Skip to main content

Thank you for visiting 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.

Proteasome inhibitors induce nucleolar aggregation of proteasome target proteins and polyadenylated RNA by altering ubiquitin availability


The ubiquitin–proteasome pathway is essential for most cellular processes, including protein quality control, cell cycle, transcription, signaling, protein transport, DNA repair and stress responses. Hampered proteasome activity leads to the accumulation of polyubiquitylated proteins, endoplastic reticulum (ER) stress and even cell death. The ability of chemical proteasome inhibitors (PIs) to induce apoptosis is utilized in cancer therapy. During PI treatment, misfolded proteins accrue to cytoplasmic aggresomes. The formation of aggresome-like structures in the nucleus has remained obscure. We identify here a nucleolus-associated RNA-protein aggregate (NoA) formed by the inhibition of proteasome activity in mammalian cells. The aggregate forms within the nucleolus and is dependent on nucleolar integrity, yet is a separate structure, lacking nucleolar marker proteins, ribosomal RNA (rRNA) and rRNA synthesis activity. The NoAs contain polyadenylated RNA, conjugated ubiquitin and numerous nucleoplasmic proteasome target proteins. Several of these are key factors in oncogenesis, including transcription factors p53 and retinoblastoma protein (Rb), several cell cycle-regulating cyclins and cyclin-dependent kinases (CDKs), and stress response kinases ataxia-telangiectasia mutated (ATM) and Chk1. The aggregate formation depends on ubiquitin availability, as shown by modulating the levels of ubiquitin and deubiquitinases. Furthermore, inhibition of chromosome region maintenance 1 protein homolog (CRM1) export pathway aggravates the formation of NoAs. Taken together, we identify here a novel nuclear stress body, which forms upon proteasome inactivity within the nucleolus and is detectable in mammalian cell lines and in human tissue. These findings show that the nucleolus controls protein and RNA surveillance and export by the ubiquitin pathway in a previously unidentified manner, and provide mechanistic insight into the cellular effects of PIs.

This is a preview of subscription content

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8


Act D:

actinomycin D


ataxia telangiectasia mutated


cyclin-dependent kinase


chromosome region maintenance 1 protein homolog


endoplastic reticulum


external transcribed spacer








herpes virus-associated ubiquitin-specific protease


leptomycin B


locked nucleic acid


nucleolar aggregate




proteasome inhibitor



RNA pol I:

RNA polymerase I


polyadenylated RNA






Triton X-100


upstream binding factor


  • Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI et al. (2005). Nucleolar proteome dynamics. Nature 433: 77–83.

    CAS  Article  Google Scholar 

  • Bennett EJ, Bence NF, Jayakumar R, Kopito RR . (2005). Global impairment of the ubiquitin–proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol Cell 17: 351–365.

    CAS  Article  Google Scholar 

  • Boisvert FM, van Koningsbruggen S, Navascués J, Lamond AI . (2007). The multifunctional nucleolus. Nat Rev Mol Cell Biol 8: 574–585.

    CAS  Article  Google Scholar 

  • Boulon S, Verheggen C, Jady BE, Girard C, Pescia C, Paul C et al. (2004). PHAX and CRM1 are required sequentially to transport U3 snoRNA to nucleoli. Mol Cell 16: 777–787.

    CAS  Article  Google Scholar 

  • Carter S, Bischof O, Dejean A, Vousden KH . (2007). C-terminal modifications regulate MDM2 dissociation and nuclear export of p53. Nat Cell Biol 9: 428–435.

    CAS  Article  Google Scholar 

  • Carter S, Vousden KH . (2008). p53-Ubl fusions as models of ubiquitination, sumoylation and neddylation of p53. Cell Cycle 7: 2519–2528.

    CAS  Article  Google Scholar 

  • Chen ZJ, Sun LJ . (2009). Nonproteolytic functions of ubiquitin in cell signaling. Mol Cell 33: 275–286.

    CAS  Article  Google Scholar 

  • Cummins JM, Rago C, Kohli M, Kinzler KW, Lengauer C, Vogelstein B . (2004). Tumour suppression: disruption of HAUSP gene stabilizes p53. Nature 428: 486–487.

    Article  Google Scholar 

  • Daelemans D, Costes SV, Lockett S, Pavlakis GN . (2005). Kinetic and molecular analysis of nuclear export factor CRM1 association with its cargo in vivo. Mol Cell Biol 25: 728–739.

    CAS  Article  Google Scholar 

  • Dundr M, Misteli T, Olson MO . (2000). The dynamics of postmitotic reassembly of the nucleolus. J Cell Biol 150: 433–446.

