Molecular Cell, Volume 76

Supplemental Information

Nascent Pre-rRNA Sorting via Phase Separation

Drives the Assembly of Dense Fibrillar Components

in the Human Nucleolus

Run-Wen Yao, Guang Xu, Ying Wang, Lin Shan, Peng-Fei Luan, Yang Wang, ManWu, Liang-Zhong Yang, Yu-Hang Xing, Li Yang, and Ling-Ling Chen

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Supplemental Information

Nascent pre-rRNA sorting via phase separation drives the assembly of dense

fibrillar components in the human nucleolus

Run-Wen Yao, Guang Xu, Ying Wang, Lin Shan, Peng-Fei Luan, Yang Wang, Man Wu, Liang-Zhong

Yang, Yu-Hang Xing, Li Yang, and Ling-Ling Chen

Supplemental Figures, S1-S7

Supplemental Table S1, title and legend

Movies S1-S5, titles and legends

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Supplemental Figures

Figure S1

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Figure S1. Alignment of 3D SIM and visualization of FC/DFC units in human cells, related to Figure 1.

(A) Alignment of 3D structured illumination microscopy (SIM). Four-color TetraSpecks at 100 nm were imaged and reconstructed by the SIM-OMX system. Reconstruction results before (top) and after (middle) the alignment are shown. Average full-width half-maximums (FWHMs) of fluorescence peaks of the TetraSpeck microspheres from images reconstructed, bottom.

(B) Cell lysates of monoclonal cell lines were immunoblotted with anti- RPA194 or FBL to confirm the CRISPR/Cas9-mediated knock-in (KI) of mEGFP-RPA194 and mEGFP-FBL in HeLa cells.

(C) Live HeLa cells were imaged by wide field microscopes (WF) with exogenous mEGFP-RPA194, mRuby3-FBL and mTagBFP2-B23. See also Figure 1C, note that SIM improves the resolution of 3 channels simultaneously.

(D) SIM achieved the same ~120 nm resolution in fixed and live cell images. Corresponding radial profile of amplitude component from the 3D FFT of reconstructed data “Fourier Transform Radial (FTR)” output from SIMcheck (Ball et al., 2015) between fixed and live cells. Amplitude is indicated in arbitrary units on the y axis, and reciprocal distance on the x axis is denoted in tenths of a micron. Red dashed line in the FTR is at 120 nm.

(E) Live cell SIM imaging for H9 cell nucleolus. Representative SIM images of H9 cells transiently expressing mEFGP-RPA194 and mRuby3-B23 (left), as well as mEGFP-FBL and mRuby3-B23 (right) are shown.

(F) Representative SIM images in fixed HEK293 (left) and H9 (right) cells. FBL was labeled with anti-FBL (green) and RPA194 (magenta) labeled with anti-RPA194.

(G) The Pol II complexes and pre-mRNA processing factors exhibit a sparse co-localization pattern. POLR2A (the largest subunit of Pol II) and four processing factors (CstF64, CPSF30, XRN2 and CPSF73) associated with pre-mRNA 3' end processing in fixed HeLa cells were imaged by SIM. Nuclei were stained by DAPI and shown in grey.

(H) RPA49 is enriched at the edge of FC at the x-y scale. Live HeLa cells with mEGFP-RPA49 expression were imaged by SIM. Pseudo-color shows relative intensity of the image.

(I) RPA194 localization is revealed by STED images. HeLa mEGFP-RPA194 KI cells were fixed for imaging and two observed patterns of RPA194 are shown. Pseudo-color shows relative intensity of the image.

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Figure S2

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Figure S2. Organization of rDNAs and pre-rRNA processing factors in the nucleolus, related to Figure 2.

(A) A representative SIM image of the actively transcribed in cis accumulated SPA1 RNA (magenta) and HP1 (green) are shown. A box plot shows that < 20% of SPA1 signals localize with HP1.

(B) Calculation of the percentage of active rDNAs per cell. A flow diagram is shown. In brief, 3D stacks of rDNA and FBL co-labeled samples were imaged by SIM. The raw SIM stacks were imported into Fiji/ImageJ. The ROI was generated by FBL channel and the integrated density of the ROI and the total regions in the rDNA channel were measured. The percentage of active rDNAs was the integrated density of ROI divided by that of the total region.

