Jacqueline Noaillac-Depeyre

Nhp2p and Nop10p are essential for the function of H/ACA snoRNPs

The small nucleolar ribonucleoprotein particles containing H/ACA‐type snoRNAs (H/ACA snoRNPs) are crucial trans‐acting factors intervening in eukaryotic ribosome biogenesis. Most of these particles generate the site‐specific pseudouridylation of rRNAs while a subset are required for 18S rRNA synthesis. To understand in detail how these particles carry out these functions, all of their protein components have to be characterized. For that purpose, we have affinity‐purified complexes containing epitope‐tagged Gar1p protein, previously shown to be part of H/ACA snoRNPs. Under the conditions used, three polypeptides of 65, 22 and 10 kDa apparent molecular weight specifically copurify with epitope‐tagged Gar1p. The 22 and 10 kDa polypeptides were identified as Nhp2p and a novel protein we termed Nop10p, respectively. Both proteins are conserved, essential and present in the dense fibrillar component of the nucleolus. Nhp2p and Nop10p are specifically associated with all H/ACA snoRNAs and are essential to the function of H/ACA snoRNPs. Cells lacking Nhp2p or Nop10p are impaired in global rRNA pseudouridylation and in the A1 and A2 cleavage steps of the pre‐rRNA required for the synthesis of mature 18S rRNA. These phenotypes are probably a direct consequence of the instability of H/ACA snoRNAs and Gar1p observed in cells deprived of Nhp2p or Nop10p. Our results suggest that Nhp2p and Nop10p, together with Cbf5p, constitute the core of H/ACA snoRNPs.

Processing of 20S pre‐rRNA to 18S ribosomal RNA in yeast requires Rrp10p, an essential non‐ribosomal cytoplasmic protein

Numerous non‐ribosomal trans‐acting factors involved in pre‐ribosomal RNA processing have been characterized, but none of them is specifically required for the last cytoplasmic steps of 18S rRNA maturation. Here we demonstrate that Rio1p/Rrp10p is such a factor. Previous studies showed that the RIO1 gene is essential for cell viability and conserved from archaebacteria to man. We isolated a RIO1 mutant in a screen for mutations synthetically lethal with a mutant allele of GAR1, an essential gene required for 18S rRNA production and rRNA pseudouridylation. We show that RIO1 encodes a cytoplasmic non‐ribosomal protein, and that depletion of Rio1p blocks 18S rRNA production leading to 20S pre‐rRNA accumulation. In situ hybridization reveals that, in Rio1p depleted cells, 20S pre‐rRNA localizes in the cytoplasm, demonstrating that its accumulation is not due to an export defect. This strongly suggests that Rio1p is involved in the cytoplasmic cleavage of 20S pre‐rRNA at site D, producing mature 18S rRNA. Thus, Rio1p has been renamed Rrp10p (ribosomal RNA processing #10). Rio1p/Rrp10p is the first non‐ribosomal factor characterized specifically required for 20S pre‐rRNA processing.

Functional compartmentalization of the nucleus in the budding yeast Saccharomyces cerevisiae

Abstract.u2002By 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. Our results also suggest that ribosomal transcripts are distributed along a nucleolar network that might include both DFC and GC. We also show that preribosomal subunits may be exported along tracks to the cytoplasm. Export takes place through all the pores of the nuclear envelope, not just those in contact with the nucleolus. Moreover, comparison of the nucleolar organization in S. cerevisiae and in Schizosaccharomyces pombe demonstrated than the distribution of the 5S genes with respect to the 35S transcription unit does not modify the organization of the nucleolus. We also report, for the first time, the ultrastructural localization of RNA polymerase II in yeast. The distribution of RNA polymerase II and morphological details that could be observed in the extra-nucleolar region of cryofixed cells provided cytological evidence of a peripheral region extending along the nuclear envelope that could correspond to heterochromatin in higher eukaryotes.

Naf1p, an essential nucleoplasmic factor specifically required for accumulation of box H/ACA small nucleolar RNPs.

