Site specific pseudouridine (Psi) on Trypanosoma brucei rRNA regulates translation: The role of individual Psi on ribosome composition

Trypanosomes are unicellular parasites that cycle between the insect and mammalian host and is the causative agent of African sleeping sickness and the wasting disease, nagana, in cattle. Previous studies from our lab suggested that individual rRNA modification such as pseudouridine (Psi) and 2-O-methylation are developmentally regulated during the complex life cycle of these parasites. In this study we identified that the Psi composition of translating ribosomes are different from rRNA derived from total RNA. Ablation of single snoRNA guiding Psi in one of the inter-subunit bridges of the ribosome affects its affinity to translate specific mRNA. Mutational analysis in the pseudouridylation pocket of this snoRNA supports for the notion that the guided modification and not the chaperonic activity, is essential for the observed phenotype. Structural probing of rRNA using DMS-seq suggested that a single modification can affect the rRNA structure locally as wells as globally. Quantitative profiling of the composition of mutant ribosomes suggested the absence of eS12 ribosomal protein in the mature ribosomes. Addback experiments validated the role of eS12 during the loss of a single Psi in these mutant cells. Orthologous experiments in related leishmania spp. also verify importance of a single Psi in the function of ribosome. Ongoing experiments intend to visualize such ribosomes lacking a single RNA modification and decipher their significance towards ribosome structure.