Angus I. Lamond

Nucleolar proteome dynamics

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.

The multifunctional nucleolus

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.

Directed Proteomic Analysis of the Human Nucleolus

BACKGROUND The nucleolus is a subnuclear organelle containing the ribosomal RNA gene clusters and ribosome biogenesis factors. Recent studies suggest it may also have roles in RNA transport, RNA modification, and cell cycle regulation. Despite over 150 years of research into nucleoli, many aspects of their structure and function remain uncharacterized. RESULTS We report a proteomic analysis of human nucleoli. Using a combination of mass spectrometry (MS) and sequence database searches, including online analysis of the draft human genome sequence, 271 proteins were identified. Over 30% of the nucleolar proteins were encoded by novel or uncharacterized genes, while the known proteins included several unexpected factors with no previously known nucleolar functions. MS analysis of nucleoli isolated from HeLa cells in which transcription had been inhibited showed that a subset of proteins was enriched. These data highlight the dynamic nature of the nucleolar proteome and show that proteins can either associate with nucleoli transiently or accumulate only under specific metabolic conditions. CONCLUSIONS 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.

Nuclear speckles: a model for nuclear organelles

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.

Retinoic acid regulates aberrant nuclear localization of PML-RARα in acute promyelocytic leukemia cells

Acute promyelocytic leukemia (APL) is characterized by a specific t(15;17) translocation that fuses the retinoic acid receptor alpha (RAR alpha) to a novel gene product, PML. The involvement of RAR alpha is particularly intriguing in view of the efficient therapeutic effect of retinoic acid (RA) in this disease. In this report, we show that PML is specifically localized within a discrete subnuclear compartment corresponding to nuclear bodies recognized by patient autoimmune sera. In APL cells, the PML-RAR alpha hybrid displays an abnormal localization and directs RXR and other nuclear antigens into aberrant structures that are tightly bound to chromatin. This suggests that the hybrid could exert a dominant negative effect by diverting a subset of proteins from their natural sites of action. Interestingly, treatment of APL cells with RA induces a complete relocalization of each of these proteins. We propose that the beneficial role of RA in promoting myeloid differentiation in APL might be related to its ability to restore a normal subnuclear organization.

Mass spectrometry and EST-database searching allows characterization of the multi-protein spliceosome complex

Many important cell mechanisms are carried out and regulated by multi-protein complexes, for example, transcription and RNA processing machinery, receptor complexes and cytoskeletal structures. Most of these complexes remain only partially characterized due to the difficulty of conventional protein analysis methods. The rapid expansion of DNA sequence databases now provides whole or partial gene sequences of model organisms, and recent advances in protein microcharacterization via mass spectrometry allow the possibility of linking these DNA sequences to the proteins in functional complexes. This approach has been demonstrated in organisms whose genomes have been sequenced, such as budding yeast. Here we report the first characterization of an entire mammalian multi-protein complex using these methods. The machinery that removes introns from mRNA precursors — the spliceosome — is a large multi-protein complex. Approximately half of the components excised from a two-dimensional gel separation of the spliceosome were found in protein sequence databases. Using nanoelectrospray mass spectrometry, the remainder were identified and cloned using public expressed sequence tag (EST) databases. Existing EST databases are thus already sufficiently complete to allow rapid characterization of large mammalian protein complexes via mass spectrometry.

Identifying specific protein interaction partners using quantitative mass spectrometry and bead proteomes

The identification of interaction partners in protein complexes is a major goal in cell biology. Here we present a reliable affinity purification strategy to identify specific interactors that combines quantitative SILAC-based mass spectrometry with characterization of common contaminants binding to affinity matrices (bead proteomes). This strategy can be applied to affinity purification of either tagged fusion protein complexes or endogenous protein complexes, illustrated here using the well-characterized SMN complex as a model. GFP is used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, can be quantitatively depleted from cell extracts, and allows the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. Proteins binding nonspecifically to the most commonly used affinity matrices were determined using quantitative mass spectrometry, revealing important differences that affect experimental design. These data provide a specificity filter to distinguish specific protein binding partners in both quantitative and nonquantitative pull-down and immunoprecipitation experiments.

