Abstract
The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2,3,4,5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3′ end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3′ external transcribed spacer (ETS) at the dense fibrillar component–PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3′ ETS. These aberrant 3′ ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.
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Data availability
All data supporting the findings of this study are available in the manuscript, Source Data, and at https://data.mendeley.com/datasets/8fmmx5vpkt/draft?a=c1a207f4-eda5-48be-99a9-92863496c868. SHAPE-MaP data with NAI and DMS, and iCLIP–seq datasets of Flag–URB1 are available at the Gene Expression Omnibus under accessions GSE194413 and GSE196625. Source data are provided with this paper.
Code availability
Custom code used in SHAPE-MaP and iCLIP–seq analysis and the ImageJ script used in image analysis in this study are available from https://github.com/YangLab/Nucleolar-URB1-ensures-3-ETS-rRNA-removal-to-prevent-exosome--surveillance.
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Acknowledgements
We thank members of the Chen laboratory for discussions; J. Zhang, B.-Y. Zou and J.-G. Li for supporting our experiments; and the core facilities of Zhejiang University Medical Center and Liangzhu Laboratory, and Optofem Technology for technical support with STED. This study was supported by the CAS Project for Young Scientists in Basic Research (YSBR-009), the National Key R&D Program of China (2021YFA1100203), the Shanghai Municipal Commission for Science and Technology (20JC1410300), the National Natural Science Foundation of China (NSFC) (31830108, 31821004, and 31725009), the Center for Excellence in Molecular Cell Science (CEMCS) (2020DF03), the HHMI International Program (55008728) and the Xplorer Prize to L.-L.C.
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L.-L.C. supervised and conceived the project. L.-L.C., L.S. and G.X. designed experiments. Y.L. performed CLEM, supervised by L. Yu. L.Z. generated Urb1−/− mice, supervised by J.L. S.-X.G. performed SILAC, supervised by C.C.L.W. P.-H.Z. performed computational analyses, supervised by L. Yang. L.S., G.X., Y.H., R.-W.Y., P.-F.L., Y.-H.P., X.G., S.L., L.-Z.Y., S.-M.C., Z.-H.Y. and Y.W. performed all other experiments and analyses, supervised by L.-L.C. L.S., G.X. and L.-L.C. drafted the manuscript. L.-L.C. edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 The schematic of pre-rRNA processing and experiment procedure of image-based nucleolar protein identification.
a. Coordination of the sub-nucleolar compartment with its function in ribosome biogenesis. b. The schematic of pre-rRNA processing in human cells. c. The workflow of live-cell imaging-based mapping of nucleolar proteins. d. Composition of nucleolar protein candidates in this paper2,7. e. Workflow of acquiring, processing and analyzing image data by Fiji/ImageJ. f. Cluster distribution of candidate nucleolar proteins. SCORENPM1 and SCOREDKC1 (see Methods for details) are shown to distinguish different nucleolar sub-regions. g. Distribution analysis procedure of candidate nucleolar proteins. h. The list of 140 proteins in different nucleolar sub-regions. i. Composition of 140 nucleolar enriched proteins from different reports2,7,10. j. Proportion of different nucleolar sub-regions (FC, DFC, PDFC, NR, GC and PNC) of the 140 nucleolar enriched proteins. k. Biological process GO-enrichment of nucleolar proteins enriched in FC, DFC, PDFC and GC. P-values are calculated by two-sided Mann-Whitney tests. l. Biological process enrichments in FC, DFC, PDFC, NR, GC and PNC. The circle areas are scaled by the number of proteins in each category.
Extended Data Fig. 2 Gallery of nucleolar protein candidates.
Representative images of all screened 200 candidate proteins by exogenous expression of EGFP tagged candidate protein in the dual-color reference knock-in cells shown in Extended Data Fig. 1c. Data was collected from n = 4 cells. Scale bar 2 μm. a. Representative images of nucleolar proteins enriched in FC. b. Representative images of nucleolar proteins enriched in PNC. c. Representative images of nucleolar proteins enriched in DFC. d. Representative images of nucleolar proteins enriched in PDFC. e. Representative images of nucleolar proteins enriched only in GC. f. Representative images of nucleolar proteins enriched in NR. g. Representative images of nucleolar proteins enriched in both GC and the nucleus. h. Representative images of nucleolar proteins enriched in both GC and the cytoplasm. i. Representative images of nucleolar proteins enriched in GC, the nucleus and the cytoplasm. j. Representative images of nucleolar proteins formed aggregations in GC. k. Representative images of protein candidates that obtained multiple distribution patterns. l. Representative images of protein candidates that did not show obvious signals in nucleoli.
