Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

NORAD-induced Pumilio phase separation is required for genome stability

Nature volume 595, pages 303–308 (2021)Cite this article

Subjects

Abstract

Liquid–liquid phase separation is a major mechanism of subcellular compartmentalization1,2. Although the segregation of RNA into phase-separated condensates broadly affects RNA metabolism3,4, whether and how specific RNAs use phase separation to regulate interacting factors such as RNA-binding proteins (RBPs), and the phenotypic consequences of such regulatory interactions, are poorly understood. Here we show that RNA-driven phase separation is a key mechanism through which a long noncoding RNA (lncRNA) controls the activity of RBPs and maintains genomic stability in mammalian cells. The lncRNA NORAD prevents aberrant mitosis by inhibiting Pumilio (PUM) proteins5,6,7,8. We show that NORAD can out-compete thousands of other PUM-binding transcripts to inhibit PUM by nucleating the formation of phase-separated PUM condensates, termed NP bodies. Dual mechanisms of PUM recruitment, involving multivalent PUM–NORAD and PUM–PUM interactions, enable NORAD to competitively sequester a super-stoichiometric amount of PUM in NP bodies. Disruption of NORAD-driven PUM phase separation leads to PUM hyperactivity and genome instability that is rescued by synthetic RNAs that induce the formation of PUM condensates. These results reveal a mechanism by which RNA-driven phase separation can regulate RBP activity and identify an essential role for this process in genome maintenance. The repetitive sequence architecture of NORAD and other lncRNAs9,10,11 suggests that phase separation may be a widely used mechanism of lncRNA-mediated regulation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: NORAD and PUM co-localize in cytoplasmic foci.
Fig. 2: NORAD induces PUM phase separation through multivalent RNA binding.
Fig. 3: Competitive, IDR-driven recruitment of PUM into NORAD–PUM condensates.
Fig. 4: PUM condensate-inducing RNAs limit PUM activity and rescue genomic instability in NORAD-deficient cells.

Similar content being viewed by others

Data availability

All data needed to evaluate the conclusions of this Article are presented in the main text or supplementary materials. RNA-seq data for circPRE-expressing HCT116 cells (Fig. 4c) is available in the GEO under accession number GSE154812. HCT116 cell RNA-seq data used to estimate the total number of PREs expressed in mRNAs per cell is available in the GEO under accession number GSE75440. Source data are provided with this paper.

References

  1. Banani, S. F., Lee, H. O., Hyman, A. A. & Rosen, M. K. Biomolecular condensates: organizers of cellular biochemistry. Nat. Rev. Mol. Cell Biol. 18, 285–298 (2017).

    Article  CAS  Google Scholar 

  2. Hyman, A. A., Weber, C. A. & Jülicher, F. Liquid–liquid phase separation in biology. Annu. Rev. Cell Dev. Biol. 30, 39–58 (2014).

    Article  CAS  Google Scholar 

  3. Corbet, G. A. & Parker, R. RNP granule formation: lessons from P-bodies and stress granules. Cold Spring Harb. Symp. Quant. Biol. 84, 203–215 (2019).

    Article  Google Scholar 

  4. Roden, C. & Gladfelter, A. S. RNA contributions to the form and function of biomolecular condensates. Nat. Rev. Mol. Cell Biol. 22, 183–195 (2021).

    Article  CAS  Google Scholar 

  5. Elguindy, M. M. et al. PUMILIO, but not RBMX, binding is required for regulation of genomic stability by noncoding RNA NORAD. eLife 8, e48625 (2019).

    Article  CAS  Google Scholar 

  6. Kopp, F. et al. PUMILIO hyperactivity drives premature aging of Norad-deficient mice. eLife 8, e42650 (2019).

    Article  Google Scholar 

  7. Lee, S. et al. Noncoding RNA NORAD regulates genomic stability by sequestering PUMILIO proteins. Cell 164, 69–80 (2016).

