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.
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- Act D:
ataxia telangiectasia mutated
chromosome region maintenance 1 protein homolog
external transcribed spacer
herpes virus-associated ubiquitin-specific protease
locked nucleic acid
- RNA pol I:
RNA polymerase I
upstream binding factor
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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).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Oncogene website
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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). https://doi.org/10.1038/onc.2010.469
- proteasome inhibitor
- polyadenylated RNA
- nuclear export
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