    CAS  Article  Google Scholar 

  • Endo A, Matsumoto M, Inada T, Yamamoto A, Nakayama KI, Kitamura N et al. (2009). Nucleolar structure and function are regulated by the deubiquitylating enzyme USP36. J Cell Sci 122: 678–686.

    CAS  Article  Google Scholar 

  • Ernoult-Lange M, Wilczynska A, Harper M, Aigueperse C, Dautry F, Kress M et al. (2009). Nucleocytoplasmic traffic of CPEB1 and accumulation in Crm1 nucleolar bodies. Mol Biol Cell 20: 176–187.

    CAS  Article  Google Scholar 

  • Fatica A, Tollervey D . (2002). Making ribosomes. Curr Opin Cell Biol 14: 313–318.

    CAS  Article  Google Scholar 

  • Finley D . (2009). Recognition and processing of ubiquitin–protein conjugates by the proteasome. Annu Rev Biochem 78: 477–513.

    CAS  Article  Google Scholar 

  • Finley D, Bartel B, Varshavsky A . (1989). The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis. Nature 338: 394–401.

    CAS  Article  Google Scholar 

  • Fornerod M, van Deursen J, van Baal S, Reynolds A, Davis D, Murti KG et al. (1997). The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88. EMBO J 16: 807–816.

    CAS  Article  Google Scholar 

  • Fujii K, Kitabatake M, Sakata T, Miyata A, Ohno M . (2009). A role for ubiquitin in the clearance of nonfunctional rRNAs. Genes Dev 23: 963–974.

    CAS  Article  Google Scholar 

  • Gadal O, Strauss D, Kessl J, Trumpower B, Tollervey D, Hurt E . (2001). Nuclear export of 60s ribosomal subunits depends on Xpo1p and requires a nuclear export sequence-containing factor, Nmd3p, that associates with the large subunit protein Rpl10p. Mol Cell Biol 21: 3405–3415.

    CAS  Article  Google Scholar 

  • Haglund K, Sigismund S, Polo S, Szymkiewicz I, Di Fiore PP, Dikic I . (2003). Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation. Nat Cell Biol 5: 461–466.

    CAS  Article  Google Scholar 

  • Hernandez-Verdun D . (2006). Nucleolus: from structure to dynamics. Histochem Cell Biol 125: 127–137.

    CAS  Article  Google Scholar 

  • Hershko A, Ciechanover A . (1998). The ubiquitin system. Annu Rev Biochem 67: 425–479.

    CAS  Article  Google Scholar 

  • Ho JH, Kallstrom G, Johnson AW . (2000). Nmd3p is a Crm1p-dependent adapter protein for nuclear export of the large ribosomal subunit. J Cell Biol 151: 1057–1066.

    CAS  Article  Google Scholar 

  • Holmberg CI, Illman SA, Kallio M, Mikhailov A, Sistonen L . (2000). Formation of nuclear HSF1 granules varies depending on stress stimuli. Cell Stress Chaperones 5: 219–228.

    CAS  Article  Google Scholar 

  • Hong S, Kim SJ, Ka S, Choi I, Kang S . (2002). USP7, a ubiquitin-specific protease, interacts with ataxin-1, the SCA1 gene product. Mol Cell Neurosci 20: 298–306.

    CAS  Article  Google Scholar 

  • Houseley J, Tollervey D . (2009). The many pathways of RNA degradation. Cell 136: 763–776.

    CAS  Article  Google Scholar 

  • Hutten S, Kehlenbach RH . (2007). CRM1-mediated nuclear export: to the pore and beyond. Trends Cell Biol 17: 193–201.

    CAS  Article  Google Scholar 

  • Ikeda F, Dikic I . (2008). Atypical ubiquitin chains: new molecular signals. ‘Protein Modifications: beyond the usual suspects’ review series. EMBO Rep 9: 536–542.

    CAS  Article  Google Scholar 

  • Johnston JA, Ward CL, Kopito RR . (1998). Aggresomes: a cellular response to misfolded proteins. J Cell Biol 143: 1883–1898.

    CAS  Article  Google Scholar 

  • Karni-Schmidt O, Friedler A, Zupnick A, McKinney K, Mattia M, Beckerman R et al. (2007). Energy-dependent nucleolar localization of p53 in vitro requires two discrete regions within the p53 carboxyl terminus. Oncogene 26: 3878–3891.

