Novel trypanosomatid small nucleolar RNAs that guide methylation: their genome organization, expression and potential use to direct specific methylation on target RNA molecules

Trypanosomatids are the causative agent of several major parasitic diseases including African trypanosomiasis, American trypanosomiasis, and leishmani asis. These parasites possess unique RNA-processing mechanisms including tr ans-splicing of pre-mRNA and RNA editing of mitochondrial transcripts. In t his study, we identified a trypanosomatid novel group of small nucleolar RN As that belong to the box C/D snoRNA, which were shown to guide ribose meth ylation on rRNA. Three snoRNA genes were identified; snoRNA-2 carrying a si ngle snoRNA and g2 and b2 coding for a single or multiple snoRNAs, respecti vely. Mapping of the methylation sites guided by snoRNA-2 using two differe nt approaches suggest that snoRNA-2 has the potential to guide methylation on both 5.8S and 18S rRNAs. The trypanosomes follow the same guide-methylat ion rule established for yeast and for mammals. As a first attempt to chang e the methylation pattern of target RNAs, we generated transgenic parasites carrying the B2 and snoRNA-2, which were engineered to shift the methylati on site on rRNA. Despite efficient expression of these tagged snoRNAs, the novel methylation site was not generated. However, efficient expression of tagged snoRNAs in transgenic parasites opens the possibility of engineering novel methylation sites on different target RNAs in vivo.