Treacle is a scaffold protein for nucleolar FCs and 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 were analyzed by laser scanning confocal microscopy. Representative images of cells and nucleoli (magnified images) are shown. Co-localization analysis was performed on the merged images. Graphs illustrate quantification in arbitrary units of fluorescence distribution along the lines shown in the figures. (B) HeLa cells were transfected with a construct coding CRISPR/Cas9 and sgRNA to the TCOF1 gene. After 7-10 days after transfection, the cells were fixed and co-immunostained with Treacle and either RPA194, UBF, Fibrillarin, B23 or Nucleolin antibodies. DNA was stained with DAPI (gray). Cells were analyzed by laser scanning confocal microscopy. Representative images of Treacle-negative cells and nucleoli (magnified images) are shown. (C) HeLa cells were transfected with a construct coding CRISPR/Cas9 and sgRNA to the TCOF1 gene (experiment). After 7-10 days after transfection cell were fixed, immunostained with Treacle antibodies and subjected to cell sorting in the fluorescent analysis mode to obtain Treacle-negative populations. Intact HeLa cells (control) were fixed, immunostained with IgG antibodies, passed through all FACS-related procedure in the light scattering analysis mode and used as a control (Treacle-positive). The sorted cell fractions were used for chromatin immunoprecipitation (ChIP) analysis with either Treacle, RPA194 or UBF antibodies. ChIP was followed by qPCR using the d0, d1, d3, d5, d7 primers to the rRNA gene (positioned as indicated on the scheme). Data are represented relative to the input. Values are means ±SD from at least three independent replicates (**, p < 0.01; *, p < 0.1; by unpaired t test). (D) HeLa cells were transfected with a construct coding CRISPR/Cas9 and sgRNA to the TCOF1 gene. After 7-10 days after transfection the cells were pulsed with EU (100 μM for 2 hr), fixed and immunostained with Treacle antibodies. EU (green) was revealed by click chemistry. The DNA was stained with DAPI (gray). Representative images of Treacle-positive and Treacle-negative HeLa cells are shown. Quantification of EU fluorescence intensities in Treacle-positive and Treacle-negative HeLa cells are show in the right panel (**, p < 0.01 by unpaired t test; n>500). (E) HeLa cells were transfected with a construct coding CRISPR/Cas9 and sgRNA to the TCOF1 gene (experiment). After 7-10 days after transfection the cells were fixed, immunostained with Treacle antibodies and subjected to cell sorting in the fluorescent analysis mode to obtain Treacle-negative populations. Intact HeLa cells (control) were fixed, stained with IgG antibodies, passed through all FACS-related procedure in the light scattering analysis mode and used as a control (Treacle-positive). The sorted cell fractions were used for RNA extraction. RT-qPCR was performed; it shows levels of 47S pre-rRNA normalized to GAPDH mRNA. Normalized pre-rRNA level in Treacle-positive cells is set to 1. Values are mean ± SD. The calculation is presented for 5 biological replicates (**, p < 0.01 by unpaired t test). (F) Hela cells were processed as described in (E). It shows levels of A’-site contained unprocessed rRNA normalized to GAPDH mRNA. Normalized unprocessed rRNA level in Treacle-positive cells is set to 1. Values are mean ± SD. The calculation is presented for 5 biological replicates (**, p < 0.01 by unpaired t test).

