Showing papers on "Nucleolus published in 2000"

Cajal Bodies: The First 100 Years

Abstract: ▪ Abstract Cajal bodies are small nuclear organelles first described nearly 100 years ago by Ramon y Cajal in vertebrate neural tissues. They have since been found in a variety of animal and plant nuclei, suggesting that they are involved in basic cellular processes. Cajal bodies contain a marker protein of unknown function, p80-coilin, and many components involved in transcription and processing of nuclear RNAs. Among these are the three eukaryotic RNA polymerases and factors required for transcribing and processing their respective nuclear transcripts: mRNA, rRNA, and pol III transcripts. A model is discussed in which Cajal bodies are the sites for preassembly of transcriptosomes, unitary particles involved in transcription and processing of RNA. A parallel is drawn to the nucleolus and the preassembly of ribosomes, which are unitary particles involved in translation of proteins.

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.

Abstract: The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19ARF and human p14ARF bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19ARF are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14ARF protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19ARF were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19ARF are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19ARF-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.

The Dynamics of Postmitotic Reassembly of the Nucleolus

Abstract: Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.

Compartmentalization of transcription and translation in Bacillus subtilis.

Abstract: Using fusions of green fluorescent protein to subunits of RNA polymerase (RNAP) and ribosomes, we have investigated the subcellular localization of the transcriptional and translational machinery in the bacterium Bacillus subtilis. Unexpectedly, we found that RNAP resides principally within the nucleoid. Conversely, ribosomes localized almost exclusively outside the nucleoid, concentrating particularly towards sites of cell division. This zonal localization was not dependent on cell division and is probably due, at least in part, to exclusion from the nucleoid. Dual labelling of RNAP and ribosomes was used to confirm the spatial separation of the two processes. We conclude that, even in the absence of a nuclear membrane, transcription and translation occur predominantly in separate functional domains. At higher growth rates, concentrations of RNAP developed, probably representing the sites of rRNA synthesis. These may represent a further spatial specialization, possibly equivalent to the eukaryotic nucleolus.

Gemin4 A Novel Component of the Smn Complex That Is Found in Both Gems and Nucleoli

Abstract: The survival of motor neurons (SMN) protein, the product of the neurodegenerative disease spinal muscular atrophy (SMA) gene, is localized both in the cytoplasm and in discrete nuclear bodies called gems. In both compartments SMN is part of a large complex that contains several proteins including Gemin2 (formerly SIP1) and the DEAD box protein Gemin3. In the cytoplasm, the SMN complex is associated with snRNP Sm core proteins and plays a critical role in spliceosomal snRNP assembly. In the nucleus, SMN is required for pre-mRNA splicing by serving in the regeneration of spliceosomes. These functions are likely impaired in cells of SMA patients because they have reduced levels of functional SMN. Here, we report the identification by nanoelectrospray mass spectrometry of a novel component of the SMN complex that we name Gemin4. Gemin4 is associated in vivo with the SMN complex through a direct interaction with Gemin3. The tight interaction of Gemin4 with Gemin3 suggests that it could serve as a cofactor of this DEAD box protein. Gemin4 also interacts directly with several of the Sm core proteins. Monoclonal antibodies against Gemin4 efficiently immunoprecipitate the spliceosomal U snRNAs U1 and U5 from Xenopus oocytes cytoplasm. Immunolocalization experiments show that Gemin4 is colocalized with SMN in the cytoplasm and in gems. Interestingly, Gemin4 is also detected in the nucleoli, suggesting that the SMN complex may also function in preribosomal RNA processing or ribosome assembly.

Human H/ACA Small Nucleolar RNPs and Telomerase Share Evolutionarily Conserved Proteins NHP2 and NOP10

