Rolling circle RNA synthesis catalysed by RNA

  1. Emil Laust Kristoffersen
  2. Matthew Burman
  3. Agnes Noy
  4. Philipp Holliger  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
  2. University of York, United Kingdom

Abstract

RNA-catalysed RNA replication is widely considered a key step in the emergence of life's first genetic system. However, RNA replication can be impeded by the extraordinary stability of duplex RNA products, which must be dissociated for re-initiation of the next replication cycle. Here we have explored rolling circle synthesis (RCS) as a potential solution to this strand separation problem. We observe sustained RCS by a triplet polymerase ribozyme beyond full-length circle synthesis with strand displacement yielding concatemeric RNA products. Furthermore, we show RCS of a circular Hammerhead ribozyme capable of self-cleavage and re-circularisation. Thus, all steps of a viroid-like RNA replication pathway can be catalysed by RNA alone. Finally, we explore potential RCS mechanisms by molecular dynamics simulations, which indicate a progressive build-up of conformational strain upon RCS' with destabilisation of nascent strand 5'- and 3'-ends. Our results have implications for the emergence of RNA replication and for understanding the potential of RNA to support complex genetic processes.

Data availability

All data generated or analyzed in this manuscript is supplied within the manuscript or supporting file; Source Data files containing original unedited gels images as well as numeric data have been provided for Figures 1,2,4 and 5, as well as figure supplements when relevant.Modelling data and sequencing data are provided as described in the data availability section in the manuscript.

The following data sets were generated

Article and author information

Author details

  1. Emil Laust Kristoffersen

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8965-8201
  2. Matthew Burman

    Department of Physics, University of York, York, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Agnes Noy

    Department of Physics, University of York, York, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0673-8949
  4. Philipp Holliger

    MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    For correspondence
    ph1@mrc-lmb.cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3440-9854

Funding

Carlsbergfondet (CF17-0809)

  • Emil Laust Kristoffersen

Medical Research Council (MC_U105178804)

  • Philipp Holliger

Engineering and Physical Sciences Research Council (EP/N027639/1)

  • Agnes Noy

Engineering and Physical Sciences Research Council (EP/R513386/1)

  • Matthew Burman

Engineering and Physical Sciences Research Council (EP/T022205/1)

  • Agnes Noy

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: November 1, 2021
  2. Preprint posted: November 30, 2021 (view preprint)
  3. Accepted: February 1, 2022
  4. Accepted Manuscript published: February 2, 2022 (version 1)
  5. Version of Record published: March 21, 2022 (version 2)
  6. Version of Record updated: March 22, 2022 (version 3)

Copyright

© 2022, Kristoffersen 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.

Metrics

  • 3,563
    Page views
  • 567
    Downloads
  • 6
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Emil Laust Kristoffersen
  2. Matthew Burman
  3. Agnes Noy
  4. Philipp Holliger
(2022)
Rolling circle RNA synthesis catalysed by RNA
eLife 11:e75186.
https://doi.org/10.7554/eLife.75186
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Neuroscience
    Jinli Geng, Yingjun Tang ... Xiaodong Liu
    Research Article Updated

    Dynamic Ca2+ signals reflect acute changes in membrane excitability, and also mediate signaling cascades in chronic processes. In both cases, chronic Ca2+ imaging is often desired, but challenged by the cytotoxicity intrinsic to calmodulin (CaM)-based GCaMP, a series of genetically-encoded Ca2+ indicators that have been widely applied. Here, we demonstrate the performance of GCaMP-X in chronic Ca2+ imaging of cortical neurons, where GCaMP-X by design is to eliminate the unwanted interactions between the conventional GCaMP and endogenous (apo)CaM-binding proteins. By expressing in adult mice at high levels over an extended time frame, GCaMP-X showed less damage and improved performance in two-photon imaging of sensory (whisker-deflection) responses or spontaneous Ca2+ fluctuations, in comparison with GCaMP. Chronic Ca2+ imaging of one month or longer was conducted for cultured cortical neurons expressing GCaMP-X, unveiling that spontaneous/local Ca2+ transients progressively developed into autonomous/global Ca2+ oscillations. Along with the morphological indices of neurite length and soma size, the major metrics of oscillatory Ca2+, including rate, amplitude and synchrony were also examined. Dysregulations of both neuritogenesis and Ca2+ oscillations became discernible around 2–3 weeks after virus injection or drug induction to express GCaMP in newborn or mature neurons, which were exacerbated by stronger or prolonged expression of GCaMP. In contrast, neurons expressing GCaMP-X were significantly less damaged or perturbed, altogether highlighting the unique importance of oscillatory Ca2+ to neural development and neuronal health. In summary, GCaMP-X provides a viable solution for Ca2+ imaging applications involving long-time and/or high-level expression of Ca2+ probes.

    1. Biochemistry and Chemical Biology
    2. Chromosomes and Gene Expression
    Radhika A Varier, Theodora Sideri ... Folkert Jacobus van Werven
    Research Article

    N6-methyladenosine (m6A) RNA modification impacts mRNA fate primarily via reader proteins, which dictate processes in development, stress, and disease. Yet little is known about m6A function in Saccharomyces cerevisiae, which occurs solely during early meiosis. Here we perform a multifaceted analysis of the m6A reader protein Pho92/Mrb1. Cross-linking immunoprecipitation analysis reveals that Pho92 associates with the 3’end of meiotic mRNAs in both an m6A-dependent and independent manner. Within cells, Pho92 transitions from the nucleus to the cytoplasm, and associates with translating ribosomes. In the nucleus Pho92 associates with target loci through its interaction with transcriptional elongator Paf1C. Functionally, we show that Pho92 promotes and links protein synthesis to mRNA decay. As such, the Pho92-mediated m6A-mRNA decay is contingent on active translation and the CCR4-NOT complex. We propose that the m6A reader Pho92 is loaded co-transcriptionally to facilitate protein synthesis and subsequent decay of m6A modified transcripts, and thereby promotes meiosis.