THE RICH REPERTOIRE OF SMALL NUCLEOAR RNA AND THE ROLE OF PSEUDOURIDYLATION DURING THE LIFE CYCLE OF TRYPANOSOMA BRUCEI

Among the developmentally regulated small RNAs are the small nucleolar RNAs (snoRNA), which guide the two major base modifications, namely 2’-O-methylations (Nm) and pseudouridylation by C/D and H/ACA snoRNAs, respectively. Using RNA-seq of snoRNAs associated with C/D and H/ACA RNP proteins we identified the complete repertoire of these RNAs, in both Trypanosoma brucei and Leishmania major. We will present evidence for the intriguing possibility that pseudouridylation of rRNA plays an important role in the capacity of the parasite to transit between the insect midgut and the mammalian bloodstream. Briefly, we mapped pseudouridines (Ψ) on rRNA by Ψ-seq in procyclic form (PCF) and bloodstream form (BSF) trypanosomes. We detected 68 Ψs on rRNA, which are guided by H/ACA small nucleolar RNAs (snoRNA). The small RNome of both life cycle stages was determined by HiSeq and 83 H/ACAs were identified. We observed an elevation of 14 Ψs modifications in BSF as a result of increased levels of the guiding snoRNAs. Interestingly, these modifications are predicted to significantly alter the secondary structure of the large subunit (LSU) rRNA in helix 69 and the peptidyl-transferase center on helix 89, suggesting that these hypermodified positions influence ribosome function in the bloodstream form trypanosomes. The majority of H/ACA snoRNA are predicted to guide modifications on rRNA. However, will present data demonstrating that many of the H/ACA RNAs have dual function and can guide modification on both rRNA and snRNAs. This novel mechanism may compensate for the presence of only a single hairpin H/ACA in trypanosomes compared to two hairpin H/ACA RNAs present in most eukaryotic H/ACA RNAs.