Stamatopoulou, Vassiliki
[ULB]
Parisot, Pascaline
[UCL]
De Vleeschouwer, Christophe
[UCL]
Lafontaine, Denis L. J.
[ULB]
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.
- Boulon Séverine, Westman Belinda J., Hutten Saskia, Boisvert François-Michel, Lamond Angus I., The Nucleolus under Stress, 10.1016/j.molcel.2010.09.024
- Boisvert François-Michel, van Koningsbruggen Silvana, Navascués Joaquín, Lamond Angus I., The multifunctional nucleolus, 10.1038/nrm2184
- Nicolas Emilien, Parisot Pascaline, Pinto-Monteiro Celina, de Walque Roxane, De Vleeschouwer Christophe, Lafontaine Denis L. J., Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress, 10.1038/ncomms11390
- Derenzini Massimo, Montanaro Lorenzo, Treré Davide, What the nucleolus says to a tumour pathologist, 10.1111/j.1365-2559.2008.03168.x
- Salvetti Anna, Greco Anna, Viruses and the nucleolus: The fatal attraction, 10.1016/j.bbadis.2013.12.010
- Buchwalter Abigail, Hetzer Martin W., Nucleolar expansion and elevated protein translation in premature aging, 10.1038/s41467-017-00322-z
- Fulka Helena, Kyogoku Hirohisa, Zatsepina Olga, Langerova Alena, Fulka Josef, Can Nucleoli Be Markers of Developmental Potential in Human Zygotes?, 10.1016/j.molmed.2015.09.005
- Hernandez-Verdun Danièle, Roussel Pascal, Thiry Marc, Sirri Valentina, Lafontaine Denis L. J., The nucleolus: structure/function relationship in RNA metabolism : Nucleolus, 10.1002/wrna.39
- Lafontaine, D., De Vleeschouwer, C., Nicolas, E. & Parisot, P. Nucleolar structure evaluation and manipulation. European patent WO2017191187A2 (2016) (16168087.1-1408).
- Feric Marina, Vaidya Nilesh, Harmon Tyler S., Mitrea Diana M., Zhu Lian, Richardson Tiffany M., Kriwacki Richard W., Pappu Rohit V., Brangwynne Clifford P., Coexisting Liquid Phases Underlie Nucleolar Subcompartments, 10.1016/j.cell.2016.04.047
- Mitrea Diana M., Cika Jaclyn A., Stanley Christopher B., Nourse Amanda, Onuchic Paulo L., Banerjee Priya R., Phillips Aaron H., Park Cheon-Gil, Deniz Ashok A., Kriwacki Richard W., Self-interaction of NPM1 modulates multiple mechanisms of liquid–liquid phase separation, 10.1038/s41467-018-03255-3
- Thiry Marc, Lafontaine Denis L.J., Birth of a nucleolus: the evolution of nucleolar compartments, 10.1016/j.tcb.2005.02.007
- Sobecki Michal, Mrouj Karim, Camasses Alain, Parisis Nikolaos, Nicolas Emilien, Llères David, Gerbe François, Prieto Susana, Krasinska Liliana, David Alexandre, Eguren Manuel, Birling Marie-Christine, Urbach Serge, Hem Sonia, Déjardin Jérôme, Malumbres Marcos, Jay Philippe, Dulic Vjekoslav, Lafontaine Denis LJ, Feil Robert, Fisher Daniel, The cell proliferation antigen Ki-67 organises heterochromatin, 10.7554/elife.13722
- Zhu Lian, Brangwynne Clifford P, Nuclear bodies: the emerging biophysics of nucleoplasmic phases, 10.1016/j.ceb.2015.04.003
- Courchaine Edward M, Lu Alice, Neugebauer Karla M, Droplet organelles?, 10.15252/embj.201593517
- Shin Yongdae, Brangwynne Clifford P., Liquid phase condensation in cell physiology and disease, 10.1126/science.aaf4382
- Weber Stephanie C., Brangwynne Clifford P., Inverse Size Scaling of the Nucleolus by a Concentration-Dependent Phase Transition, 10.1016/j.cub.2015.01.012
- Drygin Denis, Rice William G., Grummt Ingrid, The RNA Polymerase I Transcription Machinery: An Emerging Target for the Treatment of Cancer, 10.1146/annurev.pharmtox.010909.105844
- Bywater Megan J., Poortinga Gretchen, Sanij Elaine, Hein Nadine, Peck Abigail, Cullinane Carleen, Wall Meaghan, Cluse Leonie, Drygin Denis, Anderes Kenna, Huser Nanni, Proffitt Chris, Bliesath Joshua, Haddach Mustapha, Schwaebe Michael K., Ryckman David M., Rice William G., Schmitt Clemens, Lowe Scott W., Johnstone Ricky W., Pearson Richard B., McArthur Grant A., Hannan Ross D., Inhibition of RNA Polymerase I as a Therapeutic Strategy to Promote Cancer-Specific Activation of p53, 10.1016/j.ccr.2012.05.019
- Peltonen Karita, Colis Laureen, Liu Hester, Trivedi Rishi, Moubarek Michael S., Moore Henna M., Bai Baoyan, Rudek Michelle A., Bieberich Charles J., Laiho Marikki, A Targeting Modality for Destruction of RNA Polymerase I that Possesses Anticancer Activity, 10.1016/j.ccr.2013.12.009
- McCann K. L., Baserga S. J., Mysterious Ribosomopathies, 10.1126/science.1244156
