RNA-binding proteins and regulatory networks involved in life-stage, stress temperature, and drug resistance in Leishmania parasites

Abstract

Trypanosomatid parasites of the genus Leishmania rely on post-transcriptional regulation of gene expression because gene transcription is not canonically regulated at the single-gene level. RNA-binding proteins (RBPs) are central to this regulatory architecture, yet their genome-wide diversity, conservation, and condition-specific associations remain incompletely defined across the genus. Here, we combined comparative genomics and systems-level transcriptomic analyses to map the RBP repertoire in 33 strains spanning 19 Leishmania species. We also connect RBPs to developmental, stress, and drug-resistance contexts, using Leishmania braziliensis as an example. Using probabilistic domain detection and homology-based annotation, we identified 38,662 putative RBPs across all genomes, with 1,114 to 1,491 RBPs per genome. Comparative genomics analysis revealed a conserved core of 404 RBP clusters shared across all examined species, alongside lineage-restricted clusters in major Leishmania groups. We notably detected widespread conservation of enzymes linked to messenger RNA modification (for example, NAT10, TRMT6/61A, and NSUN2), but failed to identify canonical N6-methyladenosine writer orthologs, suggesting divergence of this machinery across Leishmania genomes. In L. braziliensis, expression profiling in different life-cycle stages and stress conditions highlighted stage-biased RBPs, including elevated ZC3H20 in amastigotes and increased RBP6 in metacyclic promastigotes. Finally, co-expression network analysis of trivalent antimony (SbIII) resistant genotypes identified RBPs co-expressed with genes previously associated with antimony resistance. In contrast, motif-based analysis supported a putative DRBD3-centered post-transcriptional module that includes 10 candidate stabilized transcripts in SbIII-resistant promastigotes. Together, these results provide a comparative framework to prioritize RBPs and associated regulatory programs implicated in parasite adaptation and antimony resistance. AUTHOR SUMMARYLeishmaniasis results from infection by Leishmania parasites and remains challenging to control due to limited treatments and the continual emergence of drug resistance, especially to antimonials. These parasites exhibit an unusual gene regulation method; instead of activating or deactivating transcription for individual genes, they depend heavily on proteins that bind RNA to determine which messages are retained, translated, or degraded. In this study, we created a comprehensive atlas of RNA-binding proteins across the Leishmania genus by analyzing 33 parasite strains from 19 species. We identified a large core set of conserved RNA-binding proteins shared by all species, along with lineage-specific proteins that may help different parasite groups adapt to various hosts and environments. Additionally, we mapped enzymes responsible for RNA chemical modifications and found that the typical machinery for m6A marks in many organisms appears to be divergent in Leishmania. Finally, by associating RNA-binding proteins with specific life-cycle stages, stress responses, and antimony resistance in L. braziliensis, we pinpointed candidate regulators and gene modules for further experimental validation. This resource helps prioritize regulatory factors that could drive parasite adaptation and resistance.