Specific Role for Yeast Homologs of the Diamond Blackfan Anemia-associated Rps19 Protein in Ribosome Synthesis.

Approximately 25% of cases of Diamond Blackfan anemia, a severe hypoplastic anemia, are linked to heterozygous mutations in the gene encoding ribosomal protein S19 that result in haploinsufficiency for this protein. Here we show that deletion of either of the two genes encoding Rps19 in yeast severely affects the production of 40 S ribosomal subunits. Rps19 is an essential protein that is strictly required for maturation of the 3'-end of 18 S rRNA.

The ribosomal protein Rps15p is required for nuclear exit of the 40S subunit precursors in yeast.

We have conducted a genetic screen in order to identify ribosomal proteins of Saccharomyces cerevisiae involved in nuclear export of the small subunit precursors. This has led us to distinguish Rps15p as a protein dispensable for maturation of the pre-40S particles, but whose assembly into the pre-ribosomes is a prerequisite to their nuclear exit. Upon depletion of Rps15p, 20S pre-rRNA is released from the nucleolus and retained in the nucleus, without alteration of the pre-rRNA early cleavages.

The carboxy-terminal extension of yeast ribosomal protein S14 is necessary for maturation of 43S preribosomes.

Eukaryotic ribosomal proteins are required for production of stable ribosome assembly intermediates and mature ribosomes, but more specific roles for these proteins in biogenesis of ribosomes are not known. Here we demonstrate a particular function for yeast ribosomal protein rpS14 in late steps of 40S ribosomal subunit maturation and pre-rRNA processing. Extraordinary amounts of 43S preribosomes containing 20S pre-rRNA accumulate in the cytoplasm of certain rps14 mutants.

Sequential protein association with nascent 60S ribosomal particles.

Ribosome biogenesis in eukaryotes depends on the coordinated action of ribosomal and nonribosomal proteins that guide the assembly of preribosomal particles. These intermediate particles follow a maturation pathway in which important changes in their protein composition occur. The mechanisms involved in the coordinated assembly of the ribosomal particles are poorly understood.

The pre-ribosomal network.

Recent achievements in yeast functional proteomics have significantly advanced our knowledge about ribosome biogenesis. Here, we present a program developed to integrate data from various proteome analyses with cell biological data on components present in the ribosome producing factories. This program allows users to attribute factors to certain complexes and to specific steps of ribosome biogenesis.

Loss of OPA1 perturbates the mitochondrial inner membrane structure and integrity, leading to cytochrome c release and apoptosis.

OPA1 encodes a large GTPase related to dynamins, anchored to the mitochondrial cristae inner membrane, facing the intermembrane space. OPA1 haplo-insufficiency is responsible for the most common form of autosomal dominant optic atrophy (ADOA, MIM165500), a neuropathy resulting from degeneration of the retinal ganglion cells and optic nerve atrophy. Here we show that down-regulation of OPA1 in HeLa cells using specific small interfering RNA (siRNA) leads to fragmentation of the mitochondrial network concomitantly to the dissipation of the mitochondrial membrane potential and to a drastic disorganization of the cristae.

A Noc complex specifically involved in the formation and nuclear export of ribosomal 40 S subunits.

Formation and nuclear export of 60 S pre-ribosomes requires many factors including the heterodimeric Noc1-Noc2 and Noc2-Noc3 complexes. Here, we report another Noc complex with a specific role in 40 S subunit biogenesis. This complex consists of Noc4p, which exhibits the conserved Noc domain and is homologous to Noc1p, and Nop14p, a nucleolar protein with a role in 40 S subunit formation.

The human dynamin-related protein OPA1 is anchored to the mitochondrial inner membrane facing the inter-membrane space.

Mutations in the OPA1 gene are associated with autosomal dominant optic atrophy. OPA1 encodes a dynamin-related protein orthologous to Msp1 of Schizosaccharomyces pombe and Mgm1p of Saccharomyces cerevisiae, both involved in mitochondrial morphology and genome maintenance. We present immuno-fluorescence and biochemical evidences showing that OPA1 resides in the mitochondria where it is imported through its highly basic amino-terminal extension.

Rlp7p is associated with 60S preribosomes, restricted to the granular component of the nucleolus, and required for pre-rRNA processing.

