A yeast nucleolar protein related to mammalian fibrillarin is associated with small nucleolar RNA and is essential for viability.

In order to study the structural and functional organization of the eukaryotic nucleolus, we have started to isolate and characterize nucleolar components of the yeast Saccharomyces cerevisiae. We have identified a major 38 kd nucleolar protein (NOP1), which is located within nucleolar structures resembling the dense fibrillar region of mammalian nucleoli. This 38 kd protein is conserved in evolution since affinity-purified antibodies against the yeast protein stain the nucleolus of mammalian cells in indirect immunofluorescence microscopy and the yeast protein is decorated by antibodies directed against human fibrillarin. Affinity-purified antibodies against the yeast NOP1 efficiently precipitate at least seven small nuclear RNAs involved in rRNA maturation. We have cloned the gene encoding the yeast NOP1 protein. Haploid cells carrying a disrupted copy of the gene are not viable, showing that NOP1 is essential for cell growth. The gene codes for a 34.5 kd protein which contains glycine/arginine rich sequence repeats at the amino terminus similar to those found in other nucleolar proteins. This suggests that NOP1 is in association with small nucleolar RNAs, required for rRNA processing and likely to be the homologue of the mammalian fibrillarin.     

Determination of the functional domains involved in nucleolar targeting of nucleolin.

Nucleolin (713 aa), a major nucleolar protein, presents two structural domains: a N-terminus implicated in interaction with chromatin and a C-terminus containing four RNA-binding domains (RRMs) and a glycine/arginine-rich domain mainly involved in pre-rRNA packaging. Furthermore, nucleolin was shown to shuttle between cytoplasm and nucleolus. To get an insight on the nature of nuclear and nucleolar localization signals, a set of nucleolin deletion mutants in fusion with the prokaryotic chloramphenicol acetyltransferase (CAT) were constructed, and the resulting chimeric proteins were recognized by anti-CAT antibodies. First, a nuclear location signal bipartite and composed of two short basic stretches separated by eleven residues was characterized. Deletion of either motifs renders the protein cytoplasmic. Second, by deleting one or more domains implicated in nucleolin association either with DNA, RNA, or proteins, we demonstrated that nucleolar accumulation requires, in addition to the nuclear localization sequence, at least two of the five RRMs in presence or absence of N-terminus. However, in presence of only one RRM the N-terminus allowed a partial targeting of the chimeric protein to the nucleolus.     

Identification of the Ubiquitin-like Domain of Midnolin as a New Glucokinase Interaction Partner

Glucokinase acts as a glucose sensor in pancreatic beta cells. Its posttranslational regulation is important but not yet fully understood. Therefore, a pancreatic islet yeast two-hybrid library was produced and searched for glucokinase-binding proteins. A protein sequence containing a full-length ubiquitin-like domain was identified to interact with glucokinase. Mammalian two-hybrid and fluorescence resonance energy transfer analyses confirmed the interaction between glucokinase and the ubiquitin-like domain in insulin-secreting MIN6 cells and revealed the highest binding affinity at low glucose. Overexpression of parkin, an ubiquitin E3 ligase exhibiting an ubiquitin-like domain with high homology to the identified, diminished insulin secretion in MIN6 cells but had only some effect on glucokinase activity. Overexpression of the elucidated ubiquitin-like domain or midnolin, containing exactly this ubiquitin-like domain, significantly reduced both intrinsic glucokinase activity and glucose-induced insulin secretion. Midnolin has been to date classified as a nucleolar protein regulating mouse development. However, we could not confirm localization of midnolin in nucleoli. Fluorescence microscopy analyses revealed localization of midnolin in nucleus and cytoplasm and co-localization with glucokinase in pancreatic beta cells. In addition we could show that midnolin gene expression in pancreatic islets is up-regulated at low glucose and that the midnolin protein is highly expressed in pancreatic beta cells and also in liver, muscle, and brain of the adult mouse and cell lines of human and rat origin. Thus, the results of our study suggest that midnolin plays a role in cellular signaling of adult tissues and regulates glucokinase enzyme activity in pancreatic beta cells.     

Net1, a Sir2-associated nucleolar protein required for rDNA silencing and nucleolar integrity

The Sir2 protein mediates gene silencing and repression of recombination at the rDNA repeats in budding yeast. Here we show that Sir2 executes these functions as a component of a nucleolar complex designated RENT (regulator of nucleolar silencing and telophase exit). Net1, a core subunit of this complex, preferentially cross-links to the rDNA repeats, but not to silent DNA regions near telomeres or to active genes, and tethers the RENT complex to rDNA. Net1 is furthermore required for rDNA silencing and nucleolar integrity. During interphase, Net1 and Sir2 colocalize to a subdomain within the nucleous, but at the end of mitosis a fraction of Sir2 leaves the nucleolus and disperses as foci throughout the nucleus, suggesting that the structure of rDNA silent chromatin changes during the cell cycle. Our findings suggest that a protein complex shown to regulate exit from mitosis is also involved in gene silencing.     

The nucleolus and viral infection

The nucleolus is a subnuclear structure of eukaryocytes. It was thought that nucleolus only participates in the biogenesis and processing of rRNA. However, more and more evidence shows that it has many other functions, such as tRNA precursor processing, stress sensing and it is also involved in gene silencing, senescence and cell cycle regulation. Here, we summarize the recent understandings about the nucleolar functions, the regulation of nucleolar localization of proteins and the role that the nucleolus plays in virus infection, in which some related studies of Herpes simplex virus type 1 (HSV-1) US11, UL24 and bovine herpesvirus-1 infected cell protein 27 (BICP27) carried out in our lab will also be included.     

