Nucleolus

Abstract: The nucleolus is a distinct subnuclear compartment that was first observed more than 200 years ago. Nucleoli assemble around the tandemly repeated ribosomal DNA gene clusters and 28S, 18S and 5.8S ribosomal RNAs (rRNAs) are transcribed as a single precursor, which is processed and assembled with the 5S rRNA into ribosome subunits. Although the nucleolus is primarily associated with ribosome biogenesis, several lines of evidence now show that it has additional functions. Some of these functions, such as regulation of mitosis, cell-cycle progression and proliferation, many forms of stress response and biogenesis of multiple ribonucleoprotein particles, will be discussed, as will the relation of the nucleolus to human diseases.

Abstract: Nucleolin is a 92 kd nucleolar protein implicated in regulating polymerase I transcription and binding of preribosomal RNA. Another abundant nucleolar protein of 38 kd (B23/No38) is thought to be involved in intranuclear packaging of preribosomal particles. Although both proteins have previously been detected only in nuclei, we conclude that they shuttle constantly between nucleus and cytoplasm. This conclusion is based on monitoring the equilibration of these proteins between nuclei present in interspecies heterokaryons, and on observing the antigen-mediated nuclear accumulation of cytoplasmically injected antibodies. Our unexpected results suggest a role for these major nucleolar proteins in the nucleocytoplasmic transport of ribosomal components. Moreover, they suggest that transient exposure of shuttling proteins to the cytoplasm may provide a mechanism for cytoplasmic regulation of nuclear activities.

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.

Abstract: Results: We report a proteomic analysis of human Previous work from our own laboratories and from other nucleoli. Using a combination of mass spectrometry groups has employed proteomic methods to character(MS) and sequence database searches, including online ize the protein composition of a variety of multiprotein analysis of the draft human genome sequence, 271 pro- complexes and subcellular organelles from yeast to huteins were identified. Over 30% of the nucleolar proteins man, including U1 snRNPs [16], spliceosomes [17], and were encoded by novel or uncharacterized genes, while nuclear pore complexes [18]. the known proteins included several unexpected factors The best-studied example of a membrane-free nuclear with no previously known nucleolar functions. MS analy- subdomain is the nucleolus, a “cellular factory” in which sis of nucleoli isolated from HeLa cells in which tran- 28S, 18S, and 5.8S ribosomal RNAs (rRNAs) are transcription had been inhibited showed that a subset of scribed and together with 5S rRNA are processed and proteins was enriched. These data highlight the dynamic assembled into the ribosome subunits. The nucleolus nature of the nucleolar proteome and show that proteins is a dynamic structure that disassembles and reforms can either associate with nucleoli transiently or accumu- during each cell cycle around the rRNA gene clusters late only under specific metabolic conditions. [19]. Within the nucleolus, three distinct subcompartments have been described based on their morphology in the electron microscope. These correspond to the Conclusions: This extensive proteomic analysis shows fibrillar centers (FC), dense fibrillar components (DFC), that nucleoli have a surprisingly large protein complexand granular components (GC). The current consensus

Abstract: The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.