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 immunostained with Treacle and either RPA194, UBF, Fibrillarin, B23 or Nucleolin antibodies. 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 to obtain Treacle-negative populations. The sorted cell fractions were used for ChIP analysis with either Treacle, RPA194 or UBF antibodies. Intact HeLa cells were fixed, immunostained with IgG antibodies, passed through all FACS-related procedure and used as a control (Treacle-positive). 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. (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 (n>500). (E) 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, immunostained with Treacle antibodies and subjected to cell sorting into Treacle-negative populations. The sorted cell fractions were used for RNA extraction. Intact HeLa cells were fixed, stained with IgG antibodies, passed through all FACS-related procedure and used as a control (Treacle-positive). 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. (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.

Treacle drives the formation of nuclear condensates.

(A) Purified recombinant Treacle underwent phase separation from the buffer and formed liquid droplets in the presence of 1-10% dextran as a crowding agent. The size of Treacle drops was measured (n>500). (B) Drops of recombinant Treacle were time-lapse imaged. Representative images of drop fusion events are shown. (C) HeLa cells were transfected with Treacle-FusionRed-Cry2 (opto-Treacle) construct. Formation of opto-Treacle drops was induced with blue light illumination of the cells for 10 sec. DNA was stained with Hoechst 33342 (gray). Representative images are shown. (D) HeLa cells were transfected with Treacle-Katushka2S (Treacle-2S) at a quantity of 50 ng plasmids per 2×105 cells. For low or high levels of expression analysis, the cells were fixed 16-24 or 48 h after transfection, respectively. DNA was stained with DAPI (gray). Cells were analyzed by laser scanning confocal microscopy. Surface reconstructions of Treacle-2S drops are shown in the right panel. Graphs illustrate quantification in arbitrary units of the 3D analysis of Treacle-2S drops’ shape by LasX software. Sphere form factor = sphere area/particle area. (E) 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. (F) 48 h after transfection with Treacle-2S, HeLa cells were live time-lapse imaged. Representative images of cells with Treacle-2S drops 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. (G) HeLa cells were transiently transfected with Treacle-2S. 16-24 h after transfection FRAP analysis of Treacle-labelled fibrillar center (FCs) dynamics was performed. Part of one FC was bleached, and fluorescence recovery was monitored. Representative time-lapse images of the photobleached FC are shown (magnified images). (H) 16-24 h after transfection with Treacle-2S, HeLa cells were treated with 0.05µg/ml AMD to induce the formation of nucleolar caps. Part of the nucleolar cap was bleached, and fluorescence recovery was monitored. Representative time-lapse images of photobleached nucleolar caps are shown (magnified images). (I) Graphs illustrate the quantification of the Treacle-2S FRAP analysis described in (G) and (H). Each trace represents an average of measurements for at least twenty FCs or nucleolar caps; error bars represent SD. (J) HeLa cells were transiently transfected with Treacle-2S. 48 h after transfection, FRAP analysis of Treacle-2S extranucleolar condensates dynamics was performed. Part of one drop was bleached, and fluorescence recovery was monitored. Representative time-lapse images of photobleached Treacle-2S extranucleolar condensate are shown (magnified images). (K) HeLa cells were transiently transfected with Treacle-2S. 72-96 h after transfection, HeLa cells were processed as described in (J). (L) Graphs illustrate the quantification of the Treacle-2S FRAP analysis described in (J) and (K). Each trace represents an average of measurements for at least twenty Treacle-2S condensates; error bars represent SD.

