RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes

  1. Jessica M Roberts
  2. James D Beck
  3. Tanner B Pollock
  4. Devin P Bendixsen
  5. Eric J Hayden  Is a corresponding author
  1. Boise State University, United States
  2. 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).

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Jessica M Roberts

    Biomolecular Sciences Graduate Programs, Boise State University, Boise, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3164-7256
  2. James D Beck

    Computing PhD Program, Boise State University, Boise, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9086-2653
  3. Tanner B Pollock

    Department of Biological Science, Boise State University, Boise, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Devin P Bendixsen

    Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0831-7646
  5. Eric J Hayden

    Biomolecular Sciences Graduate Programs, Boise State University, Boise, United States
    For correspondence
    erichayden@boisestate.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6078-5418

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

  1. Timothy W Nilsen, Case Western Reserve University, United States

Publication history

  1. Received: May 18, 2022
  2. Accepted: December 28, 2022
  3. Accepted Manuscript published: January 19, 2023 (version 1)

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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  1. Jessica M Roberts
  2. James D Beck
  3. Tanner B Pollock
  4. Devin P Bendixsen
  5. Eric J Hayden
(2023)
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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