(C) A workflow to illustrate the calculation of the copy number of rDNAs per cell.

(D) Examined pre-rRNA processing factors exhibit similar distribution patterns as FBL in DFC. Representative SIM images of mEGFP-FBL and mCherry-NHP2L1, as well as mEGFP-FBL and mCherry-DKC1 double KI HeLa cells under SIM.

(E) Immunoblots show the successful KI of NHP2L1 or DKC1 in HeLa cells.

(F) Statistic analyses of the number of FBL clusters in the max-cross section of DFCs. 85 DFC regions were counted under SIM.

(G) A diagram that illustrates the image alignment and average of the cross section of FBL SIM data using Fourier transform and cross-correlation. Briefly, the registered images from 0° to 359° by 1° were rotated, and all rotated registered images and reference images were fast Fourier transformed. Then the cross-correlation peak between the reference image and each rotated registered image was calculated; a rotated image, which has the best cross-correlation peak with the reference image, was then refined in the x-y scale according to the reference image and further averaged with the original reference image to generate a new reference image for subsequent analysis.

(H) Cross-correlation of aligned and averaged images shows that the max-cross sections of individual DFC regions contain six FBL clusters. 30 max-cross sections of DFC were aligned and averaged as described in (F). The minimum distance of two clusters is ~180nm, the diameter of FBL cluster is ~133 nm; the distance between the center of individual clusters and the center of DFC is ~247.5 nm; the detached distance between two adjacent PF clusters is ~180 nm.

(I) The z-section of SIM has limited resolution (~340 nm) for the 3D-reconstruction of FBL clusters.

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Figure S3

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Figure S3. Calculation of FBL clusters in the DFC in 3D, related to Figure 2.

(A) A diagram that illustrates the process to simulate the 3D assembly pattern of FBL clusters in the DFC with the real SIM images. Briefly, the center of a given number (12-30) of FBL clusters (vertices) were chosen randomly 100 times in a polyhedral hollow sphere; and each time cross-sections with an interval of 125 nm (the SIM setting) from 200 rotated angles were scanned. All the max-cross sections were collected, numbers of clusters in each section were counted and images of the max-cross section were called from 200 different randomly rotated angles. The numbers of clusters in a total of 100*200 = 20,000 max-cross sections of each polyhedral hollow sphere were counted.

(B) Examples of computational simulation results of max-cross section FBL distribution patterns (left columns) are correlated with the real images taken by SIM (right columns).

(C) Probability value and error value are derived from the comparison of the computational simulations of all polyhedral hollow sphere models in Figures S3A and S3B with SIM images. Grey lines correspond to one randomly chosen position of a given polyhedral hollow sphere model (from 12-30 clusters) from 200 rotated angles illustrated in Figure S3A. There are 100 gray lines in each given number of clusters. The red line is the average of the 100 gray lines. The blue line is the distribution of FBL clusters observed under SIM.

(D) Comparison of polyhedral hollow sphere models with 12-30 clusters that are indicative of PF clusters with direct SIM images reveals the dodecahedron-arrangement with 20~22 aggregations are the most optimal model for PFs assembly in the DFC. Probability value (left); error value (right).

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Figure S4

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Figure S4. Define parameters used in the Metropolis Hastings model, related to Figure 2.

(A) A workflow illustrates the quantification of RPA194 and FBL molecules per cell by measuring fluorescence intensities of in vitro purified mEGFP and in vivo mEGFP KI cells under microscopy. Briefly, the standard curve of the mEGFP intensity was generated by imaging the purified mEGFP at different concentrations on a confocal microscope equipped with a hybrid (HyD) detector in photon counting mode. The endogenous RPA194 or FBL were tagged by mEGFP via CRISPR/Cas9 system and imaged by the same system with the same parameters as the acquisition of the standard curve.

(B) Purification of mEGFP from E. coli, shown by SDS-PAGE and Coomassie Blue staining.

(C) Generation of the standard curve of purified mEGFP at different concentrations and their fluorescence intensities. The mean fluorescence intensities of five concentrations of the purified mEGFP were measured as mentioned above; each concentration mEGFP was measured 6 times and the standards curve was plotted.