    Article  CAS  Google Scholar 

  8. Tichon, A. et al. A conserved abundant cytoplasmic long noncoding RNA modulates repression by Pumilio proteins in human cells. Nat. Commun. 7, 12209 (2016).

    Article  ADS  CAS  Google Scholar 

  9. Pandya-Jones, A. et al. A protein assembly mediates Xist localization and gene silencing. Nature 587, 145–151 (2020).

    Article  ADS  CAS  Google Scholar 

  10. Hacisuleyman, E., Shukla, C. J., Weiner, C. L. & Rinn, J. L. Function and evolution of local repeats in the Firre locus. Nat. Commun. 7, 11021 (2016).

    Article  ADS  CAS  Google Scholar 

  11. Yamazaki, T. et al. Functional domains of NEAT1 architectural lncRNA induce paraspeckle assembly through phase separation. Mol. Cell 70, 1038–1053.e7 (2018).

    Article  CAS  Google Scholar 

  12. Lin, Y., Protter, D. S., Rosen, M. K. & Parker, R. Formation and maturation of phase-separated liquid droplets by RNA-binding proteins. Mol. Cell 60, 208–219 (2015).

    Article  CAS  Google Scholar 

  13. Quinn, J. J. & Chang, H. Y. Unique features of long non-coding RNA biogenesis and function. Nat. Rev. Genet. 17, 47–62 (2016).

    Article  CAS  Google Scholar 

  14. Kopp, F. & Mendell, J. T. Functional classification and experimental dissection of long noncoding RNAs. Cell 172, 393–407 (2018).

    Article  CAS  Google Scholar 

  15. Yao, R. W., Wang, Y. & Chen, L. L. Cellular functions of long noncoding RNAs. Nat. Cell Biol. 21, 542–551 (2019).

    Article  CAS  Google Scholar 

  16. Goldstrohm, A. C., Hall, T. M. T. & McKenney, K. M. Post-transcriptional regulatory functions of mammalian Pumilio proteins. Trends Genet. 34, 972–990 (2018).

    Article  CAS  Google Scholar 

  17. Khong, A. et al. The stress granule transcriptome reveals principles of mRNA accumulation in stress granules. Mol. Cell 68, 808–820.e5 (2017).

    Article  CAS  Google Scholar 

  18. Namkoong, S., Ho, A., Woo, Y. M., Kwak, H. & Lee, J. H. Systematic characterization of stress-induced RNA granulation. Mol. Cell 70, 175–187.e8 (2018).

    Article  CAS  Google Scholar 

  19. Zhang, H. et al. RNA controls polyQ protein phase transitions. Mol. Cell 60, 220–230 (2015).

    Article  CAS  Google Scholar 

  20. Edwards, T. A., Pyle, S. E., Wharton, R. P. & Aggarwal, A. K. Structure of Pumilio reveals similarity between RNA and peptide binding motifs. Cell 105, 281–289 (2001).

    Article  CAS  Google Scholar 

  21. Litke, J. L. & Jaffrey, S. R. Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nat. Biotechnol. 37, 667–675 (2019).

    Article  CAS  Google Scholar 

  22. Gennarino, V. A. et al. A mild PUM1 mutation is associated with adult-onset ataxia, whereas haploinsufficiency causes developmental delay and seizures. Cell 172, 924–936.e11 (2018).

    Article  CAS  Google Scholar 

  23. Gennarino, V. A. et al. Pumilio1 haploinsufficiency leads to SCA1-like neurodegeneration by increasing wild-type Ataxin1 levels. Cell 160, 1087–1098 (2015).

    Article  CAS  Google Scholar 

  24. D’Amico, D. et al. The RNA-binding protein PUM2 impairs mitochondrial dynamics and mitophagy during aging. Mol. Cell 73, 775–787.e10 (2019).

    Article  Google Scholar 

  25. Klosin, A. et al. Phase separation provides a mechanism to reduce noise in cells. Science 367, 464–468 (2020).

    Article  ADS  CAS  Google Scholar 

  26. Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq. Genome Res. 21, 1160–1167 (2011).

