Quilles, J. C. | bioRxiv Science

bioRxiv2024

A short noncoding RNA modulates gene expression and affects stress response and parasite differentiation in Leishmania braziliensis

The protozoan parasite Leishmania spp. is a causative agent of leishmaniasis, a disease that affects millions of people in more than 80 countries worldwide. Apart from its medical relevance, this organism has a genetic organization that is unique among eukaryotes. Studies of the mechanisms regulating gene expression in Leishmania led us to investigate noncoding RNAs (ncRNAs) as regulatory elements. We previously identified differentially expressed (DE) ncRNAs in Leishmania braziliensis with potential roles in the parasite biology and development. Herein, we present a functional analysis of one such DE ncRNA, the 147-nucleotide-long transcript ncRNA97, which is preferentially expressed in amastigotes, the replicative form within mammalian phagocytes. By RT-qPCR the ncRNA97 was detected in greater quantities in the nucleus under physiological conditions and in the cytoplasm under nutritional stress. Interestingly, the transcript is protected at the 5 end but is not processed by the canonical trypanosomatid trans-splicing mechanism, according to the RNA circularization assay. ncRNA97 knockout (KO) and addback (AB) transfectants were generated and subjected to phenotypic analysis, which revealed that ncRNA97 impairs the starvation response and differentiation to the infective form. Comparative transcriptomics of ncRNA97KO and parental cells revealed that transcripts encoding amastigote-specific proteins were affected. This pioneering work demonstrates that ncRNAs contribute to the developmental regulatory mechanisms of Leishmania.

molecular biology↗

bioRxiv2021

Functional study of Leishmania braziliensis 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 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.

molecular biology↗