Psiscan: a computational approach to identify H/ACA-like and AGA-like non-coding RNA in trypanosomatid genomes

Detection of non coding RNA (ncRNA) molecules is a major bioinformatics challenge. This challenge is particularly difficult when attempting to detect H/ACA molecules which are involved in converting uridine to pseudouridine on rRNA in trypanosomes, because these organisms have unique H/ACA molecules (termed H/ACA-like) that lack several of the features that characterize H/ACA molecules in most other organisms.

We present here a computational tool called Psiscan, which was designed to detect H/ACA-like molecules in trypanosomes. We started by analyzing known H/ACA-like molecules and characterized their crucial elements both computationally and experimentally. Next, we set up constraints based on this analysis and additional phylogenic and functional data to rapidly scan three trypanosome genomes (T. brucei, T. cruzi and L. major) for sequences that observe these constraints and are conserved among the species. In the next step, we used minimal energy calculation to select the molecules that are predicted to fold into a lowest energy structure that is consistent with the constraints. In the final computational step, we used a Support Vector Machine that was trained on known H/ACA-like molecules as positive examples and on negative examples of molecules that were identified by the computational analyses but were shown experimentally not to be H/ACA-like molecules. The leading candidate molecules predicted by the SVM model were then subjected to experimental validation.

The experimental validation showed 11 molecules to be expressed (4 out of 25 in the intermediate stage and 7 out of 19 in the final validation after the machine learning stage). Five of these 11 molecules were further shown to be bona fide H/ACA-like molecules. As snoRNA in trypanosomes are organized in clusters, the new H/ACA-like molecules could be used as starting points to manually search for additional molecules in their neighbourhood. All together this study increased our repertoire by fourteen H/ACA-like and six C/D snoRNAs molecules from T. brucei and L. Major. In addition the experimental analysis revealed that six ncRNA molecules that are expressed are not downregulated in CBF5 silenced cells, suggesting that they have structural features of H/ACA-like molecules but do not have their standard function. We termed this novel class of molecules AGA-like, and we are exploring their function. This study demonstrates the power of tight collaboration between computational and experimental approaches in a combined effort to reveal the repertoire of ncRNA molecles.