    Article  CAS  Google Scholar 

  27. Marinov, G. K. et al. From single-cell to cell-pool transcriptomes: stochasticity in gene expression and RNA splicing. Genome Res. 24, 496–510 (2014).

    Article  CAS  Google Scholar 

  28. Shapiro, E., Biezuner, T. & Linnarsson, S. Single-cell sequencing-based technologies will revolutionize whole-organism science. Nat. Rev. Genet. 14, 618–630 (2013).

    Article  CAS  Google Scholar 

  29. Golden, R. J. et al. An Argonaute phosphorylation cycle promotes microRNA-mediated silencing. Nature 542, 197–202 (2017).

    Article  ADS  CAS  Google Scholar 

  30. Randolph, L. N., Bao, X., Zhou, C. & Lian, X. An all-in-one, Tet-On 3G inducible PiggyBac system for human pluripotent stem cells and derivatives. Sci. Rep. 7, 1549 (2017).

    Article  ADS  Google Scholar 

  31. Mito, M., Kawaguchi, T., Hirose, T. & Nakagawa, S. Simultaneous multicolor detection of RNA and proteins using super-resolution microscopy. Methods 98, 158–165 (2016).

    Article  CAS  Google Scholar 

  32. Bolte, S. & Cordelières, F. P. A guided tour into subcellular colocalization analysis in light microscopy. J. Microsc. 224, 213–232 (2006).

    Article  MathSciNet  CAS  Google Scholar 

  33. Costes, S. V. et al. Automatic and quantitative measurement of protein-protein colocalization in live cells. Biophys. J. 86, 3993–4003 (2004).

    Article  ADS  CAS  Google Scholar 

  34. Wheeler, J. R., Matheny, T., Jain, S., Abrisch, R. & Parker, R. Distinct stages in stress granule assembly and disassembly. eLife 5, e18413 (2016).

    Article  Google Scholar 

  35. Frankish, A. et al. GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res. 47, D766–D773 (2019).

    Article  CAS  Google Scholar 

  36. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  Google Scholar 

  37. Liao, Y., Smyth, G. K. & Shi, W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 30, 923–930 (2014).

    Article  CAS  Google Scholar 

  38. McCarthy, D. J., Chen, Y. & Smyth, G. K. Differential expression analysis of multifactor RNA-seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012).

    Article  CAS  Google Scholar 

  39. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank M. Rosen, D. Trono, S. Qi, J. Weissman, F. Zhang, X. Lian, and S. Jaffrey for plasmids; H. Zhang for bioinformatics support; S. Nakagawa for technical assistance with RNA FISH; M. Rosen, W. Peeples, J. Han, and B. Sabari for discussions; K. Jancynzka for experimental assistance; A. Mobley and UTSW Flow Cytometry Core for assistance with FACS; and K. O’Donnell, Y. Meleis, and members of the Mendell laboratory for comments on the manuscript. This work was supported by grants from the NIH (R35CA197311 to J.T.M.; P30CA142543 to J.T.M.; and P50CA196516 to J.T.M.) and the Welch Foundation (I-1961 to J.T.M.). J.T.M. is an Investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Mahmoud M. Elguindy & Joshua T. Mendell

  2. Medical Scientist Training Program, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Mahmoud M. Elguindy

  3. Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Joshua T. Mendell

  4. Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Joshua T. Mendell

  5. Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA

    Joshua T. Mendell

Authors
  1. Mahmoud M. Elguindy
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joshua T. Mendell
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

M.M.E. and J.T.M designed experiments. M.M.E performed experiments. M.M.E. and J.T.M. wrote the manuscript.