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 disordered protein with N-terminal (ND, 1–83 aa), C-terminal (CD, 1121–1488 aa) regions, and central repeated domain (RD, 83–1121 aa) consisting of 15 low complexity regions (LCR) interspersed with disordered linker sequences. (B) The purified recombinant fragment of Treacle undergoes condensation in vitro in the presence of 5% polyethylene glycol. (C) Condensates of recombinant Treacle’s fragment were time-lapse imaged. Representative images of condensates fusion events are shown. (D) HeLa cells were transfected with Treacle-FusionRed-Cry2 (opto-Treacle) construct. DNA was stained with Hoechst 33342 (gray). Formation of Treacle condensates was induced with blue light illumination of the cells for 10 sec. Representative images are shown. (E) HeLa cells were transfected with Treacle-Katushka2S (Treacle-2S) at a quantity of 50 ng plasmids per 2×105 cells. For low levels of expression analysis, the cells were fixed 16-24 hours after transfection. For high levels of expression analysis, the cells were fixed 48 hours after transfection. DNA was stained with DAPI (gray). Cells were analyzed by laser scanning confocal microscopy. Surface reconstructions of Treacle-2S FCs or extranucleolar condensates are shown in the right panels. Graphs illustrate quantification in arbitrary units of the 3D analysis of Treacle-2S condensate’s shape by LasX software. Sphere form factor = sphere area/particle area. (F) 16-24 h after transfection with Treacle-2S, HeLa cells were treated with 0.05µg/ml actinomycin D (AMD) to induce rDNA transcriptional repression and time-lapse imaged for 120 minutes. DNA was stained with Hoechst 33342 (gray). (G) 48 h after transfection with Treacle-2S, HeLa cells were live time-lapse imaged for 12 hours. DNA was stained with Hoechst 33342 (gray). Representative images of cells with Treacle-2S condensates fusion events are shown. Graphs illustrate the frequency of fusion Treacle condensates per cell for 12 hours. The calculation is presented for 6 biological replicates of 10 cells each. (H) HeLa cells were transiently transfected with Treacle-2S. 16-24 h after transfection half-FRAP analysis of Treacle-labelled fibrillar center (FCs) was performed. Half of the one FC was bleached, and fluorescence recovery was monitored. Representative time-lapse images of the photobleached FC are shown (magnified images). DNA was stained with Hoechst 33342 (gray). (I) Graphs illustrate the quantification of the FCs half-FRAP analysis described in (H). Half-FRAP curves represent the normalized intensity in the bleached half (blue) and the non-bleached half (green). Each trace represents an average of measurements for at least twenty FCs; error bars represent SD. (J) 16-24 h after transfection with Treacle-2S, HeLa cells were treated with 0.05µg/ml AMD to induce the formation of nucleolar caps. Half-FRAP analysis of Treacle-labelled nucleolar caps was performed. Representative time-lapse images of photobleached nucleolar caps are shown (magnified images). DNA was stained with Hoechst 33342 (gray). (K) Graphs illustrate the quantification of the nucleolar caps half-FRAP analysis described in (J). Half-FRAP curves represent the normalized intensity in the bleached half (blue) and the non-bleached half (green). Each trace represents an average of measurements for at least twenty caps; error bars represent SD. (L) HeLa cells were transiently transfected with Treacle-2S. 48 h after transfection, half-FRAP analysis of Treacle-2S extranucleolar condensates was performed. Half of one condensate was bleached, and fluorescence recovery was monitored. The analysis included condensates with diameters of up to 3 µm (type 1), as well as the largest condensates exceeding 5 µm in diameter (type 2). Representative time-lapse images of photobleached Treacle-2S extranucleolar condensates of both categories are shown (magnified images). DNA was stained with Hoechst 33342 (gray). (M) Graphs illustrate the quantification of the nucleolar caps half-FRAP analysis described in (L). Half-FRAP curves represent the normalized intensity in the bleached half (blue) and the non-bleached half (green). Each trace represents an average of measurements for at least twenty extranucleolar condensates of both categories; error bars represent SD. DNA was stained with Hoechst 33342 (gray).

