In eukaryotes, the two major base modifications are 2’-O-methylations (Nm) and pseudouridylation guided by C/D and H/ACA snoRNAs, respectively. Each RNP is bound by distinct subset of proteins and the modification is mediated by enzymes bound to the RNP; Fibrillarin (NOP1) for Nm and pseudouridine synthase (CBF5) for H/ACA RNA. By affinity-selection of snoRNA bound to a snoRNP core proteins and deep-sequencing we identified more than 80 C/D and the same number of H/ACA in L. major. Interestingly, the majority of these RNAs are guiding modifications on rRNA. To identify snoRNAs that guide modifications on snRNAs we developed the deep walk methodology. The analysis revealed dual function snoRNA in a single hairpin H/ACA molecules which target modification on both snRNA and rRNA. Such a possibility was never reported before in nature. Our study identified highly abundant snoRNAs which are implicated to direct trypanosome-specific rRNA processing. Using snoRNAi in T. brucei we were able to assign a function to each of these 15 molecules. Together, this study sheds light on the evolution of these important non-coding RNAs, revealing a much richer repertoire than many other unicellular eukaryotes, possibly because of these direct modifications that stabilize the ribosomes while cycling between the two hosts.