Treacle’s phase separation is regulated by its central and C-terminal domains

(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 14 low complexity regions (LCR). (B) HeLa cells were transfected with 2S-fused full-length Treacle. 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 DNA and subsequent nucleolar cap formation. Cells were fixed and analyzed by laser scanning confocal microscopy. DNA was stained with DAPI (gray). Representative images of full-length Treacle-2S condensate are shown. (C) HeLa cells were transiently transfected 2S-fused full-length Treacle and cultivated as described in (B). FRAP analysis of 2S-fused full-length Treacle condensates dynamics was performed. Graphs illustrate quantification of FRAP analysis for each type of Treacle-formed condensates (FCs, nucleolar caps, extranucleolar condensates). Each trace represents an average of measurements for at least twenty for each type of condensates; error bars represent SD. (D) HeLa cells were transfected with 2S-fused Treacle Δ1-83 deletion mutant and processed as described in (B). (E) HeLa cells were transfected with 2S-fused Treacle Δ1-83 deletion mutant and cultivated as described in (B). FRAP analysis of 2S-fused Treacle Δ1-83 condensates dynamics was performed as described in (C). (F) HeLa cells were transfected with 2S-fused Treacle Δ83-1121 deletion mutant and processed as described in (B). (G) HeLa cells were transfected with 2S-fused Treacle Δ83-1121 deletion mutant and cultivated as described in (B). FRAP analysis of 2S-fused Treacle Δ83-1121 condensates dynamics was performed as described in (C). (H) HeLa cells were transfected with Treacle Δ1121-1488 deletion mutant fused with 2S and 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. (I) Purified recombinant Treacle underwent phase separation in the presence of 10% 1,6-hexandiol (1,6-HEX) or 500 mM sodium chloride. In each case, 10% dextran was used as a crowding agent. The size of Treacle drops was measured (n>500). (J) HeLa cells were transfected with 2S-fused full-length Treacle. 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. (K) Charge plots of full-length Treacle (top panel) and charge-scrambled Treacle (Treacle CS) form (bottom 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 the third LCR of RD for the full Treacle and Treacle CS. (L) HeLa cells were transfected with charge-scrambled 2S-fused Treacle (Treacle-2S CS) and processed as described in (B). (M) HeLa cells were transfected with Treacle-2S CS and cultivated as described in (B). FRAP analysis of Treacle-2S CS condensates dynamics was performed as described in (C). (N) HeLa cells were transfected with 2S-fused Treacle Δ1350-1488 deletion mutant (Treacle-2S ΔNOLC) and processed as described in (B). (O) HeLa cells were transfected with Treacle-2S ΔNOLC and cultivated as described in (B). FRAP analysis of Treacle-2S ΔNOLC condensates dynamics was performed as described in (C).

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 2S-fused full-length Treacle (Treacle-2S full), charge-scrambled Treacle (Treacle-2S CS) or Treacle Δ83-1121 deletion mutant (Treacle-2S Δ83-1121). Cells were fixed 16-24 after transfection and immunostained with anti-UBF 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 UBF 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 immunostained with Treacle and UBF antibodies. Cells were analyzed by laser scanning confocal microscopy. Representative images of Treacle-negative nucleolus (magnified images) are shown. (C) HeLa cells were processed as described in (A) and immunostained with Fibrillarin antibodies. (D) HeLa cells were processed as described in (B) and immunostained with Fibrillarin and UBF antibodies. Cells were analyzed by laser scanning confocal microscopy. Representative images of Treacle-negative nucleolus (magnified images) are shown. (E) HeLa cells were processed as described in (A). Cells were fixed 16-24 after transfection and subjected to cell sorting into 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 2S-fused full-length Treacle-positive cells is set to 1. Values are mean ± SD. The calculation is presented for 5 biological replicates. (F) 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. (G) HeLa cells were processed as described in (A). Cells were fixed 16-24 after transfection and stained for 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. (H) HeLa cells were processed as described in (B) and stained with UBF antibodies and smFISH. Representative images of Treacle-negative nucleolus (magnified images) are shown. (I) HeLa cells were processed as described in (A). Cells were fixed 16-24 after transfection and subjected to cell sorting into 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 2S-fused full-length Treacle-positive cells is set to 1. Values are mean ± SD. The calculation is presented for 5 biological replicates.

Treacle phase separation is essential for DDR activation in ribosomal genes under genotoxic stress conditions

(A) Control (DMSO-treated) and VP16-treated (90 μM, 30 min) HeLa cells were stained for Treacle (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. (B) Control (DMSO-treated) and VP16-treated (90 μM, 30 min) HeLa cells were subjected to 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) Сontrol (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) Endogenous Treacle was depleted by siRNA-mediated knockdown of the TCOF1 gene (Treacle kd). Next, Treacle-depleted cells were transfected with plasmid constructs encoding siRNA-resistant 2S-fused full-length Treacle, 2S-fused Treacle Δ83-1121, or 2S-fused charge-scrambled mutant Treacle (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). (E) Endogenous Treacle was depleted by siRNA-mediated knockdown of the TCOF1 gene (Treacle kd). Treacle-depleted cells were transfected with plasmid constructs encoding siRNA-resistant 2S-fused full-length Treacle, 2S-fused Treacle Δ83-1121, or 2S-fused charge-scrambled mutant Treacle (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 into 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. (F) 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 (E). (G) HeLa cells were processed as described in (E). 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.