Nucleolar proteome dynamics

Abstract

The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.

Subcellular localization and nucleocytoplasmic transport of the chromosomal passenger proteins before nuclear envelope breakdown

“…Borealin also contains functional NLSs (Gassman et al, 2004) and, after translocation into the nucleus, it accumulates in the nucleolus. Although not depicted in the model, the presence of Aurora B and INCENP (but not of Survivin) in the nucleolus has also been reported (Andersen et al, 2005). INCENP modulates the nucleocytoplasmic distribution of Aurora B (probably through increased nuclear import and/or reduced nuclear export/enhanced retention of the kinase in the nucleus), but not of Survivin.…”

“…The nucleolar localization of GFP-Borealin was considered a non-physiological feature in the initial report, because the authors did not detect endogenous Borealin in this organelle using immunocytochemistry (ICC) (Gassman et al, 2004). More recently, however, a highly sensitive proteomics approach using mass spectrometry has revealed the presence of endogenous Borealin in the nucleolus (Andersen et al, 2005, see the Nucleolar Proteome Database available at http:// www.lamondlab.com/NOPdb/), suggesting that Borealin is a bona fide nucleolar protein. In fact, not only Borealin but also Aurora B and INCENP were found to copurify with human nucleoli.…”

The NEDD8 inhibitor MLN4924 increases the size of the nucleolus and activates p53 through the ribosomal-Mdm2 pathway

“…What is also evident is that the nucleolus is not a static subnuclear structure but responds to a great variety of cellular stresses. DNA damage induced by ionizing radiation, UV or chemotherapeutic drugs, inhibition of RNA Pol I transcription by nutrient starvation or Actinomycin D (ActD), proteasome inhibition and viral infection, can all change nucleolar structure and alter the nucleolar proteome 18,19,20,21,22,23 .…”

Enhanced NOLC1 promotes cell senescence and represses hepatocellular carcinoma cell proliferation by disturbing the organization of nucleolus

“…Human diploid fibroblast 2BS cells have been well characterized as a model of replicative senescence (Andersen et al ., 2005). Based on our present results in mouse tissue, we propose that the effect of CSIG on cell senescence may depend on the NOLC1–rRNA pathway.…”

“…The nucleolus is a key organelle that coordinates the synthesis of rRNA and the assembly of ribosomal subunits (Andersen et al ., 2005), which play an important role in protein metabolism (Boisvert et al ., 2007). NOLC1 was first identified as a nuclear localization signal‐binding protein that also functions as a chaperone for shuttling between the nucleolus and cytoplasm (Meier & Blobel, 1990; Meier & Blobel, 1992).…”