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: 6.56). 07/2011; 10(10):M111.009241. DOI: 10.1074/mcp.M111.009241
Source: PubMed


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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    • "There is wide variation in nucleolar response to stress and DNA damage, although it is generally considered the hub for directing the stress response [27]. For example, in response to cellular UV exposure, the nucleolus segments into nucleolar caps [55]. In our studies, the nucleolus was sensitive to even low levels of dnTRF2 expression as early as 12 hours after induction, as exhibited by nucleolar fragmentation and necklace formation. "
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    ABSTRACT: The short arms of the ten acrocentric human chromosomes share several repetitive DNAs, including ribosomal RNA genes (rDNA). The rDNA arrays correspond to nucleolar organizing regions that coalesce each cell cycle to form the nucleolus. Telomere disruption by expressing a mutant version of telomere binding protein TRF2 (dnTRF2) causes non-random acrocentric fusions, as well as large-scale nucleolar defects. The mechanisms responsible for acrocentric chromosome sensitivity to dysfunctional telomeres are unclear. In this study, we show that TRF2 normally associates with the nucleolus and rDNA. However, when telomeres are crippled by dnTRF2 or RNAi knockdown of TRF2, gross nucleolar and chromosomal changes occur. We used the controllable dnTRF2 system to precisely dissect the timing and progression of nucleolar and chromosomal instability induced by telomere dysfunction, demonstrating that nucleolar changes precede the DNA damage and morphological changes that occur at acrocentric short arms. The rDNA repeat arrays on the short arms decondense, and are coated by RNA polymerase I transcription binding factor UBF, physically linking acrocentrics to one another as they become fusogenic. These results highlight the importance of telomere function in nucleolar stability and structural integrity of acrocentric chromosomes, particularly the rDNA arrays. Telomeric stress is widely accepted to cause DNA damage at chromosome ends, but our findings suggest that it also disrupts chromosome structure beyond the telomere region, specifically within the rDNA arrays located on acrocentric chromosomes. These results have relevance for Robertsonian translocation formation in humans and mechanisms by which acrocentric-acrocentric fusions are promoted by DNA damage and repair.
    PLoS ONE 03/2014; 9(3):e92432. DOI:10.1371/journal.pone.0092432 · 3.23 Impact Factor
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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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    ABSTRACT: Sirtuins are NAD(+)-dependent protein deacetylases that connect metabolism and cellular homeostasis. Here we show that the nuclear Sirtuin SIRT7 targets PAF53, a subunit of RNA polymerase I (Pol I). Acetylation of PAF53 at lysine 373 by CBP and deacetylation by SIRT7 modulate the association of Pol I with DNA, hypoacetylation correlating with increased rDNA occupancy of Pol I and transcription activation. SIRT7 is released from nucleoli in response to different stress conditions, leading to hyperacetylation of PAF53 and decreased Pol I transcription. Nucleolar detention requires binding of SIRT7 to nascent pre-rRNA, linking the spatial distribution of SIRT7 and deacetylation of PAF53 to ongoing transcription. The results identify a nonhistone target of SIRT7 and uncover an RNA-mediated mechanism that adapts nucleolar transcription to stress signaling.
    Molecular cell 11/2013; 52(3):303-13. DOI:10.1016/j.molcel.2013.10.010 · 14.02 Impact Factor
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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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    ABSTRACT: Mature ribosomal RNAs (rRNAs) are produced from polycistronic precursors following complex processing. Precursor (pre)-rRNA processing has been extensively characterized in yeast and was assumed to be conserved in humans. We functionally characterized 625 nucleolar proteins in HeLa cells and identified 286 required for processing, including 74 without a yeast homolog. For selected candidates, we demonstrated that pre-rRNA processing defects are conserved in different cell types (including primary cells), defects are not due to activation of a p53-dependent nucleolar tumor surveillance pathway, and they precede cell-cycle arrest and apoptosis. We also investigated the exosome's role in processing internal transcribed spacers (ITSs) and report that 3' end maturation of 18S rRNA involves EXOSC10/Rrp6, a yeast ITS2 processing factor. We conclude that human cells adopt unique strategies and recruit distinct trans-acting factors to carry out essential processing steps, posing fundamental implications for understanding ribosomopathies at the molecular level and developing effective therapeutic agents.
    Molecular cell 08/2013; 51(4):539-51. DOI:10.1016/j.molcel.2013.08.011 · 14.02 Impact Factor
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