RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes
- Boise State University, United States
- University of Edinburgh, United Kingdom
Abstract
Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence to function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heat-map based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.
Data availability
Sequencing reads in FastQ format are available at ENA (PRJEB52899 and PRJEB51631). Sequences, activity data, and computer code is available at GitLab ( https://gitlab.com/bsu/biocompute-public/mut_12).
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RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymesEuropean Nucleotide Archive, PRJEB52899.
Article and author information
Author details
Funding
National Science Foundation (OIA-1738865)
- Eric J Hayden
National Science Foundation (OIA-1826801)
- Eric J Hayden
National Aeronautics and Space Administration (80NSSC17K0738)
- Eric J Hayden
Human Frontier Science Program (RGY0077/2019)
- Eric J Hayden
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Timothy W Nilsen, Case Western Reserve University, United States
Version history
- Preprint posted: May 17, 2022 (view preprint)
- Received: May 18, 2022
- Accepted: December 28, 2022
- Accepted Manuscript published: January 19, 2023 (version 1)
- Version of Record published: February 6, 2023 (version 2)
Copyright
© 2023, Roberts et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes
eLife 12:e80360.
https://doi.org/10.7554/eLife.80360
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