Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
The nucleolus is a non-membrane bound organelle central to ribosome biogenesis. The nucleolus contains a mix of proteins and RNA and has 3 known nucleolar compartments: the fibrillar center (FC), the dense fibrillar component (DFC), and the granular component (GC). The spatial organization of the nucleolus is influenced by the phase separation properties of nucleolar proteins, the presence of RNA, protein modification, and cellular activity. Many nucleolar proteins appear to concentrate within the borders of the compartments. We investigated whether the intrinsically disordered regions from several proteins provided the information needed to establish specific compartment localization using Xenopus laevis oocytes. For the proteins we tested, the disordered regions were not sufficient to direct specific domain localization and appear dispensable with respect to compartmentalization. Among the proteins that colocalize to the DFC are the quartet that comprise the box H/ACA pseudouridylation complex. In contrast to the insufficiency of IDRs to direct compartment localization, we found that the DFC accumulation of 2 box H/ACA proteins, Gar1 and Nhp2, was disrupted by mutations that were previously shown to reduce their ability to join the box H/ACA complex. Using a nanobody to introduce novel binding to a different DFC localized protein, we restored the localization of the mutated forms of Gar1 and Nhp2.
Fig 1. IDRs in nucleolar localization.
(A) Model of the 3 nucleolar subdomains or compartments. (B) Biological variation of wild-type Ncl-GFP co-expressed with Fbl-mRed, shown as expressed in four different frogs, showing that Ncl is always in the GC and sometimes spreads into the DFC. (C) Schematic of the protein fusions shown in D-F. Each of these fusions also contains a green or red fluorescent protein as indicated in B that allowed detection. The IDR-Ncl has a net negative charge of −40, the IDR-Fbl has a net positive charge of +17, and the N terminal IDR-Gar1 has a net positive charge of +7. (D–F) Representative images of nucleoli with IDR chimera proteins fused with a fluorescent protein co-expressed with a DFC nucleolar domain marker (Fbl). (D) Representative images showing the intrinsically disordered regions of Fbl (IDR-Fbl-GFP) and Ncl (IDR-Ncl-GFP) co-expressed with Fbl-mRed. (E) Representative images showing the fusions of IDR-Ncl and Fbl, showing IDR-Ncl-Fbl-eGFP, IDR-Ncl-ΔNFbl-eGFP, and ΔNFbl-eGFP all co-expressed with Fbl-mRed. *IDR-Ncl-Fbl-eGFP images were taken at a lower exposure in the green channel due to it being far brighter than the other constructs in this set. (F) Representative images showing the fusions of IDR-Ncl and Gar1, showing the localization of Gar1-mCherry (WT that localizes to the DFC), IDR-Ncl-Gar1-mCherry, IDR-Ncl-ΔNGar1-mCherry, and ΔNGar1-mCherry all co-expressed with Fbl-eGFP. Scale 10 μm. Statistical analysis is shown in S1 Fig and S2 Data. DFC, dense fibrillar component; IDR, intrinsically disordered region.
Fig 2. Nucleolar localization after binding disruption.
(A) Model of the general layout of the box H/ACA complex proteins and a schematic showing the mutation sites to disrupt binding. (B) Representative images of nucleoli expressing wild-type Gar1-mCherry and Gar1 with a mutated binding site to DKC1 (Gar1M1-mCherry) along with the DFC marker Fbl-eGFP. Also shown are wild-type Nhp2-mRed and Nhp2 with a point mutation that disrupts its binding to Nop10 (Nhp2P83A-mRed) with DFC marker Fbl-eGFP. Isolated nuclei were incubated in OR2 for 20 min prior to imaging. Scale: 10 μm. (C) Quantification of the colocalization of Gar1M1-mCherry or Gar1-mCherry with Fbl-eGFP and of Nhp2-RFP or Nhp2P83A-RFP with Fbl-eGFP shown with Pearson’s coefficient. Significance was determined using a t test (p < 0.05) with N = 30 (Gar1) and 26 (Gar1M1) for the Gar1 set and N = 24 (Nhp2) and 21 (Nhp2P83A) for the Nhp2 set. These experiments were repeated with nucleoli from the oocytes of 3 different frogs and were significant each time. The underlying data can be found in S1 Data. (D) Images of Nhp2-mRed and Nhp2P83A-mRed showing extra-nucleolar deposits of Nhp2P83A (arrows) that were not present for the wild type. Scale: 20 μm. DFC, dense fibrillar component.