Extended Data Fig. 3 Validation of nucleolar sub-regions.
a. Representative wide-field images of each individual PDFC protein in fixed HeLa cells with DKC1/NPM1 as markers of DFC/GC, respectively. Data was collected from n = 4 cells. Scale bar 2 μm. b. CLEM images of EGFP-KI URB1. Left, the merged image of EGFP-URB1 acquired by light microscopy and correlative nucleolus acquired by TEM. Right, the zoomed-in image from the white square. c. Validation of PDFC in H9 cells. Representative SIM image of PDFC (URB1) and DFC (FBL) by direct anti-URB1 and anti-FBL IF in H9 cells. d. Validation of PDFC in different cell types. Representative wide-field images of PDFC marker proteins URB1 and URB2 in K562, U2OS and HEK293 cells by exogenous expression of EGFP-tagged individual candidate proteins, mRuby3-DKC1and mTagBFP2-NPM1. Data was collected from n = 4 cells. Scale bar 2 μm. e. Diameters of different nucleolar compartments. RPA194, DKC1, URB1, and NPM1 were used as FC, DFC, PDFC and GC marker proteins, respectively, n = 60. f. Representative wide-field images of classical and non-classical nucleolar sub-regions. Left panel, classical nucleolar sub-regions are represented by exogenous expression of UBTF (FC marker), NOP56 (DFC marker) and DDX18 (GC marker) in DKC1/NPM1-KI dual-color cells as described in Extended Data Fig. 1c. Non-classical nucleolar sub-regions are marked by exogenous expression of URB1 (PDFC marker), PTBP1 (PNC marker) and MKI67 (NR marker). Data was collected from n = 4 cells. Scale bar 2 μm. g. Validation of GC condensates in different cell types. Representative wide-field images of GC condensates in K562, U2OS and HEK293 cells by exogenous expression of EGFP-RBM4, mRuby3-DKC1and mTagBFP2-NPM1. Data was collected from n = 4 cells. Scale bar 2 μm.
Extended Data Fig. 4 Characterization of nucleolar PDFC proteins.
a. The disorder tendency of 12 PDFC proteins calculated by IUPred2A. b. The rank of PDFC proteins about the disorder sequence content. URB1 and URB2 obtain the lowest disorder sequence content among all PDFC proteins. c. Two different perspectives of the URB1 and URB2 protein structures predicted by AlphaFold2. The predicted RNA binding domains are shown in magenta. d. Representative images of PDFC (URB1) and DFC (FBL) in HeLa cells treated with FBS starvation (24 h and 48 h), or UV radiation (60J). Data was collected from n = 4 cells. Scale bar 2 μm. e. URB2 surrounds DFC under different treatments. Representative images of DKC1 (DFC), URB2 (PDFC), and NPM1 (GC) proteins under DMSO (control), ActD, cx-5461 and DRB treatments in live HeLa cells. Of note, mRuby3-DKC1/mTagBFP2-NPM1 knock-in HeLa cells were introduced with EGFP-URB2 expression, followed by different treatments (ActD: 10 ng/mL, 3 h; DRB: 50 μM, 1.5 h; CX-5461:2 μM, 1 h). f. PDFC proteins surrounds DFC under Pol I inhibition. Representative images of DFC, PDFC (DDX47, NCL and DHX9) and GC proteins under ActD treatment in live HeLa cells. Data was collected from n = 4 cells. Scale bar 2 μm. g. RT-qPCR validation of the shRNA knockdown efficiency in URB1 KD and NCL KD cells. Data are presented as mean values +/− SEM. P-values are calculated by two-tailed Student’s t-test. n = 3 independent experiments.
