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.

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.

Cells typically respond quickly to stress, altering their metabolism to compensate. In mammalian cells, stress signaling usually leads to either cell-cycle arrest or apoptosis, depending on the severity of the insult and the ability of the cell to recover. Stress also often leads to reorganization of nuclear architecture, reflecting the simultaneous inhibition of major nuclear pathways (e.g., replication and transcription) and activation of specific stress responses (e.g., DNA repair). In this review, we focus on how two nuclear organelles, the nucleolus and the Cajal body, respond to stress. The nucleolus senses stress and is a central hub for coordinating the stress response. We review nucleolar function in the stress-induced regulation of p53 and the specific changes in nucleolar morphology and composition that occur upon stress. Crosstalk between nucleoli and CBs is also discussed in the context of stress responses.

Speckles are subnuclear structures that are enriched in pre-messenger RNA splicing factors and are located in the interchromatin regions of the nucleoplasm of mammalian cells. At the fluorescence-microscope level they appear as irregular, punctate structures, which vary in size and shape, and when examined by electron microscopy they are seen as clusters of interchromatin granules. Speckles are dynamic structures, and both their protein and RNA-protein components can cycle continuously between speckles and other nuclear locations, including active transcription sites. Studies on the composition, structure and behaviour of speckles have provided a model for understanding the functional compartmentalization of the nucleus and the organization of the gene-expression machinery.

This extensive proteomic analysis shows that nucleoli have a surprisingly large protein complexity. The many novel factors and separate classes of proteins identified support the view that the nucleolus may perform additional functions beyond its known role in ribosome subunit biogenesis. The data also show that the protein composition of nucleoli is not static and can alter significantly in response to the metabolic state of the cell.

In a previous proteomic study of the human spliceosome, we identified 42 spliceosome-associated factors, including 19 novel ones. Using enhanced mass spectrometric tools and improved databases, we now report identification of 311 proteins that copurify with splicing complexes assembled on two separate pre-mRNAs. All known essential human splicing factors were found, and 96 novel proteins were identified, of which 55 contain domains directly linking them to functions in splicing/RNA processing. We also detected 20 proteins related to transcription, which indicates a direct connection between this process and splicing. This investigation provides the most detailed inventory of human spliceosome-associated factors to date, and the data indicate a number of interesting links coordinating splicing with other steps in the gene expression pathway.Biogenesis of proteins in eukaryotes is a multistep process that involves the concerted action of several complex machineries. Multiprotein complexes containing RNA polymerase II are involved in transcribing genes into pre-messenger RNA. Most human genes contain introns that are removed by splicing, a process orchestrated and catalyzed by the large multiprotein/ RNA complex termed the spliceosome. Polyadenylation of the mRNA is also catalyzed by a complex processing machinery before mRNAs are exported to the cytosol, where translation by ribosomes takes place. Although much is known about the individual processes in protein biogenesis, how the separate steps are integrated is much less clear.The spliceosome is comprised of five small nuclear RNAs (snRNAs)-U1, U2, U4, U5, and U6 snRNA-as well as many protein factors (Staley and Guthrie 1998). Some of these proteins are tightly associated with the snRNAs, forming small nuclear ribonucleoproteins (snRNPs) that are thought to assemble in a stepwise manner onto the pre-mRNA to form the spliceosome. Work over the last decade has elucidated the temporal sequence of recognition of the splice sites by the respective snRNPs and protein factors (Hastings and Krainer 2001). Interestingly, the view of stepwise assembly of the spliceosome has recently been challenged in favor of a more concerted mechanism involving preformed spliceosomes (Stevens et al. 2002). Besides the snRNP subunits, a large number of non-snRNP proteins are known, which perform various functions during the splicing reaction. For example, multiple members of the DEAD-box helicase family are thought to control RNA base-pairing interactions at different stages of spliceosome assembly and catalysis, whereas members of the SR motif family are believed to be link factors promoting protein-protein interactions during spliceosome assembly. In all, ∼100 different proteins have been linked to splicing through biochemical and/or genetic evidence (for review, see Will and Lührmann 1997). However, it remains unclear how complete this list might be.In an alternative systematic approach to the traditional characterization of single splicing factors, the spliceosome can be purified and...

