Nicole Gas

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. Kinetic and steady‐state measurements of the levels of pre‐rRNAs in Nog2p‐depleted cells showed a defect in 5.8S and 25S maturation and a concomitant increase in the levels of both 27SBS and 7SS precursors. We found Nog2p physically associated with large pre‐60S complexes highly enriched in the 27SB and 7S rRNA precursors. These complexes contained, besides a subset of ribosomal proteins, at least two additional factors, Nog1p, another putative GTP‐binding protein, and Rlp24p (Ylr009wp), which belongs to the Rpl24e family of archaeal and eukaryotic ribosomal proteins. In the absence of Nog2p, the pre‐60S ribosomal complexes left the nucleolus, but were retained in the nucleoplasm. These results suggest that transient, possibly GTP‐dependent association of Nog2p with the pre‐ribosomes might trigger late rRNA maturation steps in ribosomal large subunit biogenesis.

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. Mutation of each Noc protein impairs intranuclear transport of 60S subunits at different stages and inhibits pre-rRNA processing. Overexpression of a conserved domain common to Noc1p and Noc3p is dominant-negative for cell growth, with a defect in nuclear 60S subunit transport, but no inhibition of pre-rRNA processing. We propose that the dynamic interaction of Noc proteins is crucial for intranuclear movement of ribosomal precursor particles, and, thereby represent a prerequisite for proper maturation.

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. Depletion of Rps19 results in the accumulation of aberrant pre-40 S particles retained in the nucleus that fail to associate with pre-ribosomal factors involved in late maturation steps, including Enp1, Tsr1, and Rio2. When introduced in yeast Rps19, amino acid substitutions found in Diamond Blackfan anemia patients induce defects in the processing of the pre-rRNA similar to those observed in cells under-expressing Rps19. These results uncover a pivotal role of Rps19 in the assembly and maturation of the pre-40 S particles and demonstrate for the first time the effect of Diamond Blackfan anemia-associated mutations on the function of Rps19, strongly connecting the pathology to ribosome biogenesis.

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. Moreover, noc4 thermosensitive mutants are defective in 40 S biogenesis, and rRNA processing is inhibited at early cleavage sites A0, A1, and A2. Using a fluorescence-based visual assay for 40 S subunit export, we observe a strong nucleolar accumulation of the Rps2p-green fluorescent protein reporter in noc4 ts mutants, but 60 S subunit export was normal. Thus, Noc4p and Nop14p form a novel Noc complex with a specific role in nucleolar 40 S subunit formation and subsequent export to the cytoplasm.

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. The rix9-1 (rlp7-1) mutation blocks the late pre-RNA cleavage at site C2 in ITS2, which separates the precursors to the 5.8S and 25S rRNAs. Consistent with this, synthesis of the mature 5.8S and 25S rRNAs was blocked in the rlp7-1 strain at nonpermissive temperature, whereas 18S rRNA synthesis continued. Moreover, pre-rRNA containing ITS2 accumulates in the nucleolus of rix9-1 cells as revealed by in situ hybridization. Finally, tagged Rlp7p was shown to associate with a pre-60S particle, and fluorescence microscopy and immuno-EM localized Rlp7p to a subregion of the nucleolus, which could be the granular component (GC). All together, these data suggest that pre-rRNA cleavage at site C2 specifically requires Rlp7p and occurs within pre-60S particles located in the GC region of the nucleolus.

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.

Correlation between rDNA transcription and distribution of a 100 kD nucleolar protein in CHO cells

A major nucleolar protein with a molecular weight of 100 kD is directly implicated in the transcription of pre-ribosomal RNA (pre-rRNA) and appears to be cleaved into specific maturation products during pre-ribosome biogenesis. Polyclonal antibodies which recognize the 100 kD protein and its products were used to determine the correlation between rDNA transcription and these proteins. Actinomycin D (AMD) was used to block selectively rDNA transcription (AMD 0.1 microgram/ml). Immunoperoxidase and immunogold staining were carried out in untreated and treated cells. Digitalization allowed the quantification of label according to the nucleolar components and the cellular areas. In exponentially growing cells, the dense fibrillar component was shown to contain more 100 kD protein than the granular RNP component but both nucleolar components were positively immunostained. The distribution of the 100 kD protein was rapidly modified by AMD: loss of label occurred first in the dense fibrillar zone of the nucleolus, demonstrating the correlation between rDNA transcription and the presence of this protein. However, one part of the protein remains in the segregated nucleolus after 1 h of AMD treatment, thus supporting the structural function of this protein.

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

Summary— In mammalian cells, the nucleoli disintegrate during mitosis and some nucleolar proteins disperse at the periphery of allchromosomes 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,. ii) Immunoprecipitation data suggest that they are distributed at the chromosome periphery in association with U3 small nucleolar RNA (snoRNA). In addition, the distribution of U3 snoRNA visualized by in situ hybridization, is similar to that observed for the perichromosomal proteins. iii) In cells which possess a nucleolar remnant during mitosis, U3 snoRNA and perichromosomal proteins were found both in the perichromosomal layer and in the nucleolar remnant. iv) Some of these proteins are conserved from yeast to man such as fibrillarin and a protein of 52 kDa. v) The location of these proteins observed in yeast by confocal microscopy shows that they are not dispersed during mitosis. Their partition between the two daughter cells is performed by scission of nucleolar structures forming a rod during the budding process. Therefore RNP complexes related to the processing steps of ribosome biogenesis in mammalian cells quit the nucleolus in late G2 and associate with the chromosome periphery until late telophase. They associate in the perichromosomal layer in human and PtK1 cells and both in the perichromosomal layer and the nucleolar remnant in CHO cells.

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.