    CAS  Article  Google Scholar 

  • Kisselev AF, Goldberg AL . (2001). Proteasome inhibitors: from research tools to drug candidates. Chem Biol 8: 739–758.

    CAS  Article  Google Scholar 

  • Kiviharju-af Hällström TM, Jäämaa S, Mönkkönen M, Peltonen K, Andersson LC, Medema RH et al. (2007). Human prostate epithelium lacks Wee1A-mediated DNA damage-induced checkpoint enforcement. Proc Natl Acad Sci USA 104: 7211–7216.

    Article  Google Scholar 

  • Klement IA, Skinner PJ, Kaytor MD, Yi H, Hersch SM, Clark HB et al. (1998). Ataxin-1 nuclear localization and aggregation: role in polyglutamine-induced disease in SCA1 transgenic mice. Cell 95: 41–53.

    CAS  Article  Google Scholar 

  • Klibanov SA, O'Hagan HM, Ljungman M . (2001). Accumulation of soluble and nucleolar-associated p53 proteins following cellular stress. J Cell Sci 114: 1867–1873.

    CAS  PubMed  Google Scholar 

  • Kopito RR . (2000). Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol 10: 524–530.

    CAS  Article  Google Scholar 

  • Kurki S, Peltonen K, Laiho M . (2004). Nucleophosmin, HDM2 and p53: players in UV damage incited nucleolar stress response. Cell Cycle 3: 976–979.

    CAS  Article  Google Scholar 

  • Lafontaine DL, Tollervey D . (2001). The function and synthesis of ribosomes. Nat Rev Mol Cell Biol 2: 514–520.

    CAS  Article  Google Scholar 

  • Lam YW, Lamond AI, Mann M, Andersen JS . (2007). Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins. Curr Biol 17: 749–760.

    CAS  Article  Google Scholar 

  • Latonen L, Kurki S, Pitkänen K, Laiho M . (2003). p53 and MDM2 are regulated by PI-3-kinases on multiple levels under stress induced by UV radiation and proteasome dysfunction. Cell Signal 15: 95–102.

    CAS  Article  Google Scholar 

  • Leary DJ, Huang S . (2001). Regulation of ribosome biogenesis within the nucleolus. FEBS Lett 509: 145–150.

    CAS  Article  Google Scholar 

  • Li M, Brooks CL, Wu-Baer F, Chen D, Baer R, Gu W . (2003). Mono- versus polyubiquitination: differential control of p53 fate by Mdm2. Science 302: 1972–1975.

    CAS  Article  Google Scholar 

  • Li M, Chen D, Shiloh A, Luo J, Nikolaev AY, Qin J et al. (2002). Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416: 648–653.

    CAS  Article  Google Scholar 

  • Li LB, Yu Z, Teng X, Bonini NM . (2008). RNA toxicity is a component of ataxin-3 degeneration in Drosophila. Nature 453: 1107–1111.

    CAS  Article  Google Scholar 

  • Matafora V, D'Amato A, Mori S, Blasi F, Bachi A . (2009). Proteomics analysis of nucleolar SUMO-1 target proteins upon proteasome inhibition. Mol Cell Proteomics 8: 2243–2255.

    CAS  Article  Google Scholar 

  • Matsumoto M, Hatakeyama S, Oyamada K, Oda Y, Nishimura T et al. (2005). Large-scale analysis of the human ubiquitin-related proteome. Proteomics 5: 4145–4151.

    CAS  Article  Google Scholar 

  • Mattsson K, Pokrovskaja K, Kiss C, Klein G, Szekely L . (2001). Proteins associated with the promyelocytic leukemia gene product (PML)-containing nuclear body move to the nucleolus upon inhibition of proteasome-dependent protein degradation. Proc Natl Acad Sci USA 98: 1012–1017.

    CAS  Article  Google Scholar 

  • Nagai T, Ibata K, Park ES, Kubota M, Mikoshiba K, Miyawaki A . (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat Biotechnol 20: 87–90.

    CAS  Article  Google Scholar 

  • Navon A, Ciechanover A . (2009). The 26 S proteasome: from basic mechanisms to drug targeting. J Biol Chem 284: 33713–33718.

    CAS  Article  Google Scholar 

  • Olson MO, Dundr M, Szebeni A . (2000). The nucleolus: an old factory with unexpected capabilities. Trends Cell Biol 10: 189–196.

    CAS  Article  Google Scholar 

  • Reyes-Turcu FE, Ventii KH, Wilkinson KD . (2009). Regulation and cellular roles of ubiquitin-specific deubiquitinating enzymes. Annu Rev Biochem 78: 363–397.

