Authors

C A Marques¹; L Glover²; A Cassidy³; D Horn¹;  ¹ Division of Biological Chemistry and Drug Discovery, School of Life Sciences, University of Dundee;  ² Institut Pasteur, Paris, France;  ³ Tayside Centre for Genomic Analysis, School of Medicine, University of Dundee, UK

Discussion

RNA interference (RNAi) library screening in Trypanosoma brucei has proven to be a powerful approach for determining which proteins are, for example, required for the parasite�s viability. Proteins that are essential for viability can then be considered genetically validated as potential drug-targets. Major challenges remain however, in terms of developing a deeper understanding of how these proteins impact trypanosome biology and viability. To gain further insight into protein function at a genomic scale, we have now carried out a screen for cell cycle defects. A genome-wide bloodstream form RNAi library was induced for 24 h and ~10⁸ cells, stained with propidium iodide, were isolated by fluorescence-activated cell sorting (FACS) according to DNA content. Sub-G1, G1, S, G2/M and >G2/M populations were recovered and subjected to RNAi Target sequencing (RIT-seq). In this approach, each read serves as a barcode to report the relative representation of each gene-knockdown in each of the five isolated populations. As expected, we find that cell-cycle perturbations are enriched for gene-knockdowns previously associated with a fitness-cost. One particularly prominent feature is a >G2 enrichment profile that emerges for cohorts of genes associated with the cytoskeleton and motility. Another notable feature is the association of proteasome components knockdowns with an accumulation at G2/M. We are currently analysing the datasets and will present findings in relation to factors involved in DNA-replication, chromosome segregation, phosphorylation and RNA-binding, among others.