Extended Data Fig. 5 Generation of URB1 depleted zebrafish and mouse models.
a. The conservation analysis of URB1 between human, mouse and zebrafish. URB1 has two conserved domains in N- and C- terminus. The conserved amino acids are shown in red according to conservative degrees. b. urb1 is barely expressed in zebrafish embryos at 8-cell and 4dpf stages, as shown by WISH with either the sense (control, upper panels) or the antisense urb1 (bottom panels) probe. Scale bar 100 μm. c. The schematic of the MO targeting site and KD efficiency detecting primers in zebrafish urb1 gene locus. d. RT-PCR validation of the MO efficiency targeting urb1. The agarose gel shows the MO KD efficiency detected by PCR using detecting primers in un-injected and urb1-MO zebrafish, n = 15. The red asterisk indicated the splicing-blocked mRNA of urb1 after MO injection. e. Statistics of small head deformity with different dosages of urb1 MO in zebrafish. f. Introducing urb1 mRNA rescues the urb1 MO phenotypes in zebrafish. Representative images of zebrafish cartilages stained with alcian blue after injection of urb1 MO alone, or urb1 MO and in vitro transcribed zebrafish urb1 mRNA. Scale bar 100 μm. g. Statistics of the head size in (f). n = 45 biologically independent animals. Data are presented as mean values +/− SEM. h. The schematic of the URB1-sgRNA targeting site in zebrafish urb1 gene locus. i. The sanger sequencing result of urb1+/− zebrafish confirmed the urb1+/− zebrafish has a 17-nucleotides deletion at exon 24, which resulted in a frameshift in the following urb1−/− embryos. j. URB1 depletion results in zebrafish cranial cartilage hypoplasia. Lateral view of alcian blue stained control and urb1−/− larvae at 3dpf and 4dpf. n = 6 animals. Scale bar 100 μm. k. URB1 depletion results in zebrafish cranial cartilage malformations. Statistics of width, length and head area (illustrated on left) in control, urb1 MO-injected and urb1−/− larvae at 4dpf. n = 62 biologically independent animals. Error bars, mean ± SEM. P-values are calculated by two-tailed Student’s t-test. l. Dorsal and lateral view of alcian blue stained control, urb1+/− and urb1−/− larvae. Ratio of observed phenotypes is labeled lower right of the panel. Scale bar 100 μm. m. Statistics of the head length, head width, length/width and head size in (j). urb1−/− larvae have a retardation in various aspects detected in head development compared to control and urb1+/− larvae. n = 102 independent animals. Data are presented as mean values +/− SEM. P-values are calculated by two-tailed Student’s t-test. n. Sanger sequencing confirmed that the Urb1+/− mouse received a 2-nucleotides insertion at exon 1, which resulted in a frameshift in Urb1−/− embryos. o. Statistics of blastocysts (E4.5) isolated from Urb1+/− intercrosses. p. URB1 depletion impaired the colony formation ability of H9 cells. Data are presented as mean values +/− SEM. P-values are calculated by two-tailed Student’s t-test.
Extended Data Fig. 6 URB1 depletion impairs 32S pre-rRNA biogenesis.
a. WB validation of URB1 depletion efficiency in URB1 KD cell lines. b. URB1 depletion affects pre-rRNA biogenesis. Left, schematic of human pre-rRNA processing and positions of probes used in Northern Blots (NB). Right, NB results of pre-rRNA intermediates in scramble shRNA-treated and URB1-KD HeLa cells, with probes recognizing 5′ ETS and ITS1, respectively. c. Quantification of pre-rRNA intermediates detected by different NB probes shown in (b). Each indicated rRNA intermediate is normalized to scramble shRNA-treated cells and shown as the relative intensities. n = 4 independent experiments. Data are presented as mean values +/− SEM. d. The aberrant 32S pre-rRNA intermediates contain the 5.8S rRNA region. Left, schematic representation of human 5.8S probe related rRNA intermediates in NB. Right, NB analyses of 5.8S probe related rRNA intermediates in scramble and URB1 KD HeLa cells. e. Aberrant 3′ 32S pre-rRNA takes up a majority of total 32S pre-rRNA after URB1 depletion. Left, schematic of human 28S-3′ ETS related rRNA intermediates in NB. Right, NB of 28S-3′ ETS probe related rRNA intermediates in scramble shRNA-treated and URB1 KD HeLa cells. f. URB1 depletion disrupts 32S pre-rRNA biogenesis in 293FT cells. NB analyses of ITS2 and 3′ ETS probe related rRNA intermediates in scramble shRNA-treated and URB1 KD 293FT cells. Red pentacle, aberrant intermediates. g. NB analyses of ITS2 probe related rRNA intermediates show no obvious reduction in 32S pre-rRNA and no accumulation of aberrant intermediates after URB2 KD compared to URB1 KD. Red pentacle, aberrant intermediates. h. NB analyses of rRNA intermediates in scramble shRNA-treated, DDX21 KD and DDX50 KD HeLa cells, respectively with 5′ ETS, ITS1 and ITS2 probe shows no obvious or subtle reduction in 32S pre-rRNA compared to URB1 KD.
