2’-O-methylation on rRNA of Trypanosoma brucei is developmentally regulated with indispensable, single modified positions

Trypanosoma brucei is the causative agent of sleeping sickness. The parasite cycles between two hosts, an insect and a mammalian host that differ in their temperature by 10^oC. Here we endeavour to study how the ribosome is adapted to function in the two hosts. rRNA is modified mainly by two modifications that contribute to RNA stability, 2’-O-methylation (Nm) and pseudouridine (Ψ). We had previously shown the presence of 68 Ψs on rRNA of which 21 are hypermodified in the bloodstream form (BSF) that are essential for growth at elevated temperature (Chikne et al., Scientific Rep 2016). In this study, we mapped the Nms, using three independent high throughput sequencing approaches (RibOxi-seq, RiboMeth-seq and 2’-OMe seq). A total of 99 Nms were mapped on T. brucei rRNA guided by eighty-five C/D snoRNAs. RiboMeth-seq showed thirty-six hypermodified sites in the procylic stage (PCF) that propagates in the insect host, and eighteen hypermodified sites in the BSF. The hypermodified sites in both stages of life are found near but not in functional domains as the Ψ hypermodified sites. Silencing of snoRNA guiding hypermodified or abundant sites show that ablation of an individual C/D snoRNA has an impact on growth, rRNA processing especially at elevated temperatures. We are currently examining the intriguing possibility that rRNA modification also contributes to stage-specific translation.