Fig 3. Nanobody binding can induce DFC localization.
(A) Representative nucleoli from oocytes expressing Gar1 and Gar1M1 nucleolar localization and the same proteins fused with a GFP nanobody (Nb). These proteins were all fused with a red fluorescent protein and were co-expressed with Fbl-eGFP, a DFC marker. (B) Nhp2 and Nhp2P83A with and without nanobody fusion co-expressed with Fbl-eGFP. (C) The localization of a monomeric fluorescent protein (mRed) and Npm1 bound to the GFP nanobody, co-expressed with Fbl-eGFP. For each condition in Fig 3, 35+ nucleoli were observed from each of 3 different injection days, representative images are shown here. Scale: 20 μm. DFC, dense fibrillar component.
Fig 4. Nuclear fusion experiments.
(A) Schematic of nuclear fusion experiments, showing nuclei being isolated in mineral oil; 2 nuclei, each expressing different fluorescently tagged proteins, being fused together; then fused nuclei being added to a microscope slide in a well of petroleum jelly for imaging. (B) Fluorescent images taken in 10-min intervals showing the diffusion of the fluorescently tagged proteins from 1 nucleus to the nucleus it is fused with. The top row shows a nucleus containing Fbl-eGFP fused with a nucleus containing Npm1-mRed. The second row shows a nucleus with Fbl-eGFP fused with a nucleus expressing Npm1-mRed-Nb. Scale: 50 μm. (C) Representative Apotome fluorescent images of nuclei found at the junction of the fused nuclei after 40 min for the proteins indicated on each image. For each condition in Fig 4, 35+ nucleoli were observed from each of 3 different injection days, representative images are shown here. Scale: 20 μm.
S1 Fig.
Statistical analysis for Fig 1.
(A) Statistical analysis using Pearson’s Coefficient to assess the overlap of fluorescently tagged Ncl co-expressed with fluorescently tagged Fbl on 4 different days using one-way ANOVA multiple comparisons test, p < 0.05. N = 15, 58, 52, and 24 for days 1–4 respectively. (B) Table showing the results of the statistical analysis comparing the coefficients of variations of the conditions indicated in the left column. Replicates “1, 2, and 3” indicate different frogs/injection days for each condition. Values from columns are not necessarily from the same day (data should be read horizontally and not vertically). P < 0.05, N values between 15–62. The underlying data can be found in S2 Data.
S2 Fig.
Response of Fbl and Gar1 to 1,6-hexanediol treatment.
(A) Representative images of nucleoli expressing Gar1-GFP and Fbl-mRed from nuclei that were isolated in OR2 and soaked in 10% 1,6-hexanediol for 10 min. (B) Analysis of co-localization using Pearson’s coefficient between Gar1 and Fbl with and without hexanediol (p < 0.05) N = 35 (control) and 33 (hexanediol). This experiment was repeated with nucleoli from the oocytes of 3 different frogs and with the fluorescent tags switched. The underlying data can be found in S3 Data.
Alberti,
Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.
2019, Pubmed
Alberti,
Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates.
2019,
Pubmed
Berry,
RNA transcription modulates phase transition-driven nuclear body assembly.
2015,
Pubmed
Boeynaems,
Spontaneous driving forces give rise to protein-RNA condensates with coexisting phases and complex material properties.
2019,
Pubmed
Bouvet,
Nucleolin interacts with several ribosomal proteins through its RGG domain.
1998,
Pubmed
Burke,
Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II.
2015,
Pubmed
Dez,
Naf1p, an essential nucleoplasmic factor specifically required for accumulation of box H/ACA small nucleolar RNPs.
2002,
Pubmed
Düster,
1,6-Hexanediol, commonly used to dissolve liquid-liquid phase separated condensates, directly impairs kinase and phosphatase activities.
2021,
Pubmed
Feric,
Coexisting Liquid Phases Underlie Nucleolar Subcompartments.
2016,
Pubmed
,
Xenbase
Gall,
Structure in the amphibian germinal vesicle.
2004,
Pubmed
,
Xenbase
Ginisty,
Nucleolin functions in the first step of ribosomal RNA processing.
1998,
Pubmed
,
Xenbase
Handwerger,
Cajal bodies, nucleoli, and speckles in the Xenopus oocyte nucleus have a low-density, sponge-like structure.
