Application of an RNA-seq-based method for the global analysis of self-cleaving ribozyme activity in Schistosoma mansoni

Abstract text

Ribozymes are RNAs that catalyze chemical reactions. Some ribozymes, so-called self-cleaving ribozymes, cut their own sugar phosphate backbone at a specific position to enable their biological functions. In the bloodfluke Schistosoma mansoni thousands of self-cleaving ribozymes are predicted. As their analysis by classic biochemical approaches is tedious, we know little about their activity in vivo.

We developed and validated a transcriptome-wide method, called cyPhyRNA-seq, to screen for active self-cleaving ribozymes in total RNA extracts. With this strategy, we successfully analyzed self-cleavage activity of more than 100 self-cleaving ribozymes from paired and unpaired schistosomes in a proof-of-principle study. We uncovered several intriguing ribozyme representatives: We found active ribozyme examples as part of mobile genetic elements such as Perere-3, W-elements and SINE retrotransposons. Furthermore, we found ribozyme sequences with mutations expected to inactivate them but that nonetheless function in vivo. For a representative of such ribozymes, we show in vitro evidence for a possible trans-cleavage mechanism, whereas all known natural self-cleaving ribozymes cleave in cis. Additionally, we identified differences in ribozyme activity between paired and unpaired schistosomes, suggesting a pairing-dependent expression and cleavage for some of the self-cleaving RNAs in Schistosoma. Lastly, we investigated a subset of the representatives in vitro using co-transcriptional cleavage assays and reverse transcription PCR to validate their cyPhyRNA-seq activity profile.

Thus, cyPhyRNA-seq is crucial for the monitoring of ribozyme activity in organisms that harbor self-cleaving RNAs and it facilitates the investigation of self-cleavage activity under different conditions, such as stress or developmental stage. Differences in ribozyme activity between investigated conditions could point to novel biological roles of these RNAs and might even suggest that self-cleaving ribozyme activity is regulated. Lastly, the application of cyPhyRNA-seq to a ribozyme-rich organism such as Schistosoma may enable the discovery of novel self-cleaving ribozymes. These research endeavors contribute to our current understanding of the versatility of RNA and its biological significance and enable a global, functional screening approach.