Paraspeckles : a novel nuclear domain

BACKGROUND The cell nucleus contains distinct classes of subnuclear bodies, including nucleoli, splicing speckles, Cajal bodies, gems, and PML bodies. Many nuclear proteins are known to interact dynamically with one or other of these bodies, and disruption of the specific organization of nuclear proteins can result in defects in cell functions and may cause molecular disease. RESULTS A proteomic study of purified human nucleoli has identified novel proteins, including Paraspeckle Protein 1 (PSP1) (see accompanying article, this issue of Current Biology). Here we show that PSP1 accumulates in a new nucleoplasmic compartment, termed paraspeckles, that also contains at least two other protein components: PSP2 and p54/nrb. A similar pattern of typically 10 to 20 paraspeckles was detected in all human cell types analyzed, including primary and transformed cells. Paraspeckles correspond to discrete bodies in the interchromatin nucleoplasmic space that are often located adjacent to splicing speckles. A stable cell line expressing YFP-PSP1 has been established and used to demonstrate that PSP1 interacts dynamically with nucleoli and paraspeckles in living cells. The three paraspeckle proteins relocalize quantitatively to unique cap structures at the nucleolar periphery when transcription is inhibited. CONCLUSIONS We have identified a novel nuclear compartment, termed paraspeckles, found in both primary and transformed human cells. Paraspeckles contain at least three RNA binding proteins that all interact dynamically with the nucleolus in a transcription-dependent fashion.

Regulation of mammalian spliceosome assembly by a protein phosphorylation mechanism.

Splicing of mRNA precursors (pre‐mRNA) is preceded by assembly of the pre‐mRNA with small nuclear ribonucleoprotein particles (snRNPs) and protein factors to form a splicesome. Here we show that stimulating Ser/Thr‐specific protein dephosphorylation selectively inhibits an early step during mammalian spliceosome assembly. Treatment of HeLa nuclear splicing extracts with human protein phosphatase 1 (PP1) expressed in Escherichia coli, or PP1 purified from rabbit skeletal muscle, prevents pre‐spliceosome E complex (early complex) formation and stable binding of U2 and U4/U6.U5 snRNPs to the pre‐mRNA. PP1 does not inhibit splicing catalysis if added after spliceosome assembly has taken place. Addition of purified SR protein splicing factors restores spliceosome formation and splicing to PP1‐inhibited extracts, consistent with SR proteins being targets regulated by phosphorylation. These data extend earlier observations showing that splicing catalysis, but not spliceosome assembly, is blocked by inhibiting protein phosphatases. It therefore appears that pre‐mRNA splicing, in common with other biological processes, can be regulated both positively and negatively by reversible protein phosphorylation.

Analysis of nucleolar protein dynamics reveals the nuclear degradation of ribosomal proteins.

Summary Background The nucleolus is a subnuclear organelle in which rRNAs are transcribed, processed, and assembled with ribosomal proteins into ribosome subunits. Mass spectrometry combined with pulsed incorporation of stable isotopes of arginine and lysine was used to perform a quantitative and unbiased global analysis of the rates at which newly synthesized, endogenous proteins appear within mammalian nucleoli. Results Newly synthesized ribosomal proteins accumulated in nucleoli more quickly than other nucleolar components. Studies involving time-lapse fluorescence microscopy of stable HeLa cell lines expressing fluorescent-protein-tagged nucleolar factors also showed that ribosomal proteins accumulate more quickly than other components. Photobleaching and mass-spectrometry experiments suggest that only a subset of newly synthesized ribosomal proteins are assembled into ribosomes and exported to the cytoplasm. Inhibition of the proteasome caused an accumulation of ribosomal proteins in the nucleus but not in the cytoplasm. Inhibition of rRNA transcription prior to proteasomal inhibition further increased the accumulation of ribosomal proteins in the nucleoplasm. Conclusions Ribosomal proteins are expressed at high levels beyond that required for the typical rate of ribosome-subunit production and accumulate in the nucleolus more quickly than all other nucleolar components. This is balanced by continual degradation of unassembled ribosomal proteins in the nucleoplasm, thereby providing a mechanism for mammalian cells to ensure that ribosomal protein levels are never rate limiting for the efficient assembly of ribosome subunits. The dual time-lapse strategy used in this study, combining proteomics and imaging, provides a powerful approach for the quantitative analysis of the flux of newly synthesized proteins through a cell organelle.