Treacle’s ability to form liquid-like phase condensates is essential for nucleolar fibrillar center assembly, efficient rRNA transcription and processing, and rRNA gene repair
- Department of Cellular Genomics, Institute of Gene Biology RAS, Russian Federation
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology RAS, Russian Federation
- Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Russian Federation
- A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Russian Federation
- Biological Faculty, Lomonosov Moscow State University, Russian Federation
Figures
Figure 1 with 2 supplements
Treacle is a scaffold protein for nucleolar fibrillar centers (FCs) and dense fibrillar components (DFCs).
(A) Intact HeLa cells (Treacle-positive) were fixed and co-immunostained with Treacle and with either RPA194, UBF, fibrillarin, B23, or nucleolin antibodies. DNA was stained with DAPI (gray). Cells …
Figure 1—figure supplement 1
Treacle depletion induces dispersion and mixing of FC and DFC within nucleoli in both HeLa cells and normal fibroblasts.
(A) HeLa cells were transfected with a construct coding CRISPR/Cas9 and single guide RNA (sgRNA) to the TCOF1 gene. 7–10 days after transfection, the cells were fixed and co-immunostained with …
Figure 1—figure supplement 2
Depletion of Treacle impairs RNA polymerase I-mediated transcription within the nucleolus.
(A) HeLa cells were transfected with a construct encoding CRISPR/Cas9 and a mock single guide RNA (sgRNA). 7–10 days post-transfection, cells were fixed, immunostained with IgG antibodies, and …
Figure 2 with 9 supplements
Treacle drives the formation of nuclear condensates.
(A) The structure of the most common isoform of Treacle. Treacle isoform d (1488 amino acids, 152 kDa, NP_001128715.1) is encoded by the Treacle transcript variant 4. It is an intrinsically …
Figure 2—figure supplement 1
Treacle is an intrinsically disordered protein that forms intranuclear condensates upon overexpression.
(A) Full-length Treacle disorder characteristics predicted by FuzDrop, Anchor2, and IUprep2. (B) HeLa cells were transfected with FusionRed-Cry2 or FUS-FusionRed-Cry2 constructs and, 24 hr after …
Figure 2—figure supplement 2
Treacle exhibits limited molecular dynamics between condensates.
Figure 2—video 1
Intranucleolar Treacle-2S fusion events (low expression, actinomycin D [AMD]-treated cells).
Figure 2—video 2
Extranucleolar Treacle-2S fusion events (high expression).
Figure 2—video 3
Treacle-2S fibrillar centers (FCs) half-fluorescence recovery after photobleaching (FRAP).
Figure 2—video 4
Treacle-2S actinomycin D (AMD) half-fluorescence recovery after photobleaching (FRAP).
Figure 2—video 5
.Treacle-2S intranucleolar condensates type 1 half-fluorescence recovery after photobleaching (FRAP).
Figure 2—video 6
Treacle-2S intranucleolar condensate type 2 half-fluorescence recovery after photobleaching (FRAP).
Figure 2—video 7
Intranucleolar Treacle-2S fusion events (low expression, VP16-treated cells).
Figure 3 with 1 supplement
Treacle’s condensation is regulated by its central and C-terminal domains.
(A) HeLa cells were transfected with Treacle-2S Δ1–83 deletion mutant. For low or high levels expression analysis cells were cultivated 16–24 or 48 hr after transfection respectively. The expression …
Figure 3—figure supplement 1
Treacle’s condensation properties are governed by its central repeating domain (RD) and C-terminal domain (CD).
(A) HeLa cells were transfected with full-length Treacle-2S. For low or high levels expression analysis cells were cultivated 16–24 or 48 hr after transfection respectively. The expression level is …
Figure 4 with 1 supplement
The condensation of Treacle is controlled by the specific charge distribution in its domains.
(A) HeLa cells were transfected with Treacle-2S. For low or high levels expression analysis, cells were cultivated 16–24 or 48 hr after transfection respectively. The expression level is indicated …
Figure 4—figure supplement 1
The condensation properties of the Treacle are determined by the charge distribution in its central repeating domain (RD).
(A) HeLa cells were transfected with Treacle-2S ΔSE mutant. For low or high levels expression analysis cells were cultivated 16–24 or 48 hr after transfection respectively. The expression level is …
Figure 5 with 1 supplement
The condensation of Treacle facilitates its functional interactions with partner proteins.
(A) Endogenous Treacle was depleted by small interfering RNA (siRNA)-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with siRNA-resistant full-length Treacle-2S …
Figure 5—figure supplement 1
Disruption of Treacle’s condensation properties alters its interaction specificity with protein partners.
