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Life stage specific poly(A) site selection regulated by Trypanosoma brucei DRBD18

ABSTRACT: In this work, we examine the function of DRBD18 in T. brucei, here using single cell RNA sequencing (scRNAseq) of uninduced and induced DRBD18 knockdowns. In insect procyclic form, we identified one major and one minor population of cells that dramatically increased upon DRBD18 knockdown. Both of these cell populations were significantly enriched for transcripts that are normally upregulated in the quiescent stumpy bloodstream form and metacyclic (MF) life cycle stages, consistent with a role for DRBD18 in life cycle progression. Furthermore, the 3’ end bias of our sequencing technique allowed us to investigate the effect of DRBD18 on poly(A) site selection, and extension of gene annotations permitted a comprehensive analysis of these changes. We found that 3’UTR shortening upon DRBD18 depletion was prevalent, with over 1500 transcripts exhibiting this this phenotype, and RIPseq analyses showed significant overlap between bound transcripts and those with 3’UTR shortening

ORGANISM(S): Trypanosoma brucei

PROVIDER: GSE255758 | GEO | 2024/07/25

REPOSITORIES: GEO

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Project description:The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence. RNA was isolated from AnTat1.1 cells grown in mice with or without doxycycline. RBP7 (Tb927.10.12100) RNAi lines were grown in mice with or without doxycycline. Since the RBP7 RNAi is leaky (six independent cell lines were analysed and all were significantly leaky), transcripts that showed elevated or reduced abundance in both RNAi induced and uninduced populations were identified in comparison to the AnTat1.1 control. A similar approach was adopted for the RBP7 over-expressing lines.