Authors

CR ESPADA¹; RM Magalhães¹; JC Quilles¹; L de Almeida¹; TP Defina¹; MJ Plevin²; PB Walrad²; C Anthon³; J Gorodkin³; AK Cruz¹;  ¹ Department of Cellular and Molecular Biology, Ribeirão Preto, Universidade de São Paulo, Brazil;  ² York Biomedical Research Institute, Department of Biology, University of York, UK;  ³ Faculty for Health and Medical Sciences of University of Copenhagen, Copenhagen, Denmark

Discussion

During its life cycle Leishmania parasites alternate between the phlebotomine vector and the mammalian host facing dramatic environmental changes which requires a rapid shift in gene expression to survive. Recently, the Cruz laboratory sequenced the transcriptomes of the three main life stages of Leishmania braziliensis, the main causative agent of tegumentary leishmaniasis in Brazil. These revealed differences not only in the expression of messenger RNAs but also of non-coding RNAs (ncRNAs). This observation raised the hypothesis that these ncRNAs could play a role in gene expression regulation in these different morphologies. To test this hypothesis, we selected 10 differentially expressed long (>200nt) presumably intergenic ncRNAs (lncRNAs) as targets. Using CRISPR/Cas9 we successfully generated knockout of 6 lncRNAs and tested their fitness in experiments mimicking key steps of the Leishmania life cycle. Four of these mutants presented significant differences in fitness compared to the parental line. For ΔlncRNA45 and ΔlncRNA66 a reduction in growth rates was observed for procyclic promastigotes in culture. For ΔlncRNA52 a reduction in metacyclogenesis rates was observed. For ΔlncRNA31 a reduction in axenic amastigotes duplication rate as well as a reduction in lesion sizes in BALB/c mice was observed. The existence and size of these four targets, precise start and end sequences as well as the presence or absence of Spliced Leader RNA (SL-RNA) and poly(A) tail were confirmed by northern blotting and circularization assays. While lncRNA45, lncRNA66 and lncRNA52 presented poly(A) tails of variable sizes, only lncRNA52 presented the SL-RNA sequence at the 5’ UTR. No poly(A) tail nor SL sequence were detected for lncRNA31. Mutations that can cause loss of secondary RNA structure were predicted in silico for lncRNA45 and lncRNA66 based on conservation. Pulldown assays using the aptamer S1m were performed using both the original and the mutated sequences of lncRNA45. These revealed distinct protein profiles interacting with WT vs mutant transcripts suggesting the secondary structure is essential for these lncRNA activity in L. braziliensis. We obtained the add back lines of these lncRNAs and they will be compared with knockout and parental fitness, to check if the parental phenotype is restored. For lncRNA45 and lncRNA66 we also generated add back lines with mutated sequence to evaluate if the loss of secondary structure can impair the fitness recovery which would strengthen the hypothesis that these structures are essential for ncRNA activity. Our results show that lncRNAs exist and are implicit in the regulation of biological processes in L. braziliensis parasites.