(A) Endogenous Treacle was depleted by small interfering RNA (siRNA)-mediated knockdown (Treacle kd). After 3–6 days, knockdown efficiencies were analyzed by western blotting. Mock, scrambled …
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Figure 5—figure supplement 1—source data 1
TIFF file containing original western blots for panel A, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/96722/elife-96722-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
Original files for western blot analysis displayed in panel A.
- https://cdn.elifesciences.org/articles/96722/elife-96722-fig5-figsupp1-data2-v1.zip
Figure 6
The condensation of Treacle is essential for the transcription and processing of ribosomal RNA (rRNA).
(A) Endogenous Treacle was depleted by small interfering RNA (siRNA)-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with siRNA-resistant full-length Treacle-2S …
Figure 7 with 3 supplements
Treacle phase separation is essential for DNA damage response (DDR) activation in ribosomal genes under genotoxic stress conditions.
(A) DMSO-treated and VP16-treated (90 μM, 30 min) HeLa cells were co-immunostained for Treacle (Treacle endogenous; magenta) and TOPBP1 (green) and analyzed by laser scanning confocal microscopy. …
Figure 7—figure supplement 1
Etoposide (VP16) induces the association of TOPBP1 with Treacle in HeLa and MCF7 cells.
(A) DMSO-treated and VP16-treated (90 μM, 30 min) HeLa cells were co-immunostained for Treacle (Treacle endogenous; magenta) and TOPBP1 (green) and analyzed by laser scanning confocal microscopy. …
Figure 7—figure supplement 2
Etoposide (VP16) promotes TOPBP1 translocation to the nucleolus in a Treacle-dependent manner.
(A) Intact HeLa cells, small interfering RNA (siRNA)-depleted for Treacle (Treacle kd) cells or CRISPR/Cas9-depleted for Treacle (Treacle kn) cells were treated with 90 μM VP16 for 30 min. Cells …
Figure 7—figure supplement 3
Disruption of Treacle’s condensation properties abrogates its interaction with TOPBP1 and impairs activation of the rDNA damage response under genotoxic stress.
(A) Endogenous Treacle was depleted by small interfering RNA (siRNA)-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with plasmid constructs encoding either …
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Figure 7—figure supplement 3—source data 1
TIFF file containing original western blots for panel B, indicating the relevant bands.
- https://cdn.elifesciences.org/articles/96722/elife-96722-fig7-figsupp3-data1-v1.zip
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Figure 7—figure supplement 3—source data 2
Original files for western blot analysis displayed in panel B.
- https://cdn.elifesciences.org/articles/96722/elife-96722-fig7-figsupp3-data2-v1.zip
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HeLa | ATCC | CCL-2, RRID:CVCL_0030 | Mycoplasma free |
| Cell line (Homo sapiens) | MCF7 | ATCC | HTB-22, RRID:CVCL_0031 | Mycoplasma free |
| Cell line (Homo sapiens) | Dermal fibroblast (normal, Adult) | Gift from Dr. M Lagarkova | Female 46XX, Mycoplasma free | |
| Chemical compound, drug | Etoposide (VP16) | Sigma-Aldrich | E1383 | |
| Chemical compound, drug | CX-5461 | Sigma-Aldrich | #5092650001 | |
| Chemical compound, drug | Actinomycin D (AMD) | Sigma-Aldrich | A1410 | |
| Chemical compound, drug | 1,6-Hexanediol (1,6-HD) | Sigma-Aldrich | #240117 | |
| Chemical compound, drug | Ammonium acetate | Serva | #39750.01 | |
| Chemical compound, drug | Hoechst 33342 | Sigma-Aldrich | #14533 | |
| Chemical compound, drug | DAPI | Sigma-Aldrich | D9542 | |
| Antibody | Anti-Treacle/TCOF1 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-374536, RRID:AB_10987865 | ICC (1:500), WB (1: 5000), ChIP-qPCR |
| Antibody | Anti-Treacle/TCOF1 (Rabbit polyclonal) | Sigma-Aldrich | HPA038237 | ICC (1:500) |
| Antibody | Anti-RPA194 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-48385, RRID:AB_675814 | ICC (1:50), ChIP-qPCR |
| Antibody | Anti-UBF1 (Rabbit polyclonal) | Thermo Fisher | PA5-82245, RRID:AB_2789405 | ICC (1:200), ChIP-qPCR |