(D) Calculation of RPA194 and FBL molecules per FC/DFC unit using mEGFP KI cell lines and mEGFP fluorescence intensities of purified proteins. DA-KI: double allelic KI; SA-KI: single allelic KI.

(E) The parameters used in the model. The diameters of the FC region marked by RPA194 and the inner/outer diameters of the DFC region marked by FBL at the x-y scale. The average diameter of FC is 246.3 nm (n=150); the average diameters of inner or outer DFC are 362 nm and 628 nm (n=120), respectively.

(F) Schematic diagram of Metropolis Hastings model. See STAR Method for details.

(G) FBL proteins (red dots) are assembled into clusters surrounding Pol I (blue dots) in DFC, revealed by Metropolis Hastings model that searches for the most optimal distribution of processing factors for the highest binding efficiency of pre-rRNAs. See also Movie S1 and STAR Method.

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Figure S5

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Figure S5. Characterization of 47S pre-rRNA and FBL in FC/DFC unit, related to Figures 3 and 4.

(A) The SSU processome assembly proceeds in a chronological and co-transcriptional manner mainly in DFC, based on the biochemical identification, purification and characterization of multi-protein complexes (Barandun et al., 2018). The current study focuses on 5' ends of 47S pre-rRNAs sorting from their transcription sites at the border of FC/DFC to the DFC for the following SSU processome assembly.

(B) The 5' ETS-1 probe-detected 47S pre-rRNAs (red) are largely distributed in DFC; whereas the 5' ETS-2 probe-detected pre-rRNAs (blue) are mainly located with RPA194, shown by the co-localization between pre-rRNAs by RNA smFISH and mEGFP-RPA194 knock-in HeLa cells under SIM.

(C) The 5' ETS-1 probe-detected 47S pre-rRNAs (red) are largely distributed in DFC; whereas the 5' ETS-2 probe-detected pre-rRNAs (blue) are mainly located within DFC, shown by the co-localization between pre-rRNAs by RNA smFISH and mEGFP-FBL knock-in HeLa cells under SIM.

(D) Individual candidates that may have an impact on nascent 47S pre-rRNA sorting (Figure 3E) was knocked down by shRNAs. The results were detected by RT-qPCR.

(E) Validation of FBL KD by shRNA. Top, Representative images of FBL in scramble (Scr.) and FBL KD cell lines. Bottom left, the histogram showed the fluorescence intensity of FBL in scramble (Scr.) and FBL KD cell lines. Mean ± SD are shown. Bottom right, FBL knockdown was verified by WB with anti-FBL.

(F) Examination of 47S pre-rRNA processing defects by NB in FBL KD cells rescued with different FBL mutants. The normal exposure of NB gels shown in Figure 4B.

(G) Wide-Field (WF) and SIM microscopies show that both the FBL-FL and the MD domain localize at DFCs while the GAR domain localizes throughout the entire nucleolus. Cells and nuclei were marked by Lifeact or Hoechst 33342 in WF microscopy; nucleoli were marked by mRuby3-B23 under SIM.

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Figure S6

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Figure S6. Schematic flow of 5' ETS-1 sorting capability using Fiji/Image J, related to Figures 3E-3H.

Schematic flow of 5' ETS-1 sorting capability using Fiji/Image J. See STAR Method for details. Chart 1 and Chart 2 are listed in Mendeley data (https://doi.org/10.17632/k2n56cvbwd.1).

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Figure S7

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Figure S7. The IDR in FBL determines the 5' end of nascent 47S pre-rRNA trafficking towards DFC, related to Figures 5, 6 and 7.

(A) Purified fluorescent FBL and mutants (mRuby3-FBL-FL, FBL-FL-Ruby3, mNeonGreen-FBL-FL, mNeonGreen-GAR56-80-MD, mNeonGreen-MD, mNeonGreen-GAR and mRuby3-GAR) from E. coli are shown by SDS-PAGE and Coomassie Blue staining.

(B) The disorder tendency of chimeric FBL mutants is shown in pink.

(C) Phase separation of mNeonGreen-FBL-FL (7μM) and mNeonGreen-GAR56-80-MD (7μM) in the absence or presence of crowding agents (10% Dextran). The statistical results of the area of each type of droplet are shown on the right.