Many analyses have examined subnucleolar structures in eukaryotic cells, but the relationship between morphological structures, pre-rRNA processing, and ribosomal particle assembly has remained unclear. Using a visual assay for export of the 60S ribosomal subunit, we isolated a ts-lethal mutation, rix9-1, which causes nucleolar accumulation of an Rpl25p-eGFP reporter construct. The mutation results in a single amino acid substitution (F176S) in Rlp7p, an essential nucleolar protein related to ribosomal protein Rpl7p.

Ultrastructural localization of rRNA shows defective nuclear export of preribosomes in mutants of the Nup82p complex.

To study the nuclear export of preribosomes, ribosomal RNAs were detected by in situ hybridization using fluorescence and EM, in the yeast Saccharomyces cerevisiae. In wild-type cells, semiquantitative analysis shows that the distributions of pre-40S and pre-60S particles in the nucleolus and the nucleoplasm are distinct, indicating uncoordinated transport of the two subunits within the nucleus. In cells defective for the activity of the GTPase Gsp1p/Ran, ribosomal precursors accumulate in the whole nucleus.

Nog2p, a putative GTPase associated with pre-60S subunits and required for late 60S maturation steps.

Eukaryotic ribosome maturation depends on a set of well ordered processing steps. Here we describe the functional characterization of yeast Nog2p (Ynr053cp), a highly conserved nuclear protein. Nog2p contains a putative GTP-binding site, which is essential in vivo.

Maturation and intranuclear transport of pre-ribosomes requires Noc proteins.

How pre-ribosomes temporally and spatially mature during intranuclear biogenesis is not known. Here, we report three nucleolar proteins, Noc1p to Noc3p, that are required for ribosome maturation and transport. They can be isolated in two distinct complexes: Noc1p/Noc2p associates with 90S and 66S pre-ribosomes and is enriched in the nucleolus, and Noc2p/Noc3p associates with 66S pre-ribosomes and is mainly nucleoplasmic.

Assembly and functional organization of the nucleolus: ultrastructural analysis of Saccharomyces cerevisiae mutants.

Using Saccharomyces cerevisiae strains with genetically modified nucleoli, we show here that changing parameters as critical as the tandem organization of the ribosomal genes and the polymerase transcribing rDNA, although profoundly modifying the position and the shape of the nucleolus, only partially alter its functional subcompartmentation. High-resolution morphology achieved by cryofixation, together with ultrastructural localization of nucleolar proteins and rRNA, reveals that the nucleolar structure, arising upon transcription of rDNA from plasmids by RNA polymerase I, is still divided in functional subcompartments like the wild-type nucleolus. rRNA maturation is restricted to a fibrillar component, reminiscent of the dense fibrillar component in wild-type cells; a granular component is also present, whereas no fibrillar center can be distinguished, which directly links this latter substructure to rDNA chromosomal organization.

Fission yeast Msp1 is a mitochondrial dynamin-related protein.

We recently identified Msp1p, a fission yeast Schizosaccharomyces pombe dynamin-related protein, which is essential for the maintenance of mitochondrial DNA. The Msp1p sequence displays typical features of a mitochondrial protein. Here we report in vitro and in vivo data that validate that prediction.

Assembly of 5S ribosomal RNA is required at a specific step of the pre-rRNA processing pathway.

A collection of yeast strains surviving with mutant 5S RNA has been constructed. The mutant strains presented alterations of the nucleolar structure, with less granular component, and a delocalization of the 25S rRNA throughout the nucleoplasm. The 5S RNA mutations affected helix I and resulted in decreased amounts of stable 5S RNA and of the ribosomal 60S subunits.

Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae.

By combining cryofixation and cryosubstitution in a structural and functional analysis of the nucleus of Saccharomyces cerevisiae, we identified morphological subcompartments in the nucleolus. These were similar to those of nucleoli of higher eukaryotes, such as the fibrillar centre (FC), the dense fibrillar component (DFC) and the granular component (GC). In situ hybridization and immunocytochemistry revealed RNA polymerase I and proteins involved in early steps of ribosomal maturation along the DFC, while the ribosomal genes were detected at the FCs.

The role of the Schizosaccharomyces pombe gar2 protein in nucleolar structure and function depends on the concerted action of its highly charged N terminus and its RNA-binding domains.

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain.

Cerium-based ultracytochemical localization of aspartate transcarbamylase activity in the cell membrane complex of Saccharomyces cerevisiae.

Aspartate transcarbamylase (ATCase) activity was localized ultracytochemically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate. We prefixed yeast cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity. Cells were washed and incubated with ATCase substrates (aspartate, carbamyl phosphate) plus cerium chloride, and postfixed by osmium tetroxide.

