Utp8p Is a Nucleolar tRNA-binding Protein That Forms a Complex with Components of the Nuclear tRNA Export Machinery in Saccharomyces cerevisiae

Utp8p is an essential nucleolar component of the nuclear tRNA export machinery in Saccharomyces cerevisiae. It is thought to act at a step between tRNA maturation/aminoacylation and translocation of the tRNA across the nuclear pore complex. To understand the function of Utp8p in nuclear tRNA export, a comprehensive affinity purification analysis was conducted to identify proteins that interact with Utp8p in vivo. In addition to finding proteins that have been shown previously to copurify with Utp8p, a number of new interactions were identified. These interactions include aminoacyl-tRNA synthetases, the RanGTPase Gsp1p, and nuclear tRNA export receptors such as Los1p and Msn5p. Characterization of the interaction of Utp8p with a subset of the newly identified proteins suggests that Utp8p most likely transfer tRNAs to the nuclear tRNA export receptors by using a channeling mechanism.

Human RBM28 protein is a specific nucleolar component of the spliceosomal snRNPs

The biogenesis of spliceosomal small nuclear RNAs (snRNAs) involves organized translocations between the cytoplasm and certain nuclear domains, such as Cajal bodies and nucleoli. Here we identify human RBM28 protein as a novel snRNP component, based on affinity selection of U6 small nuclear ribonucleoprotein (snRNP). As shown by immunofluorescence, RBM28 is a nucleolar protein. Anti-RBM28 immunoprecipitation from HeLa cell lysates revealed that this protein specifically associates with U1, U2, U4, U5, and U6 snRNAs. Our data provide the first evidence that RBM28 is a common nucleolar component of the spliceosomal ribonucleoprotein complexes, possibly coordinating their transition through the nucleolus.

NO66, a Highly Conserved Dual Location Protein in the Nucleolus and in a Special Type of Synchronously Replicating Chromatin

It has recently become clear that the nucleolus, the most prominent nuclear subcompartment, harbors diverse functions beyond its classic role in ribosome biogenesis. To gain insight into nucleolar functions, we have purified amplified nucleoli from Xenopus laevis oocytes using a novel approach involving fluorescence-activated cell sorting techniques. The resulting protein fraction was analyzed by mass spectrometry and used for the generation of monoclonal antibodies directed against nucleolar components. Here, we report the identification and molecular characterization of a novel, ubiquitous protein, which in most cell types appears to be a constitutive nucleolar component. Immunolocalization studies have revealed that this protein, termed NO66, is highly conserved during evolution and shows in most cells analyzed a dual localization pattern, i.e., a strong enrichment in the granular part of nucleoli and in distinct nucleoplasmic entities. Colocalizations with proteins Ki-67, HP1α, and PCNA, respectively, have further shown that the staining pattern of NO66 overlaps with certain clusters of late replicating chromatin. Biochemical experiments have revealed that protein NO66 cofractionates with large preribosomal particles but is absent from cytoplasmic ribosomes. We propose that in addition to its role in ribosome biogenesis protein NO66 has functions in the replication or remodeling of certain heterochromatic regions.

Behaviour of the nucleolar fibrillar centres in reproducing plant cells

The nucleolar fibrillar centres have been described recently in animal cells and in meristematic plant cells, as a constant component of the nucleolus, corresponding to the nucleolar organizing region (NOR).In order to find out if the nucleolar fibrillar centres represent a general nucleolar component, we have studied the nucleolus in several reproducing plant tissues (megasporocytes and megaspore from Pissum sativum and mono and bicellulate pollen grains from Allium cepa and Scilla non-scripta). In these cells the fibrillar centres have a different morphological behaviour during their development.Female reproductive tissueImmediately before the meiosis, the megasporocyte nucleolus has a compact and rounded aspect. The fibrillar part occupies the centre and the granular part the periphery. Inside the fibrillar part, there are clear areas containing in some cases, a dark core immersed in a grey fibrillar material (Fig. 1). A similar structure was described recently in meristematic cells. In the pachytene stage, when the nucleolus is joined to the nuclear envelope, the fibrillar centres are made up of a homogeneous fibrillar material with a medium electron density.

FLUORESCENT-PROTEIN STAINING OF NUCLEOLI IN MONOLAYER CELL CULTURES: DETERMINANTS AND SIGNIFICANCE

In order to distinguish whether fluorescent-protein staining of nucleoli in acetone-fixed cell culture monolayers is a useless artifact or whether nucleolus-globulin affinity reflects a physiologic interaction between cells and serum, the phenomenon was investigated by further characterizing the interacting components and the conditions required. Staining proved to be a time-dependent and temperature-dependent, two-step procedure: initial modification of nucleoli by smaller serum γ-globulins is required for later ionic binding to nucleoli of fluorescein-labeled, nonimmune α-, β- or larger α-globulins. The initial modification is essential only in cells grown in minimal serum and harvested toward the end of the logarithmic growth phase of the culture. The nucleolar component binding the conjugate is both heat- and pH-stable, is maintained in situ apparently by ionic bonds and most likely includes a saline-insoluble, nonhistone basic protein. Previous serum modification of nucleoli in living primarily explanted monkey kidney cells, detected with this technique, could not be demonstrated with established H-Ep 2 or HeLa cell lines of human tumor origin. These data suggest that serum-dependent nucleolar fluorescent protein staining might be a useful artifact, signifying something about nucleolar organization or content of unexplained relation to cell growth.

Fluorescence Microscope and Electron-Microscope Studies on the Effect of Trenimon on Human Tumour Cells

SummaryA report is given on various forms of administration of Trenimon for local treatment of neoplasms of the serous membranes. For supervision and adjustment of the chemotherapeutic effect supravital staining with acridin orange and phase-contrast microscopy of the untreated effusion sediment are especially suitable. This facilitates distinction between newly formed and preformed granules, which is of importance for the diagnosis. Inhibition of dye-induced production of granules is a very sensitive criterium for assessment of cell vitality. The development of regressive changes in the tumour cells caused by chemotherapy is discussed with reference to micrographs of a metastatic pleural sarcomatosis. Electron-microscope studies on the exudate suggest that prior to necrobiosis an increased transfer of the granular nucleolar component into the cytoplasm takes place. This may be caused by a true increase in the transfer of nucleolar RNA towards the cytoplasm or by an inhibition in the late S-phase.

AFFINITY OF ANIMAL CELL NUCLEOLI FOR NORMAL SERUM

Nucleoli of animal cells cultured in vitro are modified by a component of "nonimmune" animal serum. Modified nucleoli bind fluorescein-conjugated nonimmune serum proteins, as shown by calcium ion-dependent fluorescence. Analysis of serum indicates that the nucleolar-binding component is a globulin, with an electrophoretic mobility in the same region as the slow alpha-1 component in pH 8.6 Veronal buffer. The component has a low sedimentation constant (2.4S), and appears to contain glycoprotein with relatively high sialic acid content (8.5%); the latter moiety may be essential to reaction with nucleoli. The nucleolar component reacting with this alpha globulin fraction appears to be a histonelike basic protein. Primary cultures of animal cells have been supported for 1 wk through attachment, spreading, and outgrowth from colonies to confluent monolayers in medium containing a nucleolar-reactive serum fraction as the only protein supplement.