Quantitative Proteomics and Dynamic Imaging of the Nucleolus Reveal Distinct Responses to UV and Ionizing Radiation

Molecular Cancer Biology Program and Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland.
Molecular &amp Cellular Proteomics (Impact Factor: 7.25). 07/2011; 10(10):M111.009241. DOI: 10.1074/mcp.M111.009241
Source: PubMed

ABSTRACT The nucleolus is a nuclear organelle that coordinates rRNA transcription and ribosome subunit biogenesis. Recent proteomic analyses have shown that the nucleolus contains proteins involved in cell cycle control, DNA processing and DNA damage response and repair, in addition to the many proteins connected with ribosome subunit production. Here we study the dynamics of nucleolar protein responses in cells exposed to stress and DNA damage caused by ionizing and ultraviolet (UV) radiation in diploid human fibroblasts. We show using a combination of imaging and quantitative proteomics methods that nucleolar substructure and the nucleolar proteome undergo selective reorganization in response to UV damage. The proteomic responses to UV include alterations of functional protein complexes such as the SSU processome and exosome, and paraspeckle proteins, involving both decreases and increases in steady state protein ratios, respectively. Several nonhomologous end-joining proteins (NHEJ), such as Ku70/80, display similar fast responses to UV. In contrast, nucleolar proteomic responses to IR are both temporally and spatially distinct from those caused by UV, and more limited in terms of magnitude. With the exception of the NHEJ and paraspeckle proteins, where IR induces rapid and transient changes within 15 min of the damage, IR does not alter the ratios of most other functional nucleolar protein complexes. The rapid transient decrease of NHEJ proteins in the nucleolus indicates that it may reflect a response to DNA damage. Our results underline that the nucleolus is a specific stress response organelle that responds to different damage and stress agents in a unique, damage-specific manner.

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Available from: François-Michel Boisvert, Mar 11, 2014
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    • "Upon stress, SIRT7 is released from nucleoli into the nucleoplasm, leading to hyperacetylation of PAF53 and reduced association of PAF53/Pol I with rDNA. Relocalization of proteins and reorganization of nucleolar structure in response to environmental stimuli have been observed in many cases, indicating that resident molecules are in a constant influx and outflux (Kruhlak et al., 2007; Gorski et al., 2008; Moore et al., 2011). Significantly, the nucleolar localization and function of SIRT7 are linked to ongoing transcription. "
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    • "With the increased sensitivity of mass spectrometry techniques, this estimate was raised to $4,500 putative nucleolar proteins (Ahmad et al., 2009). The mammalian nucleolar proteome was first established under a near-physiological state, and it was later analyzed under stress conditions such as drug-induced transcriptional shutdown, adenovirus infection, and UV and ionizing irradiation (Andersen et al., 2005; Lam et al., 2010; Moore et al., 2011). These studies demonstrated the highly fluctuating nature of the nucleolar proteome , confirming the results from earlier light microscopy studies (Olson and Dundr, 2005; Phair and Misteli, 2000). "
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