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
Share this article
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
  2. Download BibTeX
  3. Download .RIS

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.

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.

Metrics

  • 3,219
    views
  • 512
    downloads
  • 26
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

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. 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

Categories and tags

Research organism

Further reading

    1. Cell Biology

    Spatial and temporal coordination of Duox/TrpA1/Dh31 and IMD pathways is required for the efficient elimination of pathogenic bacteria in the intestine of Drosophila larvae

    Fatima Tleiss, Martina Montanari ... C Leopold Kurz
    Research Article

    Multiple gut antimicrobial mechanisms are coordinated in space and time to efficiently fight foodborne pathogens. In Drosophila melanogaster, production of reactive oxygen species (ROS) and antimicrobial peptides (AMPs) together with intestinal cell renewal play a key role in eliminating gut microbes. A complementary mechanism would be to isolate and treat pathogenic bacteria while allowing colonization by commensals. Using real-time imaging to follow the fate of ingested bacteria, we demonstrate that while commensal Lactiplantibacillus plantarum freely circulate within the intestinal lumen, pathogenic strains such as Erwinia carotovora or Bacillus thuringiensis, are blocked in the anterior midgut where they are rapidly eliminated by antimicrobial peptides. This sequestration of pathogenic bacteria in the anterior midgut requires the Duox enzyme in enterocytes, and both TrpA1 and Dh31 in enteroendocrine cells. Supplementing larval food with hCGRP, the human homolog of Dh31, is sufficient to block the bacteria, suggesting the existence of a conserved mechanism. While the immune deficiency (IMD) pathway is essential for eliminating the trapped bacteria, it is dispensable for the blockage. Genetic manipulations impairing bacterial compartmentalization result in abnormal colonization of posterior midgut regions by pathogenic bacteria. Despite a functional IMD pathway, this ectopic colonization leads to bacterial proliferation and larval death, demonstrating the critical role of bacteria anterior sequestration in larval defense. Our study reveals a temporal orchestration during which pathogenic bacteria, but not innocuous, are confined in the anterior part of the midgut in which they are eliminated in an IMD-pathway-dependent manner.

    1. Cancer Biology
    2. Cell Biology

    Automated workflow for the cell cycle analysis of (non-)adherent cells using a machine learning approach

    Kourosh Hayatigolkhatmi, Chiara Soriani ... Simona Rodighiero
    Tools and Resources

    Understanding the cell cycle at the single-cell level is crucial for cellular biology and cancer research. While current methods using fluorescent markers have improved the study of adherent cells, non-adherent cells remain challenging. In this study, we addressed this gap by combining a specialized surface to enhance cell attachment, the FUCCI(CA)2 sensor, an automated image analysis pipeline, and a custom machine learning algorithm. This approach enabled precise measurement of cell cycle phase durations in non-adherent cells. This method was validated in acute myeloid leukemia cell lines NB4 and Kasumi-1, which have unique cell cycle characteristics, and we tested the impact of cell cycle-modulating drugs on NB4 cells. Our cell cycle analysis system, which is also compatible with adherent cells, is fully automated and freely available, providing detailed insights from hundreds of cells under various conditions. This report presents a valuable tool for advancing cancer research and drug development by enabling comprehensive, automated cell cycle analysis in both adherent and non-adherent cells.

    1. Cell Biology

    Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation

    Yue Miao, Yongtao Du ... Mei Ding
    Research Article

    The spatiotemporal transition of small GTPase Rab5 to Rab7 is crucial for early-to-late endosome maturation, yet the precise mechanism governing Rab5-to-Rab7 switching remains elusive. USP8, a ubiquitin-specific protease, plays a prominent role in the endosomal sorting of a wide range of transmembrane receptors and is a promising target in cancer therapy. Here, we identified that USP8 is recruited to Rab5-positive carriers by Rabex5, a guanine nucleotide exchange factor (GEF) for Rab5. The recruitment of USP8 dissociates Rabex5 from early endosomes (EEs) and meanwhile promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In USP8-deficient cells, the level of active Rab5 is increased, while the Rab7 signal is decreased. As a result, enlarged EEs with abundant intraluminal vesicles accumulate and digestive lysosomes are rudimentary. Together, our results reveal an important and unexpected role of a deubiquitinating enzyme in endosome maturation.