Corresponding author

Correspondence to Joshua T. Mendell.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature thanks Phillip A. Sharp and Igor Ulitsky for their contribution to the peer review of this work. Peer reviewer reports are available.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 NORAD and PUM co-localize in cytoplasmic condensates.

a, Left, confocal images of HCT116 cells co-stained for NORAD and PUM1 with or without treatment with camptothecin (Campto; 200 nM) for 24 h. Right, box plots of the mean fluorescence intensity and area of NORAD and PUM1 foci. NORAD, n = 149 foci; PUM1, n = 58 foci; NORAD + camptothecin, n = 97 foci; and PUM1 + camptothecin, n = 67 foci from at least two different fields analysed. Two-tailed t-test. b, Western blot of PUM1 or PUM2 in HCT116 cells of the indicated genotypes. Molecular weight in kDa shown on right. For gel source data, see Supplementary Fig. 1. c, Confocal images of PUM1 or PUM2 co-stained with P-body marker XRN1. d, Confocal images of PUM2 and stress granule marker G3BP1 with or without treatment with sodium arsenite (0.5 mM, 1 h incubation). e, Confocal images of NORAD and PUM localization in the indicated cell lines and genotypes. BJ-5ta cells are TERT-immortalized human fibroblasts. f, Quantification of NORAD and PUM1 co-localization (n = 20 cells for each cell line). Mean co-localization shown above box plots. a, f, Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.

Source data

Extended Data Fig. 2 NP bodies are liquid-like condensates distinct from other known cytoplasmic granules.

a, PUM1 western blot in HCT116 clones with GFP knock-in at the endogenous PUM1 locus. Three genotype-confirmed wild-type and NORAD−/− clones shown alongside parental wild-type and NORAD−/− cells. Molecular weight in kDa shown on right. For gel source data, see Supplementary Fig. 1. b, Time-lapse images showing fusion of endogenous PUM1–GFP condensates in HCT116 cells. Scale bar, 5 μm. c, Representative 3D-reconstructed live cell confocal images used to estimate the volumes of NP bodies, P-bodies, and stress granules. Endogenously tagged PUM1–GFP HCT116 cells were used for NP body and stress granule measurements (the latter after treatment with 0.5 mM sodium arsenite for 1 h). The P-body marker DCP2–GFP was used to estimate the volume of P-bodies in HCT116 cells. d, Quantification of condensate volumes (n = 1,072 NP bodies from 20 cells, 51 P-bodies from 11 cells, and 62 stress granules from 10 cells). Average volumes shown above plots.

Source data

Extended Data Fig. 3 Liquid-like properties of PUM droplets in vitro.

a, Predicted disordered regions of PUM1 and PUM2, scored by PONDR VSL2. b, Schematic (top) and Coomassie stain (bottom) of purified MBP–SNAP-PUM–His proteins used for in vitro experiments. For gel source data, see Supplementary Fig. 1. c, PUM liquid droplet formation at 5 μM upon TEV-mediated cleavage of the solubilizing MBP tag. Proteins were fluorescently labelled with SNAP488 and visualized by DIC or fluorescence microscopy. Scale bar, 10 μm. d, PUM1 liquid droplet formation at 5 μM in the presence or absence of 10% PEG3350 after 1 h incubation. e, Confocal images (left) and quantification (right) of PUM1 droplet FRAP (5 μM PUM1). Fluorescence intensities plotted relative to pre-bleach time point (t = −5 s). Data shown as mean ± s.d. (n = 3 droplets). f, Time-lapse confocal images showing fusion of PUM1 droplets (5 μM PUM1). g, DIC and fluorescence microscopy images of Cy3-labelled NORAD RNA (2.7 nM, red) after addition to preformed PUM1 or PUM2 droplets (5 μM, green). h, Fluorescence microscopy images of PUM1 droplets (5 μM, green) formed in the presence or absence of NORAD or PREmut RNA (2.7 nM, red). PREmut contains UGU to ACA mutations in all 18 NORAD PREs, which abolish PUM binding5. i, Images and analysis of PUM1–NORAD droplet FRAP. Droplets were formed with Cy3-labelled NORAD RNA (2 nM) and SNAP488-labelled PUM1 (5 μM). Data shown as mean ± s.d. (n = 3 droplets).