ABSTRACT Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. The ways in which these particles are assembled and correctly localized in the dense fibrillar component of the nucleolus remain largely unknown. Recently, the essential Saccharomyces cerevisiae Naf1p protein (encoded by the YNL124W open reading frame) was found to interact in a two-hybrid assay with two core protein components of mature H/ACA snoRNPs, Cbf5p and Nhp2p (T. Ito, T. Chiba, R. Ozawa, M. Yoshida, M. Hattori, and Y. Sakaki, Proc. Natl. Acad. Sci. USA 98:4569-4574, 2001). Here we show that several H/ACA snoRNP components are weakly but specifically immunoprecipitated with epitope-tagged Naf1p, suggesting that the latter protein is involved in H/ACA snoRNP biogenesis, trafficking, and/or function. Consistent with this, we find that depletion of Naf1p leads to a defect in 18S rRNA accumulation. Naf1p is unlikely to directly assist H/ACA snoRNPs during pre-rRNA processing in the dense fibrillar component of the nucleolus for two reasons. Firstly, Naf1p accumulates predominantly in the nucleoplasm. Secondly, Naf1p sediments in a sucrose gradient chiefly as a free protein or associated in a complex of the size of free snoRNPs, whereas extremely little Naf1p is found in fractions containing preribosomes. These results are more consistent with a role for Naf1p in H/ACA snoRNP biogenesis and/or intranuclear trafficking. Indeed, depletion of Naf1p leads to a specific and dramatic decrease in the steady-state accumulation of all box H/ACA snoRNAs tested and of Cbf5p, Gar1p, and Nop10p. Naf1p is unlikely to be directly required for the synthesis of H/ACA snoRNP components. Naf1p could participate in H/ACA snoRNP assembly and/or transport.

Accumulation of H/ACA snoRNPs depends on the integrity of the conserved central domain of the RNA-binding protein Nhp2p

Box H/ACA small nucleolar ribonucleoprotein particles (H/ACA snoRNPs) play key roles in the synthesis of eukaryotic ribosomes. How box H/ACA snoRNPs are assembled remains unknown. Here we show that yeast Nhp2p, a core component of these particles, directly binds RNA. In vitro, Nhp2p interacts with high affinity with RNAs containing irregular stem-loop structures but shows weak affinity for poly(A), poly(C) or for double-stranded RNAs. The central region of Nhp2p is believed to function as an RNA-binding domain, since it is related to motifs found in various RNA-binding proteins. Removal of two amino acids that shortens a putative beta-strand element within Nhp2p central domain impairs the ability of the protein to interact with H/ACA snoRNAs in cell extracts. In vivo, this deletion prevents cell viability and leads to a strong defect in the accumulation of H/ACA snoRNAs and Gar1p. These data suggest that proper direct binding of Nhp2p to H/ACA snoRNAs is required for the assembly of H/ACA snoRNPs and hence for the stability of some of their components. In addition, we show that converting a highly conserved glycine residue (G(59)) within Nhp2p central domain to glutamate significantly reduces cell growth at 30 and 37 degrees C. Remarkably, this modification affects the steady-state levels of H/ACA snoRNAs and the strength of Nhp2p association with these RNAs to varying degrees, depending on the nature of the H/ACA snoRNA. Finally, we show that the modified Nhp2p protein whose interaction with H/ACA snoRNAs is impaired cannot accumulate in the nucleolus, suggesting that only the assembled H/ACA snoRNP particles can be efficiently retained in the nucleolus.

Identification and localization of a nucleolin homologue in onion nucleoli

A protein homologous to nucleolin, a major nucleolar protein with multifunctional features involved in pre-rRNA synthesis and early processing, has been identified and localized in situ in onion root meristematic cells by different techniques, which have included the use of an antibody raised against hamster nucleolin. The protein was identified on Western blots of nucleolar proteins as a 64-kDa band, by means of the anti-nucleolin antibody, bismuth staining, and the silver staining-nucleolar organizer (Ag-NOR) method. The experiments also suggested that nucleolin could be a target of these two cytochemical stainings. Although the 64-kDa band corresponds to a major nucleolar protein, it is a minor one among total nuclear proteins. The same techniques were used in situ at the ultrastructural level, and the immunogold detection of the nucleolin homologue was quantitatively evaluated. The protein accumulates in the transition area from nucleolar fibrillar centers to the dense fibrillar component, which is considered to be the structural result of ribosomal gene transcription. Out of this transition area, the dense fibrillar component may be divided into two regions, proximal and distal with respect to fibrillar centers, which show, respectively, the significant and unsignificant presence of nucleolin; we interpret this fact as the expression of the topological arrangement of pre-rRNA processing. Fibrillar centers themselves showed a weak but significant labeling with the anti-nucleolin antibody. However, bismuth staining was absent from the interior of fibrillar centers, indicating that the nucleolin in them is not phosphorylated. Ag-NOR staining uniformly covered fibrillar centers and the dense fibrillar component (at least in its proximal region), but it did not stain condensed chromatin inclusions in heterogeneous fibrillar centers, showing that the binding of nucleolin to chromatin is associated with its decondensation. This work provides additional evidence of the high phylogenetic conservation of molecular motifs which take part in ribosome biogenesis.

Ultrastructural changes in theSchizosaccharomyces pombe nucleolus following the disruption of thegar2+gene, which encodes a nucleolar protein structurally related to nucleolin

The nucleolar protein gar2, from the fission yeastSchizosaccharomyces pombe, is the functional homolog of NSR1 fromSaccharomyces 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 ofS. pombe, suggesting that gar2 is involved in the transcription and/or in the early steps of maturation of the ribosomal RNAs. Since the effects of disruption of thegar2+ gene might also shed light on the role of the gar2 protein, we analyzed the ultrastructure of the nucleolus of agar2-disruption mutant. The nucleolus of thegar2-mutant is dramatically reorganized when compared with that of the wild-typegar2+strain: a truncated protein containing the NH2-terminus of the gar2 protein is accumulated in an unusual nucleolar “dense body”. Our results also suggest that the NH2-terminus might be sufficient for nucleolar localization via interaction with specific nucleolar components and support the hypothesis that gar2 in wild-typeS. pombe interacts with nascent pre-rRNA via its two RNA-binding domains in combination with the glycine/arginine-rich domain. We also report that disruption of thegar2+ gene results in a mutant that is defective in cytokinesis and nuclear division.

The effect of adenosine analogue (DRB) on a major nucleolar phosphoprotein nucleolin.

Summary— Nucleolin, a phosphorylated nucleolar protein, of 100 kDa selectively stained with bismuth tartrate and silver nitrate, is implicated in the transcription and maturation of pre‐ribosomal RNA. Nucleolin also fulfills a structural function in nucleolar organization. Using immunocytochemistry the action of 5–6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB), an inhibitor of hn/RNA synthesis known to modify the organization of the nucleolus, was studied for its effects on the distribution and the amount of nucleolin present. After DRB treatment, the morphology of the nucleolus was rapidly disturbed, but the distribution of the nucleolin remained unchanged: the dense fibrillar and the granular components were always positively immunostained. Thirty min after incubation with the drug, a strong increase of the amount of nucleolin occurred. Prolonged treatment led to a marked loss of label. Silver and bismuth staining showed that DRB does not seem to significantly affect the phosphorylation of nucleolin.

Ultrastructural study of the fission yeast Schizosaccharomyces pombe nucleolus by freeze substitution

Image processing methods are commonly used for the structure determination of biological molecules using electron microscopy. However, the first and crucial step is to convert the electron image intensities into numerical values to be fi.Jrther processed with the computer. Electron detection devices should combine high detection efficiency, linear detection characteristics over a wide dynamic range and high spatial resolution. The electron inaage fihn presents the best characteristics for image recording, but needs a digitizing step. Dedicated flat-bed densitometers, when available are often used for this purpose. Alternatively, the use of a slow-scan CCD camera linked to the microscope permits to record electron images in digital format. However, the high cost of such systems is unatbrdable to most individual investigators, and therefore often limits such systems to a core facility. In this work, we have evaluated the characteristics of different acquisition systems more frequently found in EM. laboratories, from the video camera linked to the E.M. microscope to desktop publishing scanners. hnages of catalase crystals were recorded and their power spectrums were compared to those obtained fiom optical diffraction of E.M. negatives of the same catalase crystals. ASTROCYTES-ENDOTIIELIAL CELLS INTERACTIONS DURING Ti lE ESTABLISHMENT OF THE BLOOD-BRAIN BARRIER IN THE CHICK EMBRYO. A FREEZE-FRACTURE STUDY. BOUCHAUD Claude RAISON Daniele o) o~ Centre interuniversitaire de microscopie electronique (C1ME-Jussieu) et CNRS URA1488, 7 quai St Bernard, F-75252 Paris Cedex 05. In the embryonic brain tissue, the cerebral anlage is avascular at first and angioblasts and vascular sprouts progressively invade the neurectoderm In the embryonic chick brain, the vascular sprout occurs at 4-days rE4) but a blood-brain barrier (BBB) towards macromolecules becomes effective only at El3 with faint variations according to the cerebral area (Risau W. and Wolburg H. (1990), ?INS, 13, 174-178). Briefly, the BBB is the result o f an induction whose origin is the nervous tissue and particularly the astrocytes (Janzer R. and RaffM. (1987), Nature, 325, 255-258). Since the chronology of the establishment of the BBB is well documented in the embryonic chick, we examined the interaction between astrocytic end-feet and endothelial cells during the timing of the BBB formation. Freeze-fracture electron microscopy was used tbr identifying tight junctions (T J) between contiguous endothelial cells and orthogonal arrays (OA), peculiar aggregates of intramembrane particles characterizing astrocytes (Dermietzel R. (1974), Cell Tissue Res., 149, 121-135). At El0 and until El2, the OA are rare and not very characteristic (alignment of particles). Frmn El3 when the first TJ are clearly identified confirming the presence of the BBB, OA are unfrequent but well differentiated. At El5, OA become more numerous and sometimes associated with gap junctions. The large TJ are further amplified at E l 8 At this stage, OA become numerous but their density remains faint in the astrocytic endfeet as shown at E 20 by Nico B. et al (1994), J. Submicrosc. CytoL tathol., 26, 103-109. We conclude that the establishment of the BBB is progressive, concomitant with the OA appearance. However, the density of the OA populations in the astrocytic feet contacting the endothelial cells stays low and increases progressively during embryonic life.

