes related to stress response, cytoskeleton, proteolysis, cell cycle control and proliferation, energy generation, gene transcription, RNA processing and post-transcriptional regulation have abnormal patterns of expression in the spliced leader RNA overexpressor line. The evaluation of proteolytic pathways in the mutant revealed a selective increase of cysteine protease activity and an exacerbated ubiquitin-labeled protein population. Polysome profile analysis and measurement of cellular protein aggregates showed that protein translation in the spliced leader RNA overexpressor line is increased when compared to the control line. We found that L. major promastigotes maintain homeostasis in culture when challenged with a metabolic imbalance generated by spliced leader RNA surplus through modulation of intracellular proteolysis. However, this might interfere with a fine-tuned gene expression control necessary for the amastigote multiplication in the mammalian host.