Extended Data Fig. 7 The depletion of URB1 leads to an increase in the SHAPE value of binding region and its upstream revealed by SHAPE-MaP.
a. Schematic of primers used in SHAPE-MaP experiments. b. Predicated secondary structural modes of 28S-3′ ETS region in scramble shRNA-treated and URB1 KD HeLa cells based on NAI labeling. Red region, URB1 binding region. c. The DMS scores of 28S-3′ ETS region in scramble shRNA-treated and URB1 KD HeLa cells labeled by DMS. d. Predicated secondary structure modes in (c). Red region, URB1 binding region. e. The SHAPE scores of 28S-3′ ETS region and U8-snoRNA binding region in scramble shRNA-treated and URB1 KD HeLa cells labeled by NAI.
Extended Data Fig. 8 Examined PDFC proteins prefer to bind the 28S and 3′ end regions of pre-rRNAs.
a. Schematic of pulse-chase EU labelling experiment. b. Several examined PDFC proteins preferentially bind to the 3′ end of 28S pre-rRNA analyzed by RNA Immunoprecipitation (RIP) in HeLa cells. Left, the schematic of RIP experiments. Right, the heatmap of PDFC proteins binding to the 47S pre-rRNA. Data was collected from three independent experiments; heatmap values were generated by averaging IP/Input of each examined PDFC protein, and normalized to the highest binding capability, respectively.
Extended Data Fig. 9 URB1 depletion alters the distribution of both 3′ ETS-attached pre-rRNAs and U8 snoRNAs.
a. 3′ ETS-attached pre-rRNAs localize at the periphery of DFC in H9 cells. Top, representative single-z-section SIM images of 3′ ETS-attached pre-rRNAs (red) and DFC (FBL, green). Bottom, representative averaged images. b. U8 snoRNAs localize at the periphery of DFC. Top, representative single-z-section SIM images of U8 snoRNAs (magenta) and DFC (FBL, green) in HeLa cells. Bottom, representative single-z-section SIM images of U8 snoRNA and DFC (FBL) in H9 cells. c. URB1 KD alters the distribution of U8 snoRNAs. U8 snoRNAs (magenta) detected by smFISH were aberrantly dispersed to GC after URB1 knockdown in HeLa cells. Top, representative single-z-section SIM images of U8 snoRNAs and DFC in scramble and URB1 KD cells. Bottom, averaged images shown above, n = 20. d. Statistics of the altered distributions of U8 snoRNAs in (c). The floating bar shows the range of values, and the center line represents the median. P-values are calculated by two-sided Mann-Whitney tests. e. URB1 depletion led to reduced ribosome production in 293FT cells and HeLa cells, as shown by ribosome fractionations. The position of URB1 KD is noted by the green line and the scramble is noted by the black line. f. URB1 KD leads to reduced GC volume. Representative single z-section (upper panels) and 3D-reconstructed (bottom panels) SIM images of DKC1 (magenta) and NPM1 (blue) in scramble shRNA-treated and URB1-KD HeLa cells. White arrows show that some DFCs are extruded outside of GC. g. Statistics of the GC volume, the mean distance between DFCs and DFC numbers per cell in (f), n = 15. The floating bar shows the range of values. Data are presented as mean values +/− SEM. P-values are calculated by two-tailed Student’s t-test. (NS, not significant). h. URB1 KD led to decreased mobility of NPM1 in HeLa cells. FRAP analysis of NPM1 in scramble shRNA-treated and URB1 KD HeLa cells, n = 10. i. URB1 KD led to retarded cell proliferation in HeLa cells, revealed by MTT cell proliferation assays, n = 3. Data are presented as mean values +/− SEM. j. 3′ ETS-attached pre-rRNAs and U8 snoRNAs localize at the periphery of merged FC/DFC in zebrafish embryo. U8-snoRNAs (top, red) and 3′ ETS-attached pre-rRNAs (bottom, red) detected by smFISH surround z-fbl (blue) in zebrafish 8 hpf embryos, n = 3. Data are presented as mean values +/− SEM. k. urb1−/− larvae exhibit a less-defined midbrain-hindbrain boundaryand disorganized trunk vasculature (arrowheads) compared to siblings at 24 hpf. Scale bar 100 μm. l. mUrb1 KD impairs the radial flux processing of pre-rRNAs. 3′ ETS-attached pre-rRNAs (red) detected by 3′ ETS smFISH probes were diffused to the outside of DFC (FBL detected by IF) after mUrb1 knockdown in mouse NIH-3T3 cells, n = 20. m. mUrb1 depletion impairs the radial flux processing of pre-rRNAs in blastocyst (E4.0). Representative images of 3′ ETS-attached pre-rRNAs in control and mUrb1-KO (knockout) blastocyst by smFISH showed a diffused pattern of pre-rRNAs into GC region after mUrb1 depletion, n = 3. Scale bar 10 μm.