Yeast as a model for the functional analysis of the nucleus: Contribution of the cryomethods in electron microscopy

s Trinoculaire ‘98 des Microscopies, Strasbourg-lllkirch, France, l-3 July 1998 Yeast as a model for the functional analysis of the nucleus: Contribution of the cryomethods in electron microscopy lsabelle Leger-Silvestre, Jacqueline Noaillac-Depeyre, Stkphanie Trumtel, Anne-Marie Dechampesme*, Sylvie Gamier*, H&ne Sicard, Nicole Gas LBME du CNRS 118 route de Narbonne 31062 Toulouse cedex, *Service de Biochimie et de Gkn&ique Mokculaire CEA de Saclay, 91191 Gif-w-Yvette, France 273 Although the nucleus has traditionally been viewed as relatively unstructured when compared to the cytoplasm (mainly because of the lack of intranuclear membrane-bound compartments), it is clear that functional subcompartments exist in this highly dynamic organelle. The nucleolus, which is dedicated to the major steps of ribosome biogenesis, is the most eloquent example of a functional nuclear subcompartment, and has been referred to as the “paradigm for nuclear compartimentalization” (Strouboulis J, Wolffe AP (1996). J Cell Sci 109, 1991-2000). The existence of a higher-level organization in the extra-nucleolar nucleoplasm has also been proposed for some years. Defining the functional organization of the nucleus is central to any clear understanding of the integration and co-ordination of the numerous and complex nuclear functions. Therefore, over the detection and the description of the nuclear domains, efforts now converge to understand how this spatial organization contributes to and/or reflects the various functions that take place in the nucleus. The combination at the electron microscope level of cytochemistry, immunocytochemistry and in situ hybridization has made the ultrastructural approach the only one permitting the direct association of functional domains with specific structural components (Puvion-Dutilleul F, Puvion E (1995). Microsc Res Tech& 31,22-43). In this context, our work is dedicated to the structural and the functional analysis of the nucleolus and the nucleoplasm of yeast. Yeast has a number of attributes that make it excellent for this study, namely its unicellular nature, its small genome size, the ease of mutant isolation and genetic analysis, and the availability of recombinant DNA technology. Moreover, available data indicate that the major steps in ribosome synthesis are conserved throughout eukaryotes and, human homologues of most of the components involved in ribosome biogenesis have been identified in yeast. However, yeast also has a number of intrinsic disadvantages for such an investigation. Its small size and tough cell wall considerably limited precise structural analysis of its nucleus by microscopy. In early conventional transmission electron micrographs, two regions were distinguished in the nucleus of yeast : 1) a region of low electron density representing a chromatin-rich region and 2) an electron-dense domain which occupies one-third to one half of the nuclear volume and referred to as the nucleolus. The nucleolus appeared to lack the distinct subcompartments found in vertebrate nucleoli (for a review, Mel&se T, Xue Z (1995). Curr Opin Cell Bid 7, 319-324). Cryofixation, as a physical technique to preserve cellular morphology, appeared to our mind very attractive for the study of the nucleus in yeast (Dubochet JL (1995). Trends Cell BioI 5,366-368; Steinbrecht RA, Zierold K (1987). Cryotechniques in Biological Electron Microscopy. Springer-Verlag). During cryofixation, the water of the specimen is cooled so rapidly that it is vitrified before it has time to cristallize; cryofixation therefore avoids the freezing damages caused by ice crystals. The freezing technique also avoids conventional chemical fixation that may result in morphology disruption because of its slow action and its selectivity for cellular components. Combined with cryosubstitution (substitution of the ice of the frozen specimen by a chemical fixative at low temperature), the cryofixation increases the potential of trapping native structures and distribution patterns. We analysed the structural and the functional organization of both the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae (Leger-Silvestre I et al (1997). Eur J Cell Biol 72,13-23; LRger-Silvestre I et al (1998). Chromosoma, submitted). S.pombe has