Abstract: The H/ACA small nucleolar RNAs (snoRNAs) are involved in pseudouridylation of pre-rRNAs In the yeast Saccharomyces cerevisiae, four common proteins are associated with H/ACA snoRNAs: Gar1p, Cbf5p, Nhp2p, and Nop10p In vitro reconstitution studies showed that four proteins also specifically interact with H/ACA snoRNAs in mammalian cell extracts Two mammalian proteins, NAP57/dyskerin (the ortholog of Cbf5p) and hGAR1, have been characterized In this work we describe properties of hNOP10 and hNHP2, human orthologs of yeast Nop10p and Nhp2p, respectively, and further characterize hGAR1 hNOP10 and hNHP2 complement yeast cells depleted of Nhp2p and Nop10p, respectively Immunoprecipitation experiments with extracts from transfected HeLa cells indicated that epitope-tagged hNOP10 and hNHP2 specifically associate with hGAR1 and H/ACA RNAs; they also interact with the RNA subunit of telomerase, which contains an H/ACA-like domain in its 3' moiety Immunofluorescence microscopy experiments showed that hGAR1, hNOP10, and hNHP2 are localized in the dense fibrillar component of the nucleolus and in Cajal (coiled) bodies Deletion analysis of hGAR1 indicated that its evolutionarily conserved core domain contains all the signals required for localization, but progressive deletions from either the N or the C terminus of the core domain abolish localization in the nucleolus and/or the Cajal bodies

Molecular basis of sequence-specific recognition of pre-ribosomal RNA by nucleolin.

Abstract: The structure of the 28 kDa complex of the first two RNA binding domains (RBDs) of nucleolin (RBD12) with an RNA stem–loop that includes the nucleolin recognition element UCCCGA in the loop was determined by NMR spectroscopy. The structure of nucleolin RBD12 with the nucleolin recognition element (NRE) reveals that the two RBDs bind on opposite sides of the RNA loop, forming a molecular clamp that brings the 5′ and 3′ ends of the recognition sequence close together and stabilizing the stem–loop. The specific interactions observed in the structure explain the sequence specificity for the NRE sequence. Binding studies of mutant proteins and analysis of conserved residues support the proposed interactions. The mode of interaction of the protein with the RNA and the location of the putative NRE sites suggest that nucleolin may function as an RNA chaperone to prevent improper folding of the nascent pre-rRNA.

Signal recognition particle components in the nucleolus

Abstract: The signal recognition particle (SRP) is a ribonucleoprotein composed of an Alu domain and an S domain. The S domain contains unique sequence SRP RNA and four SRP proteins: SRP19, SRP54, SRP68, and SRP72. SRP interacts with ribosomes to bring translating membrane and secreted proteins to the endoplasmic reticulum (ER) for proper processing. Additionally, SRP RNA is a member of a family of small nonribosomal RNAs found recently in the nucleolus, suggesting that the nucleolus is more plurifunctional than previously realized. It was therefore of interest to determine whether other SRP components localize to this intranuclear site. In transfected rat fibroblasts, green fluorescent protein fusions of SRP19, SRP68, and SRP72 localized to the nucleolus, as well as to the cytoplasm, as expected. SRP68 also accumulated in the ER, consistent with its affinity for the ER-bound SRP receptor. SRP54 was detected in the cytoplasm as a green fluorescent protein fusion and in immunofluorescence studies, but was not detected in the nucleolus. In situ hybridization experiments also revealed endogenous SRP RNA in the nucleolus. These results demonstrate that SRP RNA and three SRP proteins visit the nucleolus, suggesting that partial SRP assembly, or another unidentified activity of the SRP components, occurs at the nucleolus. SRP54 apparently interacts with nascent SRP beyond the nucleolus, consistent with in vitro reconstitution experiments showing that SRP19 must bind to SRP RNA before SRP54 binds. Our findings support the notion that the nucleolus is the site of assembly and/or interaction between the family of ribonucleoproteins involved in protein synthesis, in addition to ribosomes themselves.

A novel mammalian Smt3-specific isopeptidase 1 (SMT3IP1) localized in the nucleolus at interphase.

Abstract: A novel Smt3-specific isopeptidase, SMT3IP1, was cloned using a yeast two-hybrid screen with Smt3b as bait. The clone, named SMT3IP1 (Smt3-specific isopeptidase 1), which bound to Smt3b but not SUMO-1 in the two-hybrid system, was distantly related to budding yeast Saccharomyces cerevisiae Ulp1, human SENP1 or human SUSP1. The catalytic domains in the C-terminal region were very similar, but the N-terminal region was quite different to other enzymes. The cysteine, histidine and asparatic acid residues in the catalytic domains were conserved. SMT3IP1 expressed by the baculovirus-expression system had the ability to cleave SUMO-1 or Smt3b from SUMO-1/RanGAP1 or Smt3b/RanGAP1 conjugates, respectively, and the activity was a little stronger towards the Smt3b conjugate than towards the SUMO-1 conjugate. Furthermore, the enzyme bound more strongly to Smt3a and Smt3b than to SUMO-1 in vitro. The enzyme did not cleave Nedd8 from Nedd8/cullin-1. Nor did it cleave ubiquitin from ubiquitinated p53. SMT3IP1 was localized almost exclusively at the nucleolus during interphase. The N-terminal sequence was responsible for the nucleolar localization of this enzyme. Whether SMT3IP1 functions in the nucleolus or just stays there before it functions in the nucleus, as shown in the case of CDC14 phosphatase, remains to be elucidated.