Ultrastructural changes in the Schizosaccharomyces pombe nucleolus following the disruption of the gar2+ gene, which encodes a nucleolar protein structurally related to nucleolin.

The nucleolar protein gar2, from the fission yeast Schizosaccharomyces pombe, is the functional homolog of NSR1 from Saccharomyces cerevisiae, and is structurally related to nucleolin from vertebrates. By immunocytochemistry at the electron microscope level, we show that gar2 co-localizes with RNA polymerase I and the gar1 protein along the dense fibrillar component of the nucleolus in a wild-type strain of S. pombe, suggesting that gar2 is involved in the transcription and/or in the early steps of maturation of the ribosomal RNAs.

Structural and functional analysis of the nucleolus of the fission yeast Schizosaccharomyces pombe.

Yeasts are an attractive model for the study of ribosome synthesis. However, our understanding of the relationship between the structure and function of the yeast nucleolus, in which preribosomal particles are synthesized, requires further investigations using microscopic approaches and in situ molecular biology. Combining cryofixation and cryosubstitution of Schizosaccharomyces pombe, we could identify morphologically distinct substructures in the nucleolus similar to the components of nucleoli of higher eukaryotes such as the fibrillar centers (FCs), the dense fibrillar component (DFC) and the granular component (GC).

Basic fibroblast growth factor (FGF-2) is addressed to caveolae after binding to the plasma membrane of BHK cells.

bFGF endocytosis in BHK cells was examined by electron microscopy using a conjugate of recombinant human bFGF and digoxigenin (bFGF-DIG). This probe keeps the biological activity of non-labeled bFGF and can be readily detected with anti-digoxigenin antibodies (Gleizes et al., Anal.

Basic fibroblast growth factor (FGF-2) internalization through the heparan sulfate proteoglycans-mediated pathway: an ultrastructural approach.

Biochemical studies have shown that basic fibroblast growth factor (bFGF or FGF-2) is internalized by two pathways, after binding to either FGF tyrosine kinase receptors or to heparan sulfate proteoglycans (HSPG). To get insights on the HSPG-mediated pathway, we have examined by electron microscopy the intracellular route of bFGF-HRP, a monovalent conjugate of bFGF and horseradish peroxidase which was found to bind to HSPG only and was detectable by electron microscopy. bFGF-HRP association to adult bovine aortic endothelial (ABAE) cells or baby hamster kidney (BHK) cells was inhibited by a high molar excess of native bFGF, a 2 M NaCl wash at neutral pH, heparin and heparan sulfate, but not by chondroitin 4-sulfate or chondroitin 6-sulfate.

Labeling of basic fibroblast growth factor with digoxigenin: a nonradioactive probe for biochemical and cytological applications.

Digoxigenin, a 391-Da plant sterol, was conjugated to recombinant bFGF with the aim of detecting it with high specificity and sensitivity in cultured eukaryotic cells using antibodies against digoxigenin. The conjugate, bFGF-DIG, displayed a mitogenic activity on endothelial cells equivalent to that of nonlabeled bFGF. Binding of the probe on the cell surface was assessed by ELISA on cells, which allowed discrimination between low- and high-affinity bFGF binding sites.

Cell cycle redistribution of U3 snRNA and fibrillarin. Presence in the cytoplasmic nucleolus remnant and in the prenucleolar bodies at telophase.

The distribution of the U3 small nuclear RNA during the cell cycle of the CHO cell line was studied by in situ hybridization using digoxigenin-labelled oligonucleotide probes. The location of the hybrids by immunofluorescence microscopy and at the ultrastructural level was correlated with the distribution of two nucleolar proteins, nucleolin and fibrillarin. The U3 snRNA molecules persist throughout mitosis in close association with the nucleolar remnant.

Fate of specific nucleolar perichromosomal proteins during mitosis: cellular distribution and association with U3 snoRNA.

In mammalian cells, the nucleoli disintegrate during mitosis and some nucleolar proteins disperse at the periphery of all chromosomes forming a novel class of chromosomal passenger proteins. The nucleolar components which participate in the formation of this perichromosomal layer have been investigated to elucidate the role of these perichromosomal proteins in the assembly and disassembly of the nucleoli. i) Electron microscopy immunolabelling reveals that these proteins are predominantly located in the granular component of the nucleoli during interphase.