Functional Characterization of a Novel Long Non-Coding RNA in Leishmania braziliensis Identified Through Computational Screening for Conserved RNA Structures

Throughout their life cycle, Leishmania parasites, causative agents of the life-threatening leishmaniasis, alternate between the phlebotomine vector and the mammalian host, encountering significant environmental changes that require rapid adaptation in gene expression for survival. In the absence of conventional promoters for RNA polymerase II and given the polycistronic nature of transcription, gene expression regulation in Leishmania occurs primarily at the post-transcriptional level. Although putative regulatory non-coding RNAs (ncRNAs) have been detected in the transcriptomes of L. braziliensis and other trypanosomatids, their functional role remains poorly understood. Recognizing the critical role of RNA structure in cellular systems, we sought to identify evolutionarily conserved RNA structures. We performed a genome-wide alignment of L. braziliensis genome with closely related genomes, predicting 142 conserved RNA structures, among which 38 overlap with previously identified ncRNA transcripts. One of the putative ncRNAs with a conserved structure, a 407-nucleotide long ncRNA (lncRNA45), was previously shown to be differentially expressed across the lifecycle stages of L. braziliensis. Here, we conducted a functional characterization of lncRNA45 using a knockout line, demonstrating that the transcript is crucial for parasite fitness. Reintroduction of the original lncRNA45 sequence restored the fitness of the knockout cell line to parental levels, whereas a sequence carrying a single nucleotide substitution in the structured locus did not. In vitro pull-down assays with both the original and modified versions of lncRNA45 revealed differences in the profile of lncRNA-binding proteins. Our results indicate that lncRNA45 is enrolled cellular processes of L. braziliensis promastigotes, and this activity, along with its interaction with proteins, depends on its secondary structure. This study underscores the importance of structured lncRNAs in Leishmania and advocates for further research into their regulatory roles and therapeutic potential. HA tagged cell lines were submitted to immunoprecipitation by using HA magnetic beads to capture the HA-tagged proteins and the associated proteins and the RNAs. Each immunoprecipitated protein was checked by western blot and submitted to Mass Spec analysis. The associated RNA content was purified and submitted to library preparation followed by RNA-seq .