Box C/D snoRNA-associated proteins: two pairs of evolutionarily ancient proteins and possible links to replication and transcription.

Abstract: The eukaryotic nucleolus contains a diverse population of small nucleolar RNAs (snoRNAs) essential for ribosome biogenesis. The box C/D snoRNA family possesses conserved nucleotide boxes C and D that are multifunctional elements required for snoRNA processing, snoRNA transport to the nucleolus, and 2'-O-methylation of ribosomal RNA. We have previously demonstrated that the assembly of an snoRNP complex is essential for processing the intronic box C/D snoRNAs and that specific nuclear proteins associate with the box C/D core motif in vitro. Using a box C/D motif derived from mouse U14 snoRNA, we have now affinity purified and defined four mouse proteins that associate with this minimal RNA substrate. These four proteins consist of two protein pairs: members of each pair are highly related in sequence. One protein pair corresponds to the essential yeast nucleolar proteins Nop56p and Nop58p. Affinity purification of mouse Nop58 confirms observations made in yeast that Nop58 is a core protein of the box C/D snoRNP complex. Isolation of Nop56 using this RNA motif defines an additional snoRNP core protein. The second pair of mouse proteins, designated p50 and p55, are also highly conserved among eukaryotes. Antibody probing of nuclear fractions revealed a predominance of p55 and p50 in the nucleoplasm, suggesting a possible role for the p50/p55 pair in snoRNA production and/or nucleolar transport. The reported interaction of p55 with TATA-binding protein (TBP) and replication A protein as well as the DNA helicase activity of p55 and p50 may suggest the coordination of snoRNA processing and snoRNP assembly with replication and/or transcriptional events in the nucleus. Homologs for both snoRNA-associated protein pairs occur in Archaea, strengthening the hypothesis that the box C/D RNA elements and their interacting proteins are of ancient evolutionary origin.

Mutational Analysis of Fibrillarin and Its Mobility in Living Human Cells

Abstract: Cajal bodies (CBs) are subnuclear organelles that contain components of a number of distinct pathways in RNA transcription and RNA processing. CBs have been linked to other subnuclear organelles such as nucleoli, but the reason for the presence of nucleolar proteins such as fibrillarin in CBs remains uncertain. Here, we use full-length fibrillarin and truncated fibrillarin mutants fused to green fluorescent protein (GFP) to demonstrate that specific structural domains of fibrillarin are required for correct intranuclear localization of fibrillarin to nucleoli and CBs. The second spacer domain and carboxy terminal alpha-helix domain in particular appear to target fibrillarin, respectively, to the nucleolar transcription centers and CBs. The presence of the RNP domain seems to be a prerequisite for correct targeting of fibrillarin. Time-lapse confocal microscopy of human cells that stably express fibrillarin-GFP shows that CBs fuse and split, albeit at low frequencies. Recovered fluorescence of fibrillarin-GFP in nucleoli and CBs after photobleaching indicates that it is highly mobile in both organelles (estimated diffusion constant ∼0.02 μm2 s−1), and has a significantly larger mobile fraction in CBs than in nucleoli.

Initiation of Nucleolar Assembly Is Independent of RNA Polymerase I Transcription