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 h after transfection respectively. The expression level is indicated by the colored zone to the left of the cell images. HeLa cells with low expression level were additionally treated with CX-5461 to induce rDNA transcriptional repression and subsequent nucleolar cap formation. Cells were fixed and analyzed by laser scanning confocal microscopy. DNA was stained with DAPI (gray). Representative images of Treacle-2S Δ1-83 condensate are shown. (B) HeLa cells were transfected with Treacle-2S Δ1-83 deletion mutant and processed as described in (A). Partial FRAP analysis of Treacle-2S Δ1-83 condensates was performed. A part of each condensate type was photobleached, and the subsequent fluorescence recovery was monitored. Representative time-lapse images of the photobleached condensates are shown (magnified images). DNA was stained with Hoechst 33342 (gray). (C) HeLa cells were transfected with Treacle-2S Δ1-83 deletion mutant and processed as described in (A). Graphs illustrate the quantification of the Treacle-2S Δ1-83 condensates partial FRAP analysis described in (B). Each trace represents an average of measurements for at least twenty Treacle Δ1-83 condensates of each type; error bars represent SD. (D) HeLa cells were transfected with Treacle-2S Δ83-1121 deletion mutant and processed as described in (A). (E) HeLa cells were transfected with Treacle-2S Δ83-1121 deletion mutant and processed as described in (A). Partial FRAP analysis of Treacle-2S Δ83-1121 condensates dynamics was performed as described in (B). (F) HeLa cells were transfected with Treacle-2S Δ83-1121 deletion mutant and processed as described in (A). Partial FRAP analysis of 2S-fused Treacle Δ83-1121 condensates dynamics was performed as described in (B). Graphs illustrate the quantification of the Treacle-2S Δ83-1121 condensates partial FRAP analysis. Each trace represents an average of measurements for at least twenty Treacle-2S Δ83-1121 condensates of each type; error bars represent SD. (G) HeLa cells were transfected with Treacle-2S Δ1121-1488 deletion mutant fused with nuclear localization signal (NLS) from simian virus 40 (SV40). For low or high levels expression analysis cells were cultivated 16-24 or 48 h after transfection respectively. The expression level is indicated by the colored zone to the left of the cell images. Cells were fixed and analyzed by laser scanning confocal microscopy. DNA was stained with DAPI (gray). Representative images of cells are shown.

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 h after transfection respectively. The expression level is indicated by the colored zone to the left of the cell images. Cells were treated with 5% 1,6-hexandiol (1,6-HEX) for 10 min or 200 mM ammonium acetate for 5 min. Cells were fixed and analyzed by laser scanning confocal microscopy. DNA was stained with DAPI (gray). Representative images of cells are shown. (B) The purified recombinant fragment of Treacle undergoes condensation in vitro in the presence of 10% 1,6-hexandiol (1.6-HEX), 500 mM sodium chloride or buffers with different pH. In each case, 5% PEG 8000 was used as a crowding agent. (C) HeLa cells were transfected with Treacle-2S ΔSE mutant. For low or high levels expression analysis cells were cultivated 16-24 or 48 h after transfection respectively. The expression level is indicated by the colored zone to the left of the cell images. HeLa cells with low expression level were additionally treated with CX-5461 to induce rDNA transcriptional repression. Cells were fixed and analyzed by laser scanning confocal microscopy. DNA was stained with DAPI (gray). Representative images of Treacle ΔSE condensate are shown. (D) HeLa cells were transfected with Treacle-2S ΔSE mutant and processed as described in (C). Partial FRAP analysis of Treacle-2S ΔSE condensates was performed. A part of each condensate type was photobleached, and the subsequent fluorescence recovery was monitored. Representative time-lapse images of the photobleached condensates are shown (magnified images). DNA was stained with Hoechst 33342 (gray). (E) HeLa cells were transfected with Treacle-2S ΔSE mutant and processed as described in (C). Graphs illustrate the quantification of the Treacle-2S ΔSE condensates partial FRAP analysis described in (D). Each trace represents an average of measurements for at least twenty Treacle-2S ΔSE condensates of each type; error bars represent SD. (F) HeLa cells were transfected with Treacle-2S Δ1350-1488 deletion mutant (Treacle-2S ΔNoLS) and processed as described in (C). (G) HeLa cells were transfected with Treacle-2S ΔNoLS deletion mutant and processed as described in (C). Partial FRAP analysis of Treacle-2S ΔNoLS condensates was performed as described in (D). (H) HeLa cells were transfected with Treacle-2S ΔNoLS deletion mutant and processed as described in (C). Partial FRAP analysis of Treacle-2S ΔNoLS condensates dynamics was performed as described in (D). Graphs illustrate the quantification of the Treacle-2S ΔNoLS condensates partial FRAP analysis. Each trace represents an average of measurements for at least twenty Treacle ΔNoLS condensates of each type; error bars represent SD. (I) Charge plots of full-length Treacle (left panel) and charge-scrambled Treacle (Treacle CS) form (right panel) are shown. Positive and negative charge blocks are depicted by blue and red triangles, respectively. Charge distribution was calculated as the sum of the charges (Arg and Lys, +1; Glu and Asp, −1;) in the 25 amino acids window range. The center of the panel shows the aligned amino acid sequences of one of the S/E-rich LCR and its adjacent linker of RD for both full Treacle and Treacle CS. (J) HeLa cells were transfected with Treacle-2S CS mutant and processed as described in (C).

The condensation of Treacle facilitates its functional interactions with partner proteins

(A) Endogenous Treacle was depleted by siRNA-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with siRNA-resistant full-length Treacle-2S (Treacle-2S full). Cells were fixed 16-24 after transfection and immunostained with either RPA194, UBF, Fibrillarin, B23 or Nucleolin antibodies. Cells were analyzed by laser scanning confocal microscopy. Representative images of magnified nucleoli are shown. Co-localization analysis was performed on the merged images. Graphs illustrate quantification in arbitrary units of Treacle-2S variants and RPA194, UBF, Fibrillarin, B23 or Nucleolin fluorescence distribution along the lines shown in the figures. (B) Endogenous Treacle was depleted by siRNA-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with siRNA-resistant charge-scrambled Treacle-2S (Treacle-2S CS) and processed as described in (A). (C) Endogenous Treacle was depleted by siRNA-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with siRNA-resistant full-length Treacle-2S (Treacle-2S full). 16-24 h after transfection cells were treated with 0.05µg/ml actinomycin D (AMD) to induce rDNA transcriptional repression, fixed and processed as described in (A). (D) Endogenous Treacle was depleted by siRNA-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with siRNA-resistant charge-scrambled Treacle-2S (Treacle-2S CS). 16-24 h after transfection cells were treated with 0.05µg/ml actinomycin D (AMD) to induce rDNA transcriptional repression, fixed and processed as described in (A).

Treacle LLPS is essential for the transcription and processing of rRNA

(A) Endogenous Treacle was depleted by siRNA-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with siRNA-resistant full-length Treacle-2S (Treacle-2S full), charge-scrambled Treacle-2S (Treacle-2S CS) or Treacle-2S Δ83-1121 deletion mutant (Treacle-2S Δ83-1121). Cells were fixed 16-24 after transfection and subjected to cell sorting in the fluorescent analysis mode to obtain 2S-positive populations. The sorted cell fractions were used for RNA extraction. RT-qPCR was performed; it shows levels of 47S pre-rRNA normalized to GAPDH mRNA. Normalized pre-rRNA level in full-length Treacle-2S-positive cells is set to 1. Values are mean ± SD. The calculation is presented for 5 biological replicates. **, p < 0.01 by unpaired t test. (B) HeLa cells were processed as described in (A). Cells were fixed 16-24 after transfection and subjected to ChIP-seq analysis with Katushka2S antibodies. ChIP-seq signal were normalized to the input. (C) HeLa cells were processed as described in (A). Cells were fixed 16-24 after transfection and subjected to cell sorting in the fluorescent analysis mode to obtain 2S-positive populations. The sorted cell fractions were used for used for ChIP with RPA194 or UBF antibodies. ChIP was followed by qPCR using the d1 primers to the promoter of the rRNA gene (positioned as indicated on the scheme). Percentage of enrichment relative to input in full-length Treacle-2S-positive cells is set to 1.Values are mean ± SD. The calculation is presented for 5 biological replicates. **, p < 0.01 by unpaired t test. (D) HeLa cells were processed as described in (A). Cells were fixed 16-24 after transfection and stained for 47S pre-rRNA (revealed by single-molecule FISH, smFISH). Cells were analyzed by laser scanning confocal microscopy. Representative images of magnified nucleoli are shown. Co-localization analysis was performed on the merged images. Graphs illustrate quantification in arbitrary units of Treacle-2S variants and smFISH fluorescence distribution along the lines shown in the figures. (E) HeLa cells were transfected with full-length Treacle-2S. 48 h after transfection cells were fixed and stained for 47S pre-rRNA (revealed by single-molecule FISH, smFISH). Cells were analyzed by laser scanning confocal microscopy. Representative images of magnified nucleoli are shown. (F) HeLa cells were transfected with a construct coding CRISPR/Cas9 and sgRNA to the TCOF1 gene (Treacle kn). After 7-10 days after transfection, the cells were fixed and stained with UBF antibodies and 47S pre-rRNA. Representative images of Treacle-negative nucleolus (magnified images) are shown. (G) HeLa cells were processed as described in (A). Cells were fixed 16-24 after transfection and subjected to cell sorting in the fluorescent analysis mode to obtain 2S-positive populations. The sorted cell fractions were used for RNA extraction. RT-qPCR was performed; it shows levels of A’-site contained unprocessed rRNA normalized to GAPDH mRNA. Normalized unprocessed rRNA level in full-length Treacle-2S-positive cells is set to 1. Values are mean ± SD. The calculation is presented for 5 biological replicates. **, p < 0.01 by unpaired t test.

