Concerted modification of nucleotides at functional centers of the ribosome revealed by single-molecule RNA modification profiling
- University of California, Santa Cruz, United States
- Colegio Santa Francisca Romana, Colombia
- Monta Vista High School, United States
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.
Article and author information
Author details
Andrew D Bailey IV
Shreya Mantripragada
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
-
- 3,148
- views
-
- 503
- downloads
-
- 22
- 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)
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
Further reading
-
- Cell Biology
Aggregation of mutant forms of Huntingtin is the underlying feature of neurodegeneration observed in Huntington's disorder. In addition to neurons, cellular processes in non-neuronal cell types are also shown to be affected. Cells expressing neurodegeneration-associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin-mediated endocytosis (CME) is affected in Drosophila hemocytes and mammalian cells containing Huntingtin aggregates. This is also accompanied by alterations in the organization of the actin cytoskeleton resulting in increased cellular stiffness. Further, we find that Huntingtin aggregates sequester actin and actin-modifying proteins. Overexpression of Hip1 or Arp3 (actin-interacting proteins) could restore CME and cellular stiffness in cells containing Huntingtin aggregates. Neurodegeneration driven by pathogenic Huntingtin was also rescued upon overexpression of either Hip1 or Arp3 in Drosophila. Examination of other pathogenic aggregates revealed that TDP-43 also displayed defective CME, altered actin organization and increased stiffness, similar to pathogenic Huntingtin. Together, our results point to an intimate connection between dysfunctional CME, actin misorganization and increased cellular stiffness caused by alteration in the local intracellular environment by pathogenic aggregates.
-
- Cell Biology
- Developmental Biology
The bone-resorbing activity of osteoclasts plays a critical role in the life-long remodeling of our bones that is perturbed in many bone loss diseases. Multinucleated osteoclasts are formed by the fusion of precursor cells, and larger cells – generated by an increased number of cell fusion events – have higher resorptive activity. We find that osteoclast fusion and bone resorption are promoted by reactive oxygen species (ROS) signaling and by an unconventional low molecular weight species of La protein, located at the osteoclast surface. Here, we develop the hypothesis that La’s unique regulatory role in osteoclast multinucleation and function is controlled by an ROS switch in La trafficking. Using antibodies that recognize reduced or oxidized species of La, we find that differentiating osteoclasts enrich an oxidized species of La at the cell surface, which is distinct from the reduced La species conventionally localized within cell nuclei. ROS signaling triggers the shift from reduced to oxidized La species, its dephosphorylation and delivery to the surface of osteoclasts, where La promotes multinucleation and resorptive activity. Moreover, intracellular ROS signaling in differentiating osteoclasts oxidizes critical cysteine residues in the C-terminal half of La, producing this unconventional La species that promotes osteoclast fusion. Our findings suggest that redox signaling induces changes in the location and function of La and may represent a promising target for novel skeletal therapies.