Abstract: This report examines the distribution of an RNA polymerase I transcription factor (upstream binding factor; UBF), pre-rRNA processing factors (nucleolin and fibrillarin), and pre-rRNAs throughout mitosis and postmitotic nucleologenesis in HeLa cells. The results demonstrate that nucleolin, fibrillarin, and pre-rRNAs synthesized at G2/M phase of the previous cell cycle are directly recruited to UBF-associated nucleolar organizer regions (NORs) early in telophase before chromosome decondensation. Unlike the fusion of prenucleolar bodies to the nucleoli, this early recruitment of processing factors and pre-rRNAs is independent of RNA polymerase I transcription. In the absence of polymerase I transcription, the initial localization of nucleolin, fibrillarin, and pre-rRNAs to UBF-associated NORs generates segregated mininucleoli that are similar to the larger ones observed in interphase cells grown under the same conditions. Pre-rRNAs are juxtaposed to UBF-nucleolin-fibrillarin caps that may represent the segregated nucleoli observed by electron microscopy. These findings lead to a revised model of nucleologenesis. We propose that nucleolar formation at the end of mitosis results from direct recruitment of processing factors and pre-rRNAs to UBF-associated NORs before or at the onset of rDNA transcription. This is followed by fusion of prepackaged prenucleolar bodies into the nucleolus. Pre-ribosomal ribonucleoproteins synthesized in the previous cell cycle may contribute to postmitotic nucleologenesis.

The location of pKi67 in the outer dense fibrillary compartment of the nucleolus points to a role in ribosome biogenesis during the cell division cycle.

Abstract: Although widely used as a marker of cell proliferation, the biochemical properties and function of the Ki67 antigen remain poorly understood. Recent data indicate that it can interact with RNA, DNA, and a number of cellular proteins including elements of the ubiquitin proteolytic pathway and a novel kinase. The evidence for its expression only in cycling cells is extensive and it is not regulated by stress, apoptosis or DNA damage. It was reasoned that a detailed characterization of the localization of pKi67 and analysis of its spatial relationship to other nucleolar proteins may provide insights into its function. Using high-resolution laser scanning confocal microscopy with double and triple labelling, pKi67 expression in MCF7 cells has been defined in relation to the distribution of nucleolin, fibrillarin, p130 (human Nopp 140 homologue), p120 (Nol 1), RH-II/Gu helicase, and topoisomerase II beta. All of these molecules are perichromosomal during mitosis and all but fibrillarin and p130 show extra-nucleolar distribution in early G1. The majority of p120 (Nol 1) and RH-II/Gu helicase co-localize in the diffuse fibrillar centre (DFC) of nucleoli, while there is only partial overlap with nucleolin and fibrillarin. There is no co-localization between p130 and pKi67. These data refine current understanding of the distribution of pKi67 and its physical relationship with functional domains of the nucleolus and place pKi67 in a zone of the DFC associated with late rRNA processing. Taken together with recent biochemical data, these observations allow the proposal of a model of pKi67 function in which it acts as an 'efficiency factor' in ribosome biogenesis during the heavy metabolic demands placed on a cell during the cell division cycle.

Conserved composition of mammalian box H/ACA and box C/D small nucleolar ribonucleoprotein particles and their interaction with the common factor Nopp140.

Abstract: Small nucleolar ribonucleoprotein particles (snoRNPs) mainly catalyze the modification of rRNA. The two major classes of snoRNPs, box H/ACA and box C/D, function in the pseudouridylation and 29-O-methylation, respectively, of specific nucleotides. The emerging view based on studies in yeast is that each class of snoRNPs is composed of a unique set of proteins. Here we present a characterization of mammalian snoRNPs. We show that the previously characterized NAP57 is specific for box H/ACA snoRNPs, whereas the newly identified NAP65, the rat homologue of yeast Nop5/58p, is a component of the box C/D class. Using coimmunoprecipitation experiments, we show that the nucleolar and coiled-body protein Nopp140 interacts with both classes of snoRNPs. This interaction is corroborated in vivo by the exclusive depletion of snoRNP proteins from nucleoli in cells transfected with a dominant negative Nopp140 construct. Interestingly, RNA polymerase I transcription is arrested in nucleoli depleted of snoRNPs, raising the possibility of a feedback mechanism between rRNA modification and transcription. Moreover, the Nopp140snoRNP interaction appears to be conserved in yeast, because depletion of Srp40p, the yeast Nopp140 homologue, in a conditional lethal strain induces the loss of box H/ACA small nucleolar RNAs. We propose that Nopp140 functions as a chaperone of snoRNPs in yeast and vertebrate cells.

Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA

Abstract: The HIV regulatory proteins Tat and Rev have a nucleolar localization property in human cells. However, no functional role has been attributed to this localization. Recently it has been demonstrated that expression of Rev induces nucleolar relocalization of some protein factors involved in Rev export. Because the function of Rev is to bind HIV RNