Functional validation of potential non-coding RNAs differentially expressed during Leishmania viannia braziliensis life cycle

Abstract

During its life cycle, the digenetic parasite Leishmania colonizes hostile environments with different physiological characteristics, requiring precision in the modulation of gene expression. Its genetic organization with peculiar characteristics, such as the lack of canonical promoters for RNA Polymerase II, the transcription of unrelated genes in polycistronic units, and the processing of mature mRNA by trans-splicing suggests that the regulation of gene expression in these organisms results mainly from post-transcriptional modifications. Non-protein coding RNAs (ncRNAs) are known to play a key role in the regulation of gene expression in eukaryotes. These elements have been identified in the trypanosomatids genome in recent years and with the advancement of bioinformatics approaches, new putative ncRNAs have been described in these organisms. The functional role of these elements in the regulation of gene expression in these parasites, however, has not yet been properly accessed. Dr. Cruz's laboratory has sought to understand how Leishmania gene expression is regulated, with the long-term goal of finding potential targets for the development of new drugs or vaccines. Recently, 295 ncRNAs were found by the group as differentially expressed among the different life stages of L. (V.) braziliensis. In this project, we are proposing to investigate the functional role of these ncRNAs. This will be done from an initial scan of approximately 50 ncRNAs, which will be deleted from the parasite using the CRISPR/Cas9 gene editing technique. Each knockout line generated will carry a specific nucleotide sequence (barcode) and will be subjected to phenotypic characterization of survival after starvation and infectivity profile to unravel the functional role of these differentially expressed ncRNAs in parasite fitness. Since knockout of elements emerging from Untranslated Regions (UTRs) may lead to phenotypic alterations as a result of the mRNA stability or translation rate modifications, and not necessarily caused by the lack of the ncRNA, we are also proposing to develop an RNA silencing system using the CRISPR/Cas13 for trypanosomatids. This would be a valuable tool for characterizing the functional role of emerging UTR ncRNAs by acting at the mRNA level, leaving the parasite genome unchanged. Our results will help understanding the regulation of Leishmania gene expression and may result in the discovery of ncRNAs and essential parasite pathways that may be directed in the future to the discovery of new drugs or vaccines. (AU)