Extended Data Fig. 10 Loss of URB1 activates the exosome surveillance in the nucleolus.
a. The workflow of stable isotope labeling using amino acids in cell culture (SILAC) followed by mass spectrometry (MS) in scramble shRNA-treated and URB1 KD Hela cells. b. Volcano plot of the changes in protein expression upon URB1 KD. 4,688 proteins were overlapped in independent replicates. P-values are calculated by two-sided Mann-Whitney tests. c. The number of up-regulated and down-regulated proteins in URB1 KD HeLa cells. 274 proteins are up-regulated >2-fold and 170 proteins are downregulated >2-fold. d. GO/KEGG analyses of expression changed proteins upon URB1 KD in HeLa cells. Briefly, the expression levels of ribosome components (especially LSU) are down regulated and EIF4E (RNA transport factor) is up regulated. e. WB validation of the related changes in protein expression upon URB1 KD. EXOSC8 is up regulated, RPL10 and RPL23 are down regulated. f. Exosome components accumulate with 3′ ETS-attached pre-rRNAs in nucleoli upon URB1 KD. Representative images of EXOSC8, EXOSC9 and MTR4 with 3′ ETS-attached pre-rRNAs in scramble shRNA-treated and URB1 KD HeLa cells are shown by smFISH and IF. g. NB analyses of rRNA intermediates in scramble shRNA-treated, URB1, EXOSC1/3/4, EXOSC7/9/10, DIS3 and DIS3/MTR4 KD HeLa cells, respectively with ITS2 or 3′ ETS probes. Left, depletion of different exosome components led to a reduction or no change in 32S pre-rRNA expression, and no obvious aberrant intermediates were observed. Right, depletion of different exosome components did not affect 3′ ETS processing. h. Most exosome components KD rescues the abnormal exosome-dependent 32S pre-rRNA degradation. Left, the schematic of URB1 and exosome components that affect the 32S pre-rRNA degradation. Right, NB detection of the ITS2-containing pre-rRNA intermediates in scramble shRNA-treated and URB1 KD cells that were further depleted exosome components. i. RT-qPCR validation of the shRNA knockdown efficiency in cells KD of URB1 and exosome components. P-values are calculated by two-tailed Student’s t-test. j. URB1 depletion generated 3′ ETS-attached pre-rRNAs carry short poly(A) tails. NB detection using the 3′ ETS probes after oligo-dT pull-down in scramble shRNA-treated and URB1 KD HeLa cells. k. 3′ RACE analysis of pre-rRNA intermediates in scramble shRNA-treated and URB1 KD HeLa cells shows short poly(A) tails in 3′ ETS-attached pre-rRNAs.
Supplementary information
Supplementary Figure 1
The unprocessed Immunoblots associated with the data presented in main and extended data figures.
Supplementary Table 1
Oligonucleotide sequences used in this study, comprising: sheet1—primer sequences used for plasmid construction, qPCR analysis, SHAPE-MaP analysis and 3′ RACE analysis; sheet 2—sgRNA sequences used for genome editing; sheet 3—morpholino and shRNA sequences used for knockdown; and sheet 4—smFISH and northern blot probes used in this study.
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Shan, L., Xu, G., Yao, RW. et al. Nucleolar URB1 ensures 3′ ETS rRNA removal to prevent exosome surveillance. Nature 615, 526–534 (2023). https://doi.org/10.1038/s41586-023-05767-5
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DOI: https://doi.org/10.1038/s41586-023-05767-5
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