The slender lobes gene, identified by retarded mushroom body development, is required for proper nucleolar organization in Drosophila

The nucleolus dynamically alters its shape through the assembly and disassembly of a variety of nucleolar components in proliferating cells. While the nucleolus is known to function in vital cellular events, little is known about how its components are correctly assembled. Through the analysis of a Drosophila mutant that exhibits a reduced number of mushroom body (MB) neurons in the brain, we reveal that the slender lobes (sle) gene encodes a novel nuclear protein that affects nucleolar organization during development. In sle mutant neuroblasts, the nucleolus was packed more tightly, forming a dense sphere, and the nucleolar proteins fibrillarin and Nop60B were abnormally distributed in the interphase nucleolus. Moreover, another nucleolar marker, Aj1 antigen, was localized to the center of the nucleolus in a manner complementary to the Nop60B distribution, and also formed a large aggregate in the cytoplasm. While developmental defects were limited to a few tissues in sle mutants, including MBs and nurse cells, the altered organization of the nucleolar components were evident in most developing tissues. Therefore, we conclude that Sle is a general factor of nuclear architecture in Drosophila that is required for the correct organization of the nucleolus during development.     

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.     

Nuclear and nucleolar targeting sequences of c-erb-A, c-myb, N-myc, p53, HSP70, and HIV tat proteins

Protein import into the cell nucleus requires specific binding of nuclear proteins to the nuclear pore complex. Based on amino acid sequence "motifs" of known nuclear targeting signals, we identified peptides within a number of nuclear proteins with likely nuclear targeting potential and tested their function by transfecting into cells fusion genes that produce the cytoplasmic "reporter" protein, pyruvate kinase (PK), joined to the test sequence. Sequences within c-myb (PLLKKIKQ), N-myc (PPQKKIKS), p53 (PQPKKKP), and c-erb-A (SKRVAKRKL) oncoproteins that direct PK hybrids into the nucleus were identified. A peptide (GRKKRRQRRRAP) of the human immunodeficiency virus (HIV) tat protein (Tat), which contains two short basic regions, targets fusion proteins to the nucleolus. The COOH-terminal basic Tat region (QRRRAP) does not target PK hybrid proteins into the nucleus, but mutation of two basic amino acids in this region decreases but does not abolish nucleolar accumulation mediated by the entire Tat nucleolar targeting sequence. Moreover, the c-Myc nuclear targeting sequence fused to the COOH-terminal basic Tat region (PAAKRVKLDQRRRAP) effectively localizes PK hybrids to the nucleus and nucleolus. A similar sequence (FKRKHKKDISQNKRAVRR) in the human heat-shock protein HSP70 also localizes PK to the nucleus and nucleolus.     

Identification of sequences important in the nucleolar localization of human immunodeficiency virus Rev: relevance of nucleolar localization to function.

The human immunodeficiency virus rev gene product regulates the expression of viral structural genes. It was recently shown that Rev regulates the export of viral structural mRNAs from the nucleus to the cytoplasm. Analysis of Rev subcellular localization reveals marked accumulation in the nucleolus, suggesting a role for the nucleolus in this export process. We report here the identification of amino acid residues critical to the nucleolar localization of Rev. Consistent with this finding, a Rev/beta-galactosidase fusion protein, harboring this region of Rev, localized entirely within the nucleolus. Of most significance, mutations that eliminated nucleolar localization markedly diminished Rev function, even though accumulation in the nucleoplasm was retained. These findings support a model whereby Rev-induced export of human immunodeficiency virus structural mRNAs from the nucleus to the cytoplasm is likely to involve nucleolar events.     

Fibrillarin-like proteins occur in the domain Archaea.

Fibrillarin is found in the nucleolus of Eucarya and associated with small nucleolar RNAs. It is involved in the processing of precursor rRNA. Two genes, encoding fibrillarin-like proteins from Methanococcus voltae and Methanococcus vannielii, have been isolated. The genes were named flpA (fibrillarin-like protein).     

The Drosophila modifier of variegation modulo gene product binds specific RNA sequences at the nucleolus and interacts with DNA and chromatin in a phosphorylation-dependent manner

modulo belongs to the modifier of Position Effect Variegation class of Drosophila genes, suggesting a role for its product in regulating chromatin structure. Genetics assigned a second function to the gene, in protein synthesis capacity. Bifunctionality is consistent with protein localization in two distinct subnuclear compartments, chromatin and nucleolus, and with its organization in modules potentially involved in DNA and RNA binding. In this study, we examine nucleic acid interactions established by Modulo at nucleolus and chromatin and the mechanism that controls the distribution and balances the function of the protein in the two compartments. Structure/function analysis and oligomer selection/amplification experiments indicate that, in vitro, two basic terminal domains independently contact DNA without sequence specificity, whereas a central RNA Recognition Motif (RRM)-containing domain allows recognition of a novel sequence-/motif-specific RNA class. Phosphorylation moreover is shown to down-regulate DNA binding. Evidence is provided that in vivo nucleolar Modulo is highly phosphorylated and belongs to a ribonucleoprotein particle, whereas chromatin-associated protein is not modified. A functional scheme is finally proposed in which modification by phosphorylation modulates Mod subnuclear distribution and balances its function at the nucleolus and chromatin.