A single pseudouridine on rRNA matters: A specific effect of rRNA modification on translation in Trypanosoma brucei

Differential pseudouridine (Ψ) modification was suggested by us to play a role in the parasite transition between its two hosts. Here we used additional mapping methods of Ψ such as HydraPsiSeq and nanopore RNA sequencing to quantitatively determine changes in Ψ on rRNA of procyclics (PCF) and bloodstream (BSF) parasites. The hypermodified sites in BSF were localized to the H69 domain present in the inter-subunit bridge, A-site finger domain, and near the decoding center. To investigate the role of these hypermodified sites and their implications on translation, we performed CRISPR-Cas9 knock-out of the snoRNA TB11Cs6H1 guiding Ψ530 on H69. The growth of sKO was compromised and quantitative mass-spectrometry identified changes in the level of only a subset of proteins; most are developmentally regulated. Add back of TB11Cs6H1, but not a defective mutant was able to reverse the sKO phenotype. To investigate the mechanism by which a single modification affects translation, rRNA structure was determined by dimethyl sulfate (DMS) mapping in vivo and changes in the structure of H69 and other rRNA domains,were observed. Analysis of 80S ribosomal protein composition detected major reduction the level of eS12. We propose that the change in Ψ530 affected the secondary structure of the rRNA, leading to dislodging of eS12 and producing ribosomes depleted of this protein. These “altered” ribosomes result in specific changes in translation. Thus, the differential level of Ψ modification in the two life stages may contribute to stage-specific translation regulation during cycling between the hosts.