Direct testing of natural twister ribozymes from over a thousand organisms reveals a broad tolerance for structural imperfections
Lauren N McKinley, McCauley O Meyer, Aswathy Sebastian, Benjamin K Chang, Kyle J Messina, Istvan Albert, Philip C Bevilacqua, Direct testing of natural twister ribozymes from over a thousand organisms reveals a broad tolerance for structural imperfections, Nucleic Acids Research, 2024;, gkae908, https://doi.org/10.1093/nar/gkae908
Twister ribozymes are an extensively studied class of nucleolytic RNAs. Thousands of natural twisters have been proposed using sequence homology and structural descriptors. Yet, most of these candidates have not been validated experimentally. To address this gap, we developed Cleavage High-Throughput Assay (CHiTA), a high-throughput pipeline utilizing massively parallel oligonucleotide synthesis and next-generation sequencing to test putative ribozymes en masse in a scarless fashion. As proof of principle, we applied CHiTA to a small set of known active and mutant ribozymes. We then used CHiTA to test two large sets of naturally occurring twister ribozymes: over 1600 previously reported putative twisters and ∼1000 new candidate twisters. The new candidates were identified computationally in ∼1000 organisms, representing a massive increase in the number of ribozyme-harboring organisms. Approximately 94% of the twisters we tested were active and cleaved site-specifically. Analysis of their structural features revealed that many substitutions and helical imperfections can be tolerated. We repeated our computational search with structural descriptors updated from this analysis, whereupon we identified and confirmed the first intrinsically active twister ribozyme in mammals. CHiTA broadly expands the number of active twister ribozymes found in nature and provides a powerful method for functional analyses of other RNAs.
For many classes of RNA, their function is derived from their structure. Unlike DNA, which assumes a simple double-stranded helix, RNA adopts a diverse array of intramolecular structural features including hairpins, internal loops and multihelical junctions, which aid in the functionality of the RNA ( 1–3). Interestingly, RNA can tolerate imperfections such as bulges, deletions, insertions, mismatches, mutations and overhangs while maintaining its function ( 4). This structural diversity allows RNA to fulfill biological roles apart from being a conduit for genetic information ( 2, 3). Riboswitches and RNA thermometers, for instance, employ conformational switching to regulate transcription and translation in response to small molecules and temperature, respectively ( 5, 6). RNA enzymes, known as ribozymes, catalyze protein synthesis and splicing, as well as use cleavage reactions to mediate gene expression and RNA half-life ( 7).