Source data

Extended Data Fig. 4 NORAD-induced PUM phase separation at physiological concentrations in vitro.

a, Representative 3D-reconstructed confocal image of a PUM1-stained NORAD−/− HCT116 cell used to estimate cytoplasmic volume. The length (l), width (w), and depth (d) of the entire cell and its nucleus were measured and used to calculate the total cellular and nuclear volumes using the ellipsoid volume formula. Cytoplasmic volume for each cell was determined by subtracting nuclear volume from total volume. b, Box-and-whisker plot of measured cytoplasmic volumes (n = 20 cells). Mean cytoplasmic volume shown on the right. Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum. c, Schematic of wild-type and mutant NORAD transcripts. Location of repeated NORAD domains (ND1–ND5) indicated with grey boxes and mammalian sequence conservation shown in green (UCSC Genome Browser hg38 PhastCons track). Locations of PREs indicated with red and yellow arrowheads. PREmut transcript contains 18 UGU to ACA mutations in PREs (grey arrowheads). ND4 represents the most conserved segment of NORAD and contains 4 PREs. Figure modified from Elguindy et al.5. d, DIC and fluorescence microscopy images of PUM1 (150 nM, green) droplets in the presence or absence of NORAD (2 nM) or PEG3350.

Source data

Extended Data Fig. 5 PUM target transcripts do not induce PUM1 droplet formation in vitro at physiological concentrations.

a, Dot plot of PRE-containing mRNA copy numbers in HCT116 cells (estimated by RNA-seq; see Methods) versus the number of PREs in their 3′ UTRs. Transcripts selected for in vitro assays labelled in blue with the number of PREs in their 3′ UTRs in parentheses. Note that the selected mRNAs were also identified as PUM CLIP targets that were downregulated upon PUM overexpression7,39. b, Plot of transcript PRE number multiplied by estimated copy number, highlighting the unique combination of NORAD abundance and PRE valency compared to other PRE-containing RNAs. c, qRT–PCR validation of copy numbers of the indicated transcripts in HCT116 cells. Mean copy number is shown above each bar. Data shown as mean ± s.d. n = 3 biological replicates. d, Approximate cytoplasmic concentration of each PUM target transcript in HCT116 cells. e, Confocal images of PUM1 droplets, formed with 150 nM PUM1 plus 2 nM NORAD or increasing concentrations of the indicated in vitro transcribed 3′ UTR of each PUM target. Red boxes highlight assays performed at the estimated physiologic concentration of each transcript in HCT116 cells. The same PUM1 protein preparation was used for droplet formation assays with NORAD and 3′ UTRs.

Source data

Extended Data Fig. 6 PUM target mRNAs weakly co-localize with PUM1 foci.

a, Confocal images of HCT116 cells co-stained for the indicated RNA and PUM1 by RNA FISH and immunofluorescence, respectively. b, Quantification of the indicated PUM target mRNA and PUM1 co-localization (n = 20 cells for each RNA). Mean co-localization shown above box plots. Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum co-localization for each target mRNA.

Source data

Extended Data Fig. 7 Competitive, super-stoichiometric recruitment of PUM into RNA-induced droplets.

a, Quantification of the number of PUM1 molecules per PRE in droplets nucleated by NORAD (2 nM) or PRE8 oligonucleotide (10 nM) and the indicated concentration of PUM1 in vitro. Black line represents the mean (n = 101, 115, 113, 107, 114, and 115 droplets for each condition from left to right). b, Quantification of PUM1 partition coefficients formed with 150 nM PUM1 and 10 nM PRE8 RNA normalized to partition coefficient at 0 μM competitor RNA (PRE1 RNA). IC50 represents concentration of PRE1 RNA needed to reduce PUM1 partitioning by 50%. Data shown as mean ± s.d. n = 44 or more droplets analysed for each data point. Each PRE in the PRE8 RNA is approximately 9 times more efficient at PUM1 recruitment than a monovalent PRE.

