Genome-scale RNA interference profiling of Trypanosoma brucei cell cycle progression defects

Abstract

Trypanosomatids, which include major pathogens of humans and livestock, are flagellated protozoa 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 defects in Trypanosoma brucei. We induced massive parallel knockdown, sorted the perturbed population using high-throughput flow cytometry, deep-sequenced RNAi-targets from each stage and digitally reconstructed cell cycle profiles at a genomic scale; also enabling data visualisation using an online tool (https://tryp-cycle.pages.dev/). Analysis of several hundred genes that impact cell cycle progression reveals >100 flagellar component knockdowns linked to genome endoreduplication, evidence for metabolic control of the G1-S transition, surface antigen regulatory mRNA-binding protein knockdowns linked to G2M accumulation, and a putative nucleoredoxin required for both mitochondrial genome segregation and for mitosis. The outputs provide comprehensive functional genomic evidence for the known and novel machineries, pathways and regulators that coordinate trypanosome cell cycle progression.

Profiling the bloodstream form and procyclic formTrypanosoma bruceicell cycle using single cell transcriptomics

“…Yet, in BSFs CYC9 transcript depletion results in a clear cytokinesis defeat 82 . Thus, transcript FC does not necessarily correlate with functional significance, as was also noted when comparing CCR genes to cell cycle defects profiled by the genome-scale screen in BSFs 45 . Results of CYC9 RNAi depletion in PCFs is currently conflicting, possibly due to differences in knockdown efficiency, as one study observed a substantial cell cycle arrest in G2/M 77 , while another saw no specific arrest in any cell cycle phase 82 .…”

“…The additional CCR genes identified only in BSF included those linked to glycolysis, which BSFs rely on to generate ATP from the glucose energy source in the mammal 63 . Interesting, the knockdown of 11 glycolysis-associated genes was linked to cell cycle arrest in G1 45 and so further investigation may unveil if BSFs use glycolysis activity levels as a signal for re-entering the cell cycle during G1. Other genes uniquely CCR in BSFs include DNA recombination factors, RecQ helicase.…”

“…Other G2M associated genes include a putative homolog of S. cerevisiae CDC14, which has several roles in regulating mitosis 44 . Recently, a genome scale phenotypic genetic screen (RNA Interference Target sequencing, RIT-seq) was performed to identify genes associated with a cell cycle defect when transcripts were depleted by RNAi in BSF T. brucei 45 . After induction of RNAi, cells from each cell cycle phase (G1, S and G2M) were isolated based on their DNA content using FACS; sub-diploid (<2C) and over-replication (>4C) populations were also isolated and analysed.…”

“…For comparison with proteomics 13,14 , bulk transcriptomics 12 and genome scale cell cycle defect RNAi screen 45 , all of which used the TRUE927 reference genome 103 , syntenic orthologs were identified in the Lister 427 2018 reference 47 using TriTrypDB implementation of OrthoMCL 104 . For each study, CCR genes were retained as those selected by original authors.…”

Novel kinetoplastid-specific cAMP binding proteins identified by RNAi screening for cAMP resistance inT. brucei

“…4) for association with the flagellar axoneme or the flagellum, suggesting an important impact of cAMP signalling on flagellar biology. An intact and functional flagellum was previously shown to be essential for cell division of bloodstream stage trypanosomes, as RNAi repression of many genes with flagellar localization or function result in a lethal cell division phenotype (Broadhead et al ., 2006; Marquez et al, 2022). This may explain why the screen returned specifically the observed set of proteins.…”