    CAS  Article  Google Scholar 

  • Rubbi CP, Milner J . (2003). Disruption of the nucleolus mediates stabilization of p53 in response to DNA damage and other stresses. EMBO J 22: 6068–6077.

    CAS  Article  Google Scholar 

  • Saeki Y, Kudo T, Sone T, Kikuchi Y, Yokosawa H, Toh-e A et al. (2009). Lysine 63-linked polyubiquitin chain may serve as a targeting signal for the 26S proteasome. EMBO J 28: 359–371.

    CAS  Article  Google Scholar 

  • Saudou F, Finkbeiner S, Devys D, Greenberg ME . (1998). Huntingtin acts in the nucleus to induce apoptosis but death does not correlate with the formation of intranuclear inclusions. Cell 95: 55–66.

    CAS  Article  Google Scholar 

  • Sawano A, Miyawaki A . (2000). Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis. Nucleic Acids Res 28: E78.

    CAS  Article  Google Scholar 

  • Sherr CJ, Weber JD . (2000). The ARF/p53 pathway. Curr Opin Genet Dev 10: 94–99.

    CAS  Article  Google Scholar 

  • Spence J, Gali RR, Dittmar G, Sherman F, Karin M, Finley D . (2000). Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 102: 67–76.

    CAS  Article  Google Scholar 

  • Stavreva DA, Kawasaki M, Dundr M, Koberna K, Müller WG, Tsujimura-Takahashi T et al. (2006). Potential roles for ubiquitin and the proteasome during ribosome biogenesis. Mol Cell Biol 26: 5131–5145.

    CAS  Article  Google Scholar 

  • Sudha T, Tsuji H, Sameshima M, Matsuda Y, Kaneda S, Nagai Y et al. (1995). Abnormal integrity of the nucleolus associated with cell cycle arrest owing to the temperature-sensitive ubiquitin-activating enzyme E1. Chromosome Res 3: 115–123.

    CAS  Article  Google Scholar 

  • Thomas F, Kutay U . (2003). Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway. J Cell Sci 116: 2409–2419.

    CAS  Article  Google Scholar 

  • Thomsen R, Saguez C, Nasser T, Jensen TH . (2008). General, rapid, and transcription-dependent fragmentation of nucleolar antigens in S.cerevisiae mRNA export mutants. RNA 14: 706–716.

    CAS  Article  Google Scholar 

  • Weissman AM . (2001). Themes and variations on ubiquitylation. Nat Rev Mol Cell Biol 2: 169–178.

    CAS  Article  Google Scholar 

  • Wójcik C, Schroeter D, Wilk S, Lamprecht J, Paweletz N . (1996). Ubiquitin-mediated proteolysis centers in HeLa cells: indication from studies of an inhibitor of the chymotrypsin-like activity of the proteasome. Eur J Cell Biol 71: 311–318.

    PubMed  Google Scholar 

  • Xu P, Duong DM, Seyfried NT, Cheng D, Xie Y, Robert J et al. (2009). Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation. Cell 137: 133–145.

    CAS  Article  Google Scholar 

  • Yue S, Serra HG, Zoghbi HY, Orr HT . (2001). The spinocerebellar ataxia type 1 protein, ataxin-1, has RNA-binding activity that is inversely affected by the length of its polyglutamine tract. Hum Mol Genet 10: 25–30.

    CAS  Article  Google Scholar 

Download references


We thank Drs M Olson, A Miyawaki, I Dikic, B Vogelstein and A Salminen for providing reagents and cell lines. M Salo and H Liu are thanked for excellent technical assistance. Members of Laiho lab at Helsinki and Hopkins, O Matilainen and C Holmberg are thanked for helpful discussions. University of Helsinki Molecular Imaging Unit is thanked for assistance in image acquisition. University of Helsinki Advanced Imaging Unit is thanked for assistance in TEM imaging and sample preparation. This work was supported by Academy of Finland (ML Grant No. 129699, LL Grant No. 108828), Biocentrum Helsinki and Helsinki Biomedical Graduate School (HMM).

Author information

Authors and Affiliations


Corresponding author

Correspondence to M Laiho.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Oncogene website

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Latonen, L., Moore, H., Bai, B. et al. Proteasome inhibitors induce nucleolar aggregation of proteasome target proteins and polyadenylated RNA by altering ubiquitin availability. Oncogene 30, 790–805 (2011).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • proteasome inhibitor
  • proteasome
  • ubiquitin
  • polyadenylated RNA
  • nuclear export
  • aggresome

Further reading


Quick links