Nuclear speckles, also known as interchromatin granule clusters, are nuclear domains enriched in pre-mRNA splicing factors, located in the interchromatin regions of the nucleoplasm of mammalian cells. When observed by immunofluorescence microscopy, they usually appear as 20-50 irregularly shaped structures that vary in size. Speckles are dynamic structures, and their constituents can exchange continuously with the nucleoplasm and other nuclear locations, including active transcription sites. Studies on the composition, structure, and dynamics of speckles have provided an important paradigm for understanding the functional organization of the nucleus and the dynamics of the gene expression machinery.T he mammalian cell nucleus is a highly compartmentalized yet extremely dynamic organelle (reviewed in Misteli 2001a;Spector 2006;Zhao et al. 2009). Many nuclear factors are localized in distinct structures, such as speckles, paraspeckles, nucleoli, Cajal bodies, polycomb bodies, and promyelocytic leukemia bodies and show punctate staining patterns when analyzed by indirect immunofluorescence microscopy (reviewed in Lamond et al. 1998;Spector 2001;Spector 2006).In mammalian cells the pre-mRNA splicing machinery, including small nuclear ribonucleoprotein particles (snRNPs), spliceosome subunits, and other non-snRNP protein splicing factors, shows a punctate nuclear localization pattern that is usually termed "a speckled pattern" but has also been referred to as "SC35 domains (Wansink et al. 1993)" or "splicing factor compartments (Phair et al. 2000)" (Figs. 1 and 2). The first detailed description of the nuclear domains that we presently refer to as nuclear speckles was reported by Santiago Ramó n y Cajal in 1910 (Ramó n y Cajal 1910; reviewed in Lafarga et al. 2009). Ramó n y Cajal used acid aniline stains to identify structures he referred to as "grumos hialinas" (literally "translucent clumps"). The term "speckles" was first put forth in 1961 by J. Swanson Beck (Beck 1961) upon examination of rat liver sections immunolabeled with the serum of individuals with autoimmune disorders. Although the connection was not made at the time, these speckles had been identified two years earlier by Hewson Swift (Swift 1959) at the electron microscopic level and called interchromatin particles. Swift observed that these particles were not randomly distributed but that they occurred in localized "clouds," and cytochemical analysis indicated that they contained RNA (Swift 1959). However, the first link between pre-mRNA splicing and nuclear speckles or interchromatin granule clusters came from an examination of the distribution of snRNPs using anti-splicing factor-specific antibodies, demonstrating a speckled distribution pattern of snRNPs in cell nuclei (Lerner et al. 1981;Perraud et al. 1979;Spector et al. 1983).It is now clear that much of the punctate localization of splicing factors observed by immunofluorescence microscopy corresponds to the presence of these factors in nuclear speckles of variable size and irregular shape that...

Current evidence suggests that the nucleus has a distinct substructure, albeit one that is dynamic rather than a rigid framework. Viral infection, oncogene expression, and inherited human disorders can each cause profound and specific changes in nuclear organization. This review summarizes recent progress in understanding nuclear organization, highlighting in particular the dynamic aspects of nuclear structure.First described by Brown in 1831, the cell nucleus is one of the best known but least understood of cellular organelles. The structure and functional organization of the nucleus remains a subject of energetic debate. At one extreme, the nucleus has been proposed to have its own nucleoskeleton and distinct organelles. At the other, it is viewed as a largely disordered, membranebound bag of DNA and other molecules, in which all "structures" are no more than transient complexes that form and disperse as a result of transcription, replication, and RNA processing activities in various regions of the genome. Understanding in molecular detail the organizing principles of the nucleus-including the arrangement of chromosomal DNA and how the synthesis, processing, assembly, and transport of macromolecules are coordinated and regulated-is a major goal for cell biology. Compartments of the Interphase NucleusIn the interphase nucleus, individual chromosomes occupy discrete patches referred to as chromosome territories (1), which are separated by channels called the interchromosomal domain (Fig. 1). Active genes tend to be preferentially localized to the periphery of the chromosome territories (2, 3). RNA transcripts are apparently formed preferentially at the surface of the territories and are then "shed" into interchromosomal domain channels for further processing and transport. Because the volume available to factors involved in RNA transcription and processing is thereby reduced (4), this process may enhance the assembly of large macromolecular transcription and splicing complexes.This model predicts that, relative to less active chromosomes, transcriptionally active chromosomes might have more surface area in contact with the channels. This has been confirmed for human X chromosomes: The volumes of the active and inactive X chromosomes were shown to be essentially identical (5), and the inactive X chromosome was apparently no more condensed than the active X. Instead, the inactive X, with far fewer active genes, had a much reduced surface area relative to that of its active counterpart.Active and inactive regions are interspersed along the length of the chromosomes but are segregated from one another within the chromosome territories. When highly synchronous populations of Chinese hamster fibroblasts undergoing DNA replication were pulse-labeled for short periods with halogenated deoxyribonucleotide triphosphates (dNTPs), early-replicating (R band) DNA was found largely dispersed throughout the nuclear interior, whereas later-replicating (G band) DNA was concentrated near the nuclear periphery (6). A similar arrang...