Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei
ABSTRACT Trypanosoma brucei is a parasitic protozoan that undergoes a complex life cycle involving insect and mammalian hosts that present dramatically different nutritional environments. Mitochondrial metabolism and gene expression are highly regulated to accommodate these environmental changes, including regulation of mRNAs that require extensive uridine insertion/deletion (U-indel) editing for their maturation. Here, we use high throughput sequencing and a method for promoting life cycle changes in vitro to assess the mechanisms and timing of developmentally regulated edited mRNA expression. We show that edited CYb mRNA is downregulated in mammalian bloodstream forms (BSF) at the level of editing initiation and/or edited mRNA stability. In contrast, edited COIII mRNAs are depleted in BSF by inhibition of editing progression. We identify cell linespecific differences in the mechanisms abrogating COIII mRNA editing, including the possible utilization of terminator gRNAs that preclude the 3 to 5 progression of editing. By examining the developmental timing of altered mitochondrial mRNA levels, we also reveal transcript-specific developmental checkpoints in epimastigote (EMF), metacyclic (MCF), and BSF. These studies represent the first analysis of the mechanisms governing edited mRNA levels during T. brucei development and the first to interrogate Uindel editing in EMF and MCF life cycle stages
INTRODUCTION
Trypanosoma brucei is a flagellated, parasitic protozoan and the causative agent of human African trypanosomiasis and one of the parasitic trypanosomes that cause nagana in cattle and other domesticated livestock in 36 sub- SaharanAfrican countries, where over 70 million people are at risk of infection (1,2). The parasite has a digenetic life cycle in which it transitions through several developmental stages, each with distinct cell morphologies and transcriptomic, proteomic, and metabolic profiles (3,4). T. brucei is transmitted to its mammalian hosts by the bite of infected tsetse flies. Infective metacyclic form (MCF) trypomastigotes are injected into the mammalian bloodstream and subcutaneous tissues when the tsetse fly takes a blood meal. The shift in temperature and nutritional environment initiates differentiation from MCF to proliferative, slender bloodstream form (BSF) trypomastigotes. As the slender BSF parasites divide and parasitemia increases, a subpopulation of parasites responds to increasing concentrations of stumpy induction factor by differentiating into the quiescent, stumpy BSF (5–8). The stumpy BSF parasites are unable to revert back to proliferating slender BSF and are pre-adapted for survival in the tsetse fly insect vector (9–13). In the tsetse fly midgut, stumpy BSF must rapidly transition to the proliferative procyclic form (PCF) to survive the new nutritional environment and insect immune response (11,14–18). PCF trypomastigotes begin to migrate towards the salivary glands. During this journey, the PCF differentiates into the epimastigote form (EMF). EMF parasites attach to the epithelium of the salivary gland and proliferate to colonize the salivary glands. Asymmetrical cell division of the epithelium-bound EMF generates a daughter cell thatmatures into infective, quiescent MCF that are preadapted for survival in the mammalian host (19).
Smith J.T. Jr., Doleželová E., Tylec B., Bard J.E., Chen R., Sun Y., Zíková A., Read L.K. 2020: Developmental regulation of edited CYb and COIII mitochondrial mRNAs is achieved by distinct mechanisms in Trypanosoma brucei. Nucleic acids research (in press). [IF=11.501] DOI: 10.1093/nar/gkaa641