Source data

Extended Data Fig. 8 Purification and characterization of PUM1HDmut and IDR-deletion proteins.

a, Structure of the human PUM1 HD domain (PDB: 1M8X) in complex with PRE–RNA, showing mutated residues in PUM1HDmut (pink). b, EMSA demonstrating loss of PUM1HDmut RNA binding. c, Coomassie stain of purified MBP–SNAP–PUM1HDmut–His protein used for in vitro experiments. d, Droplet formation by the indicated PUM proteins in the presence or absence of PRE8 RNA oligonucleotide. e, Coomassie stain of purified MBP–SNAP–PUM1HD-WTΔIDR–His and MBP–SNAP–PUM1HDmutΔIDR–His proteins used for in vitro experiments. Gels were cropped to remove irrelevant lanes where indicated with vertical lines. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 9 Recruitment of PUM proteins into pre-formed NORAD–PUM condensates independently of RNA binding.

a, Western blot analysis of PUM1 (left) and PUM2 (right) in HCT116 cells of the indicated genotypes transduced with lentiviruses expressing PUMWT–GFP or PUMHDmut–GFP. Molecular weight in kDa shown on right. For gel source data, see Supplementary Fig. 1. b, Time-lapse live-cell confocal images showing fusion of PUM1WT–GFP or PUM1HDmut–GFP condensates in wild-type HCT116 cells. Scale bar, 5 μm. c, d, Top, images of PUM1WT–GFP and PUM1HDmut–GFP FRAP (c) or PUM2WT–GFP and PUM2HDmut–GFP FRAP (d) in wild-type HCT116 cells. Puncta undergoing photobleaching shown in dashed boxes. Bottom, FRAP quantification with fluorescence intensities plotted relative to pre-bleach time point (t = –5 s). Data shown as mean ± s.d. (c; n = 3 puncta) or mean (d; n = 2 puncta). Scale bar, 5 μm. e, Confocal images of PUM2WT–GFP (top) and PUM2HDmut–GFP (bottom) in HCT116 cells of the indicated genotypes. f, Left, confocal images of GFP-tagged full-length or IDR-deleted PUM1WT or PUM1HDmut expressed in wild-type HCT116 cells. Right, quantification of PUM1 partition coefficients, defined as the intensity of PUM1–GFP inside condensates relative to the surrounding cytoplasm. Partition coefficients were calculated for n = 175, 116, 36, or 121 condensates from 5 different cells for each protein from left to right. Black bar depicts the mean partition coefficient. Two-tailed t-test comparing each PUM1 mutant to PUM1WT. ****P < 1 × 10−15; n.s., not significant.

Source data

Extended Data Fig. 10 NORAD expression and PUM localization in circPRE-expressing cell lines.

a, Schematic of circPRE-producing constructs21, which encode the Broccoli aptamer and 0–8 PREs. b, Copy number analysis of circPRE-transcripts in HCT116 CRISPRi cells expressing control or NORAD-targeting sgRNAs. Mean copy number in sgNORAD cell lines is shown above each bar. circPRE4-low and circPRE4-mid represent distinct cell populations sorted for different circPRE copy numbers. Data shown as mean ± s.d. n = 3 biological replicates. c, qRT–PCR analysis of NORAD expression in the indicated circPRE HCT116 CRISPRi cell lines expressing control or NORAD-targeting sgRNAs. Data shown as mean ± s.d. n = 3 technical replicates. d, e, Top, confocal images of PUM1 (d) or PUM2 (e) immunofluorescence in the indicated cell lines. Bottom, quantification of the number of PUM1 (d) or PUM2 (e) foci per cell in the indicated sgControl- or sgNORAD-infected cell lines (n = 20 cells for each cell line). Mean number of foci shown above each box plot. Middle line, median; box, 25th to 75th percentiles; whiskers, minimum to maximum.

Source data

Supplementary information

Supplementary Figure 1

Gel source data.

Reporting Summary

Supplementary Table 1

A list of oligonucleotides and antibodies used in this study.

Peer Review File

Source data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Elguindy, M.M., Mendell, J.T. NORAD-induced Pumilio phase separation is required for genome stability. Nature 595, 303–308 (2021). https://doi.org/10.1038/s41586-021-03633-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41586-021-03633-w

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing