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Use of the iNo score to discriminate normal from altered nucleolar morphology, with applications in basic cell biology and potential in human disease diagnostics

Nature Protocols volume 13, pages 2387–2406 (2018)Cite this article

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Abstract

Ribosome biogenesis is initiated in the nucleolus, a cell condensate essential to gene expression, whose morphology informs cancer pathologists on the health status of a cell. Here, we describe a protocol for assessing, both qualitatively and quantitatively, the involvement of trans-acting factors in the nucleolar structure. The protocol involves use of siRNAs to deplete cells of factors of interest, fluorescence imaging of nucleoli in an automated high-throughput platform, and use of dedicated software to determine an index of nucleolar disruption, the iNo score. This scoring system is unique in that it integrates the five most discriminant shape and textural features of the nucleolus into a parametric equation. Determining the iNo score enables both qualitative and quantitative factor classification with prediction of function (functional clustering), which to our knowledge is not achieved by competing approaches, as well as stratification of their effect (severity of defects) on nucleolar structure. The iNo score has the potential to be useful in basic cell biology (nucleolar structure–function relationships, mitosis, and senescence), developmental and/or organismal biology (aging), and clinical practice (cancer, viral infection, and reproduction). The entire protocol can be completed within 1 week.

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Fig. 1: High-throughput screening pipeline for identifying factors involved in nucleolar structure maintenance.
Fig. 2: Testing the involvement of 86 abundant nucleolar proteins in nucleolar structure maintenance.
Fig. 3: Examples of validation of nucleolar morphological alterations by high-resolution imaging.

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Acknowledgements

V.S. was the recipient of a fellowship from the Fonds National de la Recherche Scientifique (F.R.S./FNRS). The De Vleeschouwer lab is supported by the Fonds National de la Recherche Scientifique (F.R.S./FNRS) and the Walloon Region (DGO6). The Lafontaine lab is supported by the Université Libre de Bruxelles (ULB), the Fonds National de la Recherche Scientifique (F.R.S./FNRS), the Walloon Region (DGO6), the Fédération Wallonie-Bruxelles, and the European Research Development Fund (ERDF) through its affiliation with the Centre for Microscopy and Molecular Imaging (CMMI). The Lafontaine Lab is also affiliated with the ULB Cancer Research Center (U-CRC).

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Authors and Affiliations

  1. Department of Biochemistry, School of Medicine, University of Patras, Patras, Greece

    Vassiliki Stamatopoulou

  2. ICTEAM-ELEN, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

    Pascaline Parisot & Christophe De Vleeschouwer

  3. RNA Molecular Biology, F.R.S./FNRS, Université Libre de Bruxelles, Charleroi-Gosselies, Belgium

    Denis L. J. Lafontaine

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  1. Vassiliki Stamatopoulou
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  2. Pascaline Parisot
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  3. Christophe De Vleeschouwer
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  4. Denis L. J. Lafontaine
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Contributions

V.S. performed the wet lab experimental work. V.S. and D.L.J.L. interpreted the data. P.P. wrote the computer script and performed the image processing analysis. C.D.V. supervised the code production and statistical analysis. D.L.J.L. designed the project, helped with code production, and integrated the data. All authors contributed to writing the manuscript.

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Correspondence to Denis L. J. Lafontaine.

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Key reference using this protocol

1. Nicolas, E. et al. Nat. Commun. 7, 11390 (2016): https://www.nature.com/articles/ncomms11390

Integrated supplementary information

Supplementary Figure 1 Analysis of the role of DDB1 and ZNF622 in pre-rRNA synthesis, pre-rRNA processing, and nucleolar stress activation.

a, b. Dynamic analysis of pre-rRNA synthesis and processing by metabolic pulse-chase labeling. Cells treated for 3 days with an siRNA targeting DDB1 a, or ZNF622 b, or with a non-targeting SCR siRNA control were labeled with tritiated methionine for 30 min and the label was then “chased”, by incubating cells with non-radioactive methionine, for the indicated times. At each time point, total RNA was extracted, resolved by denaturing agarose-gel electrophoresis and analyzed by fluorography. In order to compare RNA synthesis under different conditions, the signal was quantitated with a phosphor imager at the 0-min time point (black profile for cells treated with SCR siRNA, red profiles for DDB1-depleted and ZNF622-depleted cells). Pre-rRNA intermediates (labeled in cyan) and mature rRNAs (the 18S and 28S rRNAs, in green) are indicated. Depletion of DDB1 expression strongly influences the accumulation of the high-molecular-weight RNA species (45S, 43S, 41S). This observation confirms the PCA-based prediction that this protein is required for RNA synthesis (see also flat profile in red). By comparison, ZNF622 expression depletion influences RNA synthesis only marginally. Pre-rRNA processing remains active after depletion of either proteins (the mature rRNAs 18S and 28S are produced) but it is severely delayed after depletion of DDB1. c, In situ analysis of RNA synthesis by metabolic labeling. Cells treated for 3 days with an siRNA targeting TIF1A or DDB1 or with a non-targeting SCR siRNA control were incubated for 1 h with 5-ethynyl uridine (EU). EU-labeled RNAs were detected by chemoselective ligation (‘click’ chemistry). In cells treated with SCR, intense EU signals are detected in the DAPI-counterstained nucleoli (here the signal corresponds to robust RNA Pol I activity) and the DAPI-stained nucleoplasm (where the signal corresponds largely to transcription by RNA Pol II). In cells depleted of TIF1A or DDB1, the nucleolar signal is lost and only the nucleoplasm displays an EU signal. In control cells incubated for 2 h with α-amanitin, which inhibits RNA Pol II, the nucleoplasmic signal is lost while the nucleolar staining remains. DNA was labeled with DAPI. Scale bar: 10 µm. d, nucleolar stress activation assessed by western-blot detection of the p53 steady-state level. Three independent siRNAs (nos. 1, 2, and 3) were used to deplete the expression of target proteins DDB1 or ZNF622 for 3 days. DDB1 depletion, which severely inhibits rRNA synthesis (see panels a and c), leads to a marked activation of nucleolar stress (p53 accumulation). By contrast, ZNF622 depletion, which only marginally affects pre-rRNA synthesis and processing (panel b), does not activate nucleolar stress. As loading controls, blots were probed for β-actin.

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Stamatopoulou, V., Parisot, P., De Vleeschouwer, C. et al. Use of the iNo score to discriminate normal from altered nucleolar morphology, with applications in basic cell biology and potential in human disease diagnostics. Nat Protoc 13, 2387–2406 (2018). https://doi.org/10.1038/s41596-018-0044-3

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