Treacle phase separation is essential for 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. The DNA was stained with DAPI (gray). Co-localization analysis was performed on the merged images (magnified images). Graphs illustrate quantification in arbitrary units of Treacle and TOPBP1 fluorescence distribution along the lines shown in the figures. Percentage of cells containing TOPBP1 (TOPBP1-positive) foci within nucleoli is shown. (B) DMSO-treated and VP16-treated (90 μM, 30 min) HeLa cells were subjected to Proximity Ligation Assay (PLA) with antibodies against TOPBP1 and Treacle. DNA was stained with DAPI (gray). PLA detection of Treacle-TOPBP1 interactions is visible as distinct green fluorescent dots. (C) Mock-treated HeLa cells or cells with siRNA-mediated Treacle knockdown (Treacle kd) were treated with DMSO or VP16 (90 μM, 30 min). ChIP experiments were performed with antibodies against TOPBP1. ChIP was followed by qPCR using the d1 primers to the promoter of the rRNA gene (positioned as indicated on the scheme). Data are represented relative to the input. Values are means ±SD from at least three independent replicates. (D) Intact HeLa cells, 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 were co-immunostained for TOPBP1 (green) and Treacle (magenta) antibodies and analyzed by laser scanning confocal microscopy. The DNA was stained with DAPI (gray). (E) Endogenous Treacle was depleted by siRNA-mediated knockdown (Treacle kd). Next, Treacle-depleted cells were transfected with plasmid constructs encoding either siRNA-resistant full-length Treacle-2S (Treacle-2S full), Treacle-2S Δ83-1121 deletion mutant, or charge-scrambled mutant Treacle-2S (Treacle-2S CS). 24 h after transfection, cells were treated with VP16 (90 μM for 30 min), fixed and stained for TOPBP1 (green) and analyzed by laser scanning confocal microscopy. The DNA was stained with DAPI (gray). (F) Endogenous Treacle was depleted by siRNA-mediated knockdown (Treacle kd). Treacle-depleted cells were transfected with plasmid constructs encoding either siRNA-resistant Treacle-2S full, Treacle-2S Δ83-1121, or Treacle-2S CS. 24 h after transfection, cells were treated with DMSO or VP16 (90 μM for 30 min) and fixed. Cells were subjected to cell sorting in the fluorescent analysis mode to obtain 2S-positive populations. At least 2×106 sorted cells were used for ChIP with TOPBP1 antibodies. ChIP was followed by qPCR using the d1 primers to the promoter of the rRNA gene (positioned as indicated on the scheme). Data are represented relative to the input. Values are means ±SD from at least three independent replicates. (G) Intact HeLa cells and cells siRNA-depleted for either Treacle (Treacle kd) or TOPBP1 (TOPBP1 kd) were treated with DMSO or 90 μM VP16 for 30 min. ChIP experiments were performed with antibodies against phospho-ATR (pATR; Ser1989), phospho-ATM (pATM; Ser1981) or γH2AX antibodies. ChIP was followed as described in (F). (H) HeLa cells were processed as described in (F). At least 2×106 sorted cells were used for ChIP with phospho-ATR (pATR; Ser1989), phospho-ATM (pATM; Ser1981) or γH2AX antibodies. ChIP was followed by qPCR using the d1 primers to the promoter of the rRNA gene (positioned as indicated on the scheme). Data are represented relative to the input. Values are means ±SD from at least three independent replicates.