the advantage of being closer to higher eukaryotes with regard to the organization of the ribosome transcription unit (Schaak J et al (1982). Nucleic Acids Res 10, 2851-2864; Tabata S (1981) Nucleic Acids Res 9, 6429-6437). However ribosome biogenesis is poorly documented in S. pombe as compared to S. cerevisiae. Our results showed that the subcompartments associated to ribosome biogenesis are conserved during evolution : we identified in the nucleolus of both yeasts 274 Biology of the Cell (1998190, 247-290 morphologically distinct substructures similar to the components of nucleoli of higher eukaryotes such as fibrillar centre (FC), the dense fibrillar component (DFC) and the granular component (GC). In situ hybridization and immunocytochemistry provided evidence that transcription and early steps of maturation of ribosomal RNA take place in the DFC that extends throughout the nucleolus. Pre-ribosomal subunits appear to be exported along tracks to the cytoplasm and exit the nucleus through all the pores of the nuclear envelope and not just those in contact with the nucleolus. Moreover, comparison of the nucleolar organization in S. porn&e and S. cerez&iue demonstrated that the distribution of the 5s genes with respect to the 35s transcription unit has no effect on the organization of the nucleolus. Regarding the organization of the nucleoplasm, we reported for the first time the ultrastructural localization of the RNA polymerase II in yeast. The distribution of the RNA polymerase II and morphological details provided the cytological evidence of a peripheral region extending along the nuclear envelope that could correspond to heterochromatin of higher eukaryotes. The analysis of the nucleolar structure in mutants where genes involved in ribosome biogenesis are inactivated may bring insight into the role of some proteins in the transcription and the processing pathway leading to mature rRNA species. It may also provide information regarding events and/or molecules required for the formation and the organization of the nucleolus. We are currently combining cryomethods, in situ techniques, biochemical and genetic approaches, to study such mutants. 1) Regarding the transcription, we analysed two kind of mutants : The RNA polymerase I is likely to be an important actor in the building of nucleolus structure. Some S. pombe mutants which are defective in RNA polymerase I exhibit a strongly altered nucleolus (Hirano T et al (1989). J Cell Biol108,243-253). In S. cerevisiae strains defective for endogeneous rRNA transcription by the RNA polymerase I and surviving with rRNA synthesized by RNA polymerase II from an episomal gene, the crescent structure of the nucleolus is absent and instead mininucleolar structures appear (Oakes M et al (1993). Mol Cell Bid 13,2441-2445). We described these mininucleoli in terms of two morphological components that are dynamically reorganized when the rRNA transcription was inhibited. Our results provided evidence that the initiating event for the organization of these mininucleoli is the transcription of the rRNA and that transcription alone triggers recruitment and/or assembly of the maturation machinery. In order to investigate the role of the 5s rRNA in ribosome biogenesis we analysed yeast strains surviving with mutant 5s rRNA expressed from an episomal location. In all 5s mutants, a specific defect is observed at one stage of the processing leading to 25s and 5.8s. This is accompanied by the absence of granular component in the nucleolus and the mislocalization of the rRNA throughout the nucleus (Dechampesme A-M et al (1998). Bid Cell, 90,109). 2) Regarding the maturation, we investigated the consequences of the absence of structural domains of the non-ribosomal nucleolar gar2 protein required for 18s rRNA and 40s production in S. pornbe. Drastic functional defects correlate with dramatical nucleolar re-organization (LegerSilvestre I et al (1997). Chromosoma 105, 542-552; Sicard H et al (1998). Mol Biol Cell, submitted). By providing evidence that the nucleolus in yeast has similar organization to nucleolus in higher eukaryotes, cryomethods highlight the relevance of using yeast for the study of the ribosome biogenesis. Indeed, the combination of in situ techniques with genetic approaches, particularly easy in yeasts, opens new perspectives to reinvestigate the functions of the nucleolus.