RNA-binding proteins required for differentiation and infectivity in Leishmania mexicana parasites

Leishmania species are single celled kinetoplastid parasites and the causative agents of the neglected tropical disease, leishmaniasis. Leishmania parasites differentiate between non-infective and infective forms that differ greatly, both in gross morphology and in their molecular biology. In kinetoplastids, transcription of large polycistronic mRNA transcripts and a scarcity of classical RNA polymerase II promoters indicate that regulation is predominantly post-transcriptional. RNA-binding proteins (RBPs) are a major component of this post-transcriptional regulation in trypanosomatids, regulating mRNA stability as well as splicing, mRNA editing, and translational efficiency. A previous study conducted by the Walrad lab produced a highly descriptive RNA-binding proteome (RBPome) of Leishmania mexicana across different lifecycle stages (De Pablos et al., 2019). These data were analysed further to select a range of RBP candidates for a screen of null mutants to detect loss of fitness phenotypes. Barcoded RBP knockout lines were produced in L.mexicana using a CRISPR/Cas9 workflow, pooled and screened to assess their function in different lifecycle stages including in vivo infections in mice. Of the 67 RBPs for which deletion was attempted, many could not tolerate deletion and may have essential functions that warrant further investigation. Comparative barcode sequencing (bar-seq) of 31 null mutant lines, from multiple timepoints of the bar-seq screen, revealed stage-specific phenotypes caused by RBP deletion. Whole genome sequencing and replication of individual phenotypes were used to validate the screen. Selected RBPs were fused to small epitope tags and analysed by immunofluorescence and western blot, confirming their localisation and native size. The tagged RBP candidates will be used to identify bound RNAs with pulldown experiments in future work. The bar-seq screen data will inform future studies of RBPs in Leishmania, in particular those with phenotypes in the human infectious stages and may be linked to differentiation, infectivity or virulence.