- Narla A., Ebert B. L., Translational medicine: ribosomopathies, 10.1182/blood-2011-08-372250
- De Keersmaecker K., Sulima S. O., Dinman J. D., Ribosomopathies and the paradox of cellular hypo- to hyperproliferation, 10.1182/blood-2014-10-569616
- Yelick Pamela C, Trainor Paul A, Ribosomopathies: Global process, tissue specific defects, 10.1080/21675511.2015.1025185
- Ploton D., Menager M., Jeannesson P., Himber G., Pigeon F., Adnet J. J., Improvement in the staining and in the visualization of the argyrophilic proteins of the nucleolar organizer region at the optical level, 10.1007/bf01676192
- Trerè D, AgNOR staining and quantification, 10.1016/s0968-4328(99)00069-4
- Mais C., UBF-binding site arrays form pseudo-NORs and sequester the RNA polymerase I transcription machinery, 10.1101/gad.310705
- DERENZINI MASSIMO, PLOTON DOMINIQUE, Interphase Nucleolar Organizer Regions in Cancer Cells, International Review of Experimental Pathology (1991) ISBN:9780123649324 p.149-192, 10.1016/b978-0-12-364932-4.50008-3
- Aubele, M. et al. Guidelines of AgNOR quantitation. Committee on AgNOR quantitation within the European Society of Pathology. Zentralbl. Pathol. 140, 107–108 (1994).
- Belin Stéphane, Beghin Anne, Solano-Gonzàlez Eduardo, Bezin Laurent, Brunet-Manquat Stéphanie, Textoris Julien, Prats Anne-Catherine, Mertani Hichem C., Dumontet Charles, Diaz Jean-Jacques, Dysregulation of Ribosome Biogenesis and Translational Capacity Is Associated with Tumor Progression of Human Breast Cancer Cells, 10.1371/journal.pone.0007147
- Kodiha Mohamed, Bański Piotr, Stochaj Ursula, Computer-based fluorescence quantification: a novel approach to study nucleolar biology, 10.1186/1471-2121-12-25
- Lee HweeKuan, Chong KianT, Giron DaniloM, Cheng Li, Yap ChoonK, MKalaw Emarene, Singh Malay, Huang Chao-Hui, Law YanN, Automated image based prominent nucleoli detection, 10.4103/2153-3539.159232
- Green, M. R. & Sambrook, J. Molecular Cloning: A Laboratory Manual 4th edn (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2012).
- Tafforeau Lionel, Zorbas Christiane, Langhendries Jean-Louis, Mullineux Sahra-Taylor, Stamatopoulou Vassiliki, Mullier Romain, Wacheul Ludivine, Lafontaine Denis L.J., The Complexity of Human Ribosome Biogenesis Revealed by Systematic Nucleolar Screening of Pre-rRNA Processing Factors, 10.1016/j.molcel.2013.08.011
- Ideue T., Hino K., Kitao S., Yokoi T., Hirose T., Efficient oligonucleotide-mediated degradation of nuclear noncoding RNAs in mammalian cultured cells, 10.1261/rna.1657609
- Mitrea Diana M, Cika Jaclyn A, Guy Clifford S, Ban David, Banerjee Priya R, Stanley Christopher B, Nourse Amanda, Deniz Ashok A, Kriwacki Richard W, Nucleophosmin integrates within the nucleolus via multi-modal interactions with proteins displaying R-rich linear motifs and rRNA, 10.7554/elife.13571
- Sloan Katherine E., Bohnsack Markus T., Watkins Nicholas J., The 5S RNP Couples p53 Homeostasis to Ribosome Biogenesis and Nucleolar Stress, 10.1016/j.celrep.2013.08.049
- Zhang J., Harnpicharnchai P., Jakovljevic J., Tang L., Guo Y., Oeffinger M., Rout M. P., Hiley S. L., Hughes T., Woolford J. L., Assembly factors Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes, 10.1101/gad.1569307
- Greber Basil Johannes, Gerhardy Stefan, Leitner Alexander, Leibundgut Marc, Salem Michèle, Boehringer Daniel, Leulliot Nicolas, Aebersold Ruedi, Panse Vikram Govind, Ban Nenad, Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation, 10.1016/j.cell.2015.11.027
- Angers, S. et al. Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery. Nature 443, 590–593 (2006).
- Thiry Marc, Lamaye Françoise, Thelen Nicolas, Chatron-Colliet Aurore, Lalun Nathalie, Bobichon Hélène, Ploton Dominique, A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA, 10.1038/nprot.2008.198
Bibliographic reference |
Stamatopoulou, Vassiliki ; Parisot, Pascaline ; De Vleeschouwer, Christophe ; Lafontaine, Denis L. J.. Use of the iNo score to discriminate normal from altered nucleolar morphology, with applications in basic cell biology and potential in human disease diagnostics. In: Nature Protocols, Vol. 13, no.10, p. 2387-2406 (2018) |
Permanent URL |
http://hdl.handle.net/2078.1/203723 |