| Antibody | Anti-Fibrillarin (Rabbit monoclonal) | Abcam | ab166630, RRID:AB_2928100 | ICC (1:500) |
| Antibody | Anti-Nucleolin (Rabbit monoclonal) | Cell Signaling | 14574, RRID:AB_2798519 | ICC (1:500) |
| Antibody | Anti-B23 (Mouse monoclonal) | Sigma-Aldrich | B0556, RRID:AB_2154872 | ICC (1:300) |
| Antibody | Anti-TOPBP1 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-271043, RRID:AB_10610636 | ICC (1:200), ChIP-qPCR, WB (1:2500) |
| Antibody | Anti-BRCA1 (Mouse monoclonal) | Santa Cruz Biotechnology | sc-6954, RRID:AB_626761 | ChIP-qPCR |
| Antibody | Anti-pATM (Ser1981) (Mouse monoclonal) | Cell Signaling | 4526, RRID:AB_2062663 | ChIP-qPCR |
| Antibody | Anti-γH2AX (Ser139) (Mouse monoclonal) | Millipore | 05-636, RRID:AB_309864 | ChIP-qPCR |
| Antibody | Anti-pATR (Thr1989) (Rabbit polyclonal) | Cell Signaling | 58014, RRID:AB_2722679 | ChIP-qPCR |
| Antibody | Anti-53BP1 (Rabbit polyclonal) | Santa Cruz Biotechnology | sc-22760, RRID:AB_2256326 | ChIP-qPCR |
| Antibody | Anti-Katushka2S (Rabbit polyclonal) | Eurogene | AB233, RRID:AB_2571743 | ChIP-seq |
| Antibody | Anti-GAPDH, (Rabbit polyclonal) | Abcam | ab9485, RRID:AB_307275 | WB |
| Antibody | Polyclonal Goat anti-mouse Alexa Fluor Plus 488 | Invitrogen | A32723, RRID:AB_2633275 | ICC (1:200) |
| Antibody | Polyclonal Goat anti-mouse Alexa Fluor Plus 594 | Invitrogen | A32742, RRID:AB_2762825 | ICC (1:200) |
| Antibody | Polyclonal Goat anti-rabbit CF488A | Biotium | #20012, RRID:AB_10853801 | ICC (1:200) |
| Antibody | Polyclonal Goat anti-rabbit CF594 | Biotium | #20112, RRID:AB_10559190 | ICC (1:200) |
| Commercial assay or kit | Duolink In Situ Detection Reagents Green | Sigma-Aldrich | DUO92014 | |
| Commercial assay or kit | Click-i RNA Alexa Fluor 488 Imaging Kit | Thermo Fisher | C10329 | |
| Software, algorithm | ImageJ (version 1.44) | https://imagej.net/ij/download.html | RRID:SCR_003070 | |
| Software, algorithm | CellProfiler (version 4.2.8) | https://cellprofiler.org/releases | RRID:SCR_007358 | |
| Software, algorithm | Bowtie (version 2.2.3) | https://bowtie-bio.sourceforge.net/bowtie2/index.shtml | RRID:SCR_016368 | |
| Software, algorithm | SAMtools (version 1.5) | https://github.com/samtools/samtools | RRID:SCR_002105 | |
| Software, algorithm | deepTools (version 3.4.2) | https://github.com/deeptools/deepTools | RRID:SCR_016366 | |
| Other | Lipofectamine LTX and Plus Reagent | Invitrogen | #15338-100 | |
| Other | Lipofectamine 3000 | Invitrogen | L3000-015 |
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Abbreviations: ICC, immunocytochemistry; ChIP, chromatin immunoprecipitation; WB: western blotting.
Additional files
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Supplementary file 1
List of primers used for cloning.
- https://cdn.elifesciences.org/articles/96722/elife-96722-supp1-v1.docx
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Supplementary file 2
The amino acid substitutions and deletions engineered to generate the Treacle ΔSE and Treacle CS mutant variants.
- https://cdn.elifesciences.org/articles/96722/elife-96722-supp2-v1.docx
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Supplementary file 3
List of primers used for knockdown and knockout.
- https://cdn.elifesciences.org/articles/96722/elife-96722-supp3-v1.docx
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Supplementary file 4
List of primers used for quantitative polymerase chain reaction (qPCR).
- https://cdn.elifesciences.org/articles/96722/elife-96722-supp4-v1.docx
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Supplementary file 5
List of primers used for chromatin immunoprecipitation (ChIP)-quantitative polymerase chain reaction (qPCR).
- https://cdn.elifesciences.org/articles/96722/elife-96722-supp5-v1.docx
-
Supplementary file 6
List FITS-labeled oligonucleotides used for single-molecule fluorescence in situ hybridization (smFISH).
- https://cdn.elifesciences.org/articles/96722/elife-96722-supp6-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/96722/elife-96722-mdarchecklist1-v1.docx
