Investigation of non-coding RNA-protein interactions in Leishmania (Viannia) braziliensis: identifying ligands and dissecting binding profiles

Abstract

During its lifecycle Leishmania needs to have a rapid shift in gene expression profile in order to adapt to environmental changes. Trypanosomatids lack canonical promoters for most of their genes and thus, regulation of gene expression results mostly from post-transcriptional processes. Differentially expressed (DE) non-protein coding RNAs (ncRNAs) have been identified in the transcriptome of Leishmania braziliensis main morphologies suggesting they might play a role in regulation of gene expression in this parasite. Using reverse genetics, we have investigated the functional role of 10 of these DE long ncRNAs (lncRNAs) in L. braziliensis fitness. We found that deletion of 4 out of 10 long ncRNAs (lncRNAs) impairs promastigote growth, metacyclogenesis and amastigote reduplication. Moreover, these lncRNAs were found to undergo different kinds of processing routes regarding the presence of a spliced leader and poly-A tail processing traits. Together these results indicate that these elements indeed exist in L. braziliensis and may hold a novel regulatory function in this parasite. Our aim here is to investigate the molecular pathways in which these lncRNAs are involved. For that, we are proposing to use in vitro and in vivo methodologies to identify the binding partners of these lncRNAs, and also to unveil the basis of these interactions. The production of recombinant lncRNAs for these analyses will be done in vitro, using T7-mediated transcription, and in vivo, using an innovative tRNA scaffold methodology that allows the production of stable RNA at high yield and purity for use in pull-down assays and structural studies. In vitro, and in vivo pull-downs will be conducted in order to characterize the binding profile in WT versus mutated lncRNA sequences. Our results will allow us to identify pathways that are potentially regulated by these lncRNAs and the relevance of secondary and tertiary structures for protein-lncRNA binding. (AU)