2005,
Pubmed
,
Xenbase
Hayes,
Dual roles for ATP in the regulation of phase separated protein aggregates in Xenopus oocyte nucleoli.
2018,
Pubmed
,
Xenbase
Hayes,
Amyloids assemble as part of recognizable structures during oogenesis in Xenopus.
2016,
Pubmed
,
Xenbase
Iyer-Bierhoff,
SIRT7-Dependent Deacetylation of Fibrillarin Controls Histone H2A Methylation and rRNA Synthesis during the Cell Cycle.
2018,
Pubmed
Jumper,
Highly accurate protein structure prediction with AlphaFold.
2021,
Pubmed
Kato,
Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels.
2012,
Pubmed
Kato,
Cross-β Polymerization of Low Complexity Sequence Domains.
2017,
Pubmed
Kato,
A Solid-State Conceptualization of Information Transfer from Gene to Message to Protein.
2018,
Pubmed
Kim,
Phase transition of fibrillarin LC domain regulates localization and protein interaction of fibrillarin.
2021,
Pubmed
Koo,
Structure of H/ACA RNP protein Nhp2p reveals cis/trans isomerization of a conserved proline at the RNA and Nop10 binding interface.
2011,
Pubmed
Kosugi,
Six classes of nuclear localization signals specific to different binding grooves of importin alpha.
2009,
Pubmed
Kosugi,
Systematic identification of cell cycle-dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs.
2009,
Pubmed
Kosugi,
Design of peptide inhibitors for the importin alpha/beta nuclear import pathway by activity-based profiling.
2008,
Pubmed
Kubala,
Structural and thermodynamic analysis of the GFP:GFP-nanobody complex.
2010,
Pubmed
Kyte,
A simple method for displaying the hydropathic character of a protein.
1982,
Pubmed
Lavering,
Component analysis of nucleolar protein compartments using Xenopus laevis oocytes.
2022,
Pubmed
,
Xenbase
Levitskiĭ,
[Identification of signal sequences determining the specific nucleolar localization of fibrillarin in HeLa cells].
2004,
Pubmed
Lin,
Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers.
2016,
Pubmed
Lindström,
NPM1/B23: A Multifunctional Chaperone in Ribosome Biogenesis and Chromatin Remodeling.
2011,
Pubmed
MacNeil,
SUMOylation- and GAR1-Dependent Regulation of Dyskerin Nuclear and Subnuclear Localization.
2021,
Pubmed
Mais,
Molecular architecture of the amplified nucleoli of Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Martin,
Principles of protein targeting to the nucleolus.
2015,
Pubmed
Patel,
ATP as a biological hydrotrope.
2017,
Pubmed
Phair,
High mobility of proteins in the mammalian cell nucleus.
2000,
Pubmed
Ruff,
Advances in Understanding Stimulus-Responsive Phase Behavior of Intrinsically Disordered Protein Polymers.
2018,
Pubmed
Sanders,
Competing Protein-RNA Interaction Networks Control Multiphase Intracellular Organization.
2020,
Pubmed
Scott,
Characterization and prediction of protein nucleolar localization sequences.
2010,
Pubmed
Shan,
Nucleolar URB1 ensures 3' ETS rRNA removal to prevent exosome surveillance.
2023,
Pubmed
Shaner,
Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein.
2004,
Pubmed
Smith,
Oogenesis and oocyte isolation.
1991,
Pubmed
,
Xenbase
Spaulding,
RG/RGG repeats in the C. elegans homologs of Nucleolin and GAR1 contribute to sub-nucleolar phase separation.
2022,
Pubmed
Wang,
A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins.
2018,
Pubmed
Wang,
Architecture and assembly of mammalian H/ACA small nucleolar and telomerase ribonucleoproteins.
2004,
Pubmed
Wheeler,
Controlling compartmentalization by non-membrane-bound organelles.
2018,
Pubmed
Wühr,
Deep proteomics of the Xenopus laevis egg using an mRNA-derived reference database.
2014,
Pubmed
,
Xenbase
Xue,
PONDR-FIT: a meta-predictor of intrinsically disordered amino acids.
2010,
Pubmed
Yang,
The Shq1p.Naf1p complex is required for box H/ACA small nucleolar ribonucleoprotein particle biogenesis.
2002,
Pubmed
Zernicka-Goetz,
An indelible lineage marker for Xenopus using a mutated green fluorescent protein.
1996,
Pubmed
,
Xenbase