1. Cell Biology
  2. Computational and Systems Biology

Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling

  1. Andrew D Bailey IV  Is a corresponding author
  2. Jason Talkish
  3. Hongxu Ding
  4. Haller A Igel
  5. Alejandra Duran
  6. Shreya Mantripragada
  7. Benedict Paten  Is a corresponding author
  8. Manuel Ares Jr  Is a corresponding author
  1. University of California, Santa Cruz, United States
  2. Colegio Santa Francisca Romana, Colombia
  3. Monta Vista High School, United States
https://doi.org/10.7554/eLife.76562
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Cite this article
  1. Andrew D Bailey IV
  2. Jason Talkish
  3. Hongxu Ding
  4. Haller A Igel
  5. Alejandra Duran
  6. Shreya Mantripragada
  7. Benedict Paten
  8. Manuel Ares Jr
(2022)
Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling
eLife 11:e76562.
https://doi.org/10.7554/eLife.76562
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Abstract

Nucleotides in RNA and DNA are chemically modified by numerous enzymes that alter their function. Eukaryotic ribosomal RNA (rRNA) is modified at more than 100 locations, particularly at highly conserved and functionally important nucleotides. During ribosome biogenesis, modifications are added at various stages of assembly. The existence of differently modified classes of ribosomes in normal cells is unknown because no method exists to simultaneously evaluate the modification status at all sites within a single rRNA molecule. Using a combination of yeast genetics and nanopore direct RNA sequencing, we developed a reliable method to track the modification status of single rRNA molecules at 37 sites in 18S rRNA and 73 sites in 25S rRNA. We use our method to characterize patterns of modification heterogeneity and identify concerted modification of nucleotides found near functional centers of the ribosome. Distinct, undermodified subpopulations of rRNAs accumulate upon loss of Dbp3 or Prp43 RNA helicases, suggesting overlapping roles in ribosome biogenesis. Modification profiles are surprisingly resistant to change in response to many genetic and acute environmental conditions that affect translation, ribosome biogenesis, and pre-mRNA splicing. The ability to capture single molecule RNA modification profiles provides new insights into the roles of nucleotide modifications in RNA function.

Data availability

Fastq files from all direct RNA sequencing runs and signalAlign modification calls are publicly available in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE186634 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE186634). Fast5 and fastq files for all direct RNA sequencing are available in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB48183 (https://www.ebi.ac.uk/ena/browser/view/PRJEB48183). A detailed description of the datasets used and sequenced in this work with their corresponding ENA or GEO IDs can be found in (table supplement 7).Code availabilityDocumentation, install requirements, and analysis scripts for all work specific to this paper can be found at https://github.com/adbailey4/yeast_rrna_modification_detection. SignalAlign v1.0.0 can be found at https://github.com/UCSC-nanopore-cgl/signalAlign and embed_fast5 1.0.0 can be found https://github.com/adbailey4/embed_fast5.

The following data sets were generated

Article and author information

Author details

  1. Andrew D Bailey IV

    Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, United States
    For correspondence
    andbaile@ucsc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8304-7565

  2. Jason Talkish

    Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0260-3345

  3. Hongxu Ding

    Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, United States
    Competing interests
    The authors declare that no competing interests exist.

  4. Haller A Igel

    Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, United States
    Competing interests
    The authors declare that no competing interests exist.

  5. Alejandra Duran

    Colegio Santa Francisca Romana, Bogota, Colombia
    Competing interests
    The authors declare that no competing interests exist.

  6. Shreya Mantripragada

    Monta Vista High School, Cupertino, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6234-6176

  7. Benedict Paten

    Department of Biomolecular Engineering, University of California, Santa Cruz, Santa Cruz, United States
    For correspondence
    bpaten@ucsc.edu
    Competing interests
    The authors declare that no competing interests exist.

  8. Manuel Ares Jr

    Department of Molecular, Cell and Developmental Biology, University of California, Santa Cruz, Santa Cruz, United States
    For correspondence
    ares@ucsc.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2552-9168

Funding

National Institute of General Medical Sciences (R01 GM040478)

  • Manuel Ares Jr

National Human Genome Research Institute (R01 HG010053)

  • Manuel Ares Jr

National Human Genome Research Institute (U41HG010972)

  • Benedict Paten

National Human Genome Research Institute (R01HG010485)

  • Benedict Paten

National Human Genome Research Institute (U01HG010961)

  • Benedict Paten

NIH Office of the Director (OT2OD026682)

  • Benedict Paten

NIH Office of the Director (OT2OD026682)

  • Benedict Paten

National Heart, Lung, and Blood Institute (U01HL137183)

  • Benedict Paten

National Human Genome Research Institute (2U41HG007234)

  • Benedict Paten

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Marina V Rodnina, Max Planck Institute for Biophysical Chemistry, Germany

Version history

  1. Preprint posted: December 17, 2021 (view preprint)
  2. Received: December 21, 2021
  3. Accepted: April 5, 2022
  4. Accepted Manuscript published: April 6, 2022 (version 1)
  5. Accepted Manuscript updated: April 7, 2022 (version 2)
  6. Version of Record published: April 27, 2022 (version 3)

Copyright

© 2022, Bailey 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. Andrew D Bailey IV
  2. Jason Talkish
  3. Hongxu Ding
  4. Haller A Igel
  5. Alejandra Duran
  6. Shreya Mantripragada
  7. Benedict Paten
  8. Manuel Ares Jr
(2022)
Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling
eLife 11:e76562.
https://doi.org/10.7554/eLife.76562

Share this article

https://doi.org/10.7554/eLife.76562

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