Genome-scale functional profiling of cell cycle controls in African trypanosomes

Abstract

Trypanosomatids, which include major pathogens of humans and livestock, are divergent eukaryotes for which cell cycle controls and the underlying mechanisms are not completely understood. Here, we describe a genome-wide RNA-interference library screen for cell cycle regulators in bloodstream form Trypanosoma brucei. We induced massive parallel knockdown and sorted the perturbed population into cell cycle stages using flow cytometry. RNAi-targets were deep-sequenced from each stage and cell cycle profiles were digitally reconstructed at a genomic scale. We identify hundreds of proteins that impact cell cycle progression; glycolytic enzymes required for G1S progression, DNA replication factors, mitosis regulators, proteasome and kinetochore complex components required for G2M progression, flagellar and cytoskeletal components required for cytokinesis, mRNA-binding factors, protein kinases and many previously uncharacterised proteins. The outputs facilitate functional annotation and drug-target prioritisation and provide comprehensive functional genomic evidence for the machineries, pathways and regulators that coordinate progression through the trypanosome cell cycle.The data can be searched and browsed using an interactive, open access, online data visualization tool (https://tryp-cycle.onrender.com).

Control of Variant Surface Glycoprotein Expression by CFB2 in Trypanosoma brucei and Quantitative Proteomic Connections to Translation and Cytokinesis

“…We next assessed each strain following 24 h of knockdown by flow cytometry and microscopy. Consistent with the overrepresentation of G 2 M-phase cells reported previously in the genome-scale knockdown screen ( 11 ), we observed an overrepresentation of 4C (G 2 -phase) cells following knockdown and as assessed by flow cytometry; ratios of 4C to 2C (G 1 -phase) plus S-phase cells were increased 3-, 1.3-, and 3.3-fold following CFB2, MKT1, and PBP1 knockdown, respectively ( Fig. 1C ).…”

“…We reasoned that the depletion of mRNA binding proteins that positively control either VSG mRNA stability or translation would trigger a similar phenotype. To prioritize such candidate VSG regulators, we used genome-scale RNA interference (RNAi) screening data sets from bloodstream-form T. brucei , reporting a relative loss of fitness ( 10 ) or relative G 2 M accumulation precytokinesis ( 11 ). All T. brucei genes linked to the Gene Ontology term “mRNA binding” (GO:0003729) ( n = 178) were assessed for these phenotypes, and the top three candidates were selected for further analysis ( Fig.…”

“…1A ). These three proteins were associated with a significant loss of fitness, with an average z-score of >8 ( 10 ) and with >35% increased G 2 M-phase accumulation ( 11 ). CFB2 is a cyclin-like F-box protein (Tb927.1.4650) previously shown to be required for cytokinesis in bloodstream-form T. brucei ( 12 ).…”

“… Identification and assessment of candidate VSG regulators. (A) Plot showing data for genes linked to the Gene Ontology term mRNA binding (GO:0003729) ( n = 178) and in relation to loss of fitness and accumulation in G 2 M phase from RNA interference knockdown screens and from the sources indicated ( 10 , 11 ). Genes with high scores in both screens are highlighted.…”

“…Since VSG knockdown ( 8 ) or blocking VSG translation ( 9 ) results in a rapid growth defect and also specific precytokinesis cell cycle arrest, we reasoned that candidate VSG-positive regulators would be mRBPs associated with similar phenotypes in high-throughput knockdown screens ( 10 , 11 ). We identified three candidates using this approach, CFB2 (cyclin F-box protein 2), MKT1, and PBP1 (polyadenylate binding protein 1), all of which were recently and independently found to stabilize and/or bind VSG mRNA ( 6 ).…”