(D) Reduced IDR length of the GAR domain in FBL results in decreased sorting capability of 5' ETS-1 (80nM) to mNeonGreen-GAR56-80-MD droplets (7μM, 10% Dextran), compared to that being sorted to FBL-FL droplets (7μM, 10% Dextran). Of note, the total fluorescence intensity of Cy3-labeled 5' ETS-1 being captured by GAR56-80-MD droplets was markedly reduced, compared to that by FBL droplets. n=50 droplets.

(E) FRAP kinetics of chimeric FBL mutants (MD, GAR56-80-MD, GAR32-80-MD and FBL-FL) in FC/DFC units. Total 200 seconds images after bleaching were captured per 1 second, >10 cells were counted. For each time point, mean ± SD are shown.

(F) A schematic of chimeric FBL mutants with extended GAR domains. Their sequences are in supplemental Table S1.

(G) FRAP kinetics of chimeric FBL mutants (1.3xGAR-MD, 2xGAR-MD and 3xGAR-MD) in FC/DFC units. Total 200 seconds images after bleaching were captured per 1 second, >10 cells were counted. For each time point, mean ± SD are shown.

(H) A schematic of chimeric FBL mutants. ReGAR, amino acids of the GAR domain are reversed. MD: methyltransferase domain. H2B: a high-sequence complexity sequence from H2B. Their sequences are in supplemental Table S1. See also Figure S7I.

(I) The disorder tendency of chimeric FBL mutants in Figure S7H is shown in pink.

(J) FBL KD cells re-introduction with chimeric FBL mutants restored the 47S pre-rRNA localization in DFC. See Figure S7H and S7I for details of FBL mutants.

(K) FRAP kinetics of chimeric FBL mutants (ReGAR-MD, MD-GAR, MD-ReGAR, H2B-MD) in FC/DFC units. Total 200 seconds images after bleaching were captured per 1 second, >10 cells were counted. For each time point, mean ± SD are shown.

(L) Schematic of wild-type GAR (WT GAR), randomized GAR1 (rGAR1) and randomized GAR2 (rGAR2). Their sequences are in supplemental Table S1.

(M) FRAP kinetics of chimeric FBL mutants (RGG1-MD, RGG2-MD, rGAR1-MD and rGAR2-MD) in FC/DFC units. Total 200 seconds images after bleaching were captured per 1 second, >10 cells were counted. For each time point, mean ± SD are shown.

(N) Fusion of 2xmEGFP or mEGFP does not alter the mobility of MD. The kinetics of 2xmEGFP-MD and mEGFP-MD were measured by FRAP. Total 200s images after bleaching were captured per 1 second; 12 cells were counted. For each time point, mean ± SD are shown. 2xmEGFP-MD and mEGFP-MD are illustrated underneath.

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(O) Statistics of FBL and GAR56-80-MD clusters in the max-cross section of DFCs. More than 75 DFCs were counted by SIM under each condition. See also Figures 2N and 7C.

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Supplemental Table S1, related to Figures 1-7 and S1-S7. Primers, probes of smFISH and Northern blots, amino acid sequences used in this study.

Movie S1. Metropolis Hastings model that searches for the most optimal distribution of processing factors for the highest pre-rRNA processing efficiency, related to Figures 2 and S4. (A) The Metropolis Hastings simulation of the most optimal PF distribution for processing efficiency

from the initial random distribution to the end state as clusters.

(B) The 3D view of the end state of Metropolis Hastings simulation.

Movie S2. Cy3-labelled 5' ETS-1 (Magenta) is sorted to the mNeonGreen-FBL-FL droplets (Green) in vitro, related to Figure 5.

Movie S3. Cy3-labelled 5' ETS-1 sorting to mNeonGreen-FBL-FL droplets requires folded RNAs, related to Figure 5.

Movie S4. Cy3-labeled 5' ETS-1 (Magenta) cannot be captured by the droplets formed by mNeonGreen-GAR (Green) in vitro, related to Figure 5.

Movie S5. Stem-loop structure formed by 38 - 166 nt of 5' ETS-1 is crucial for its sorting to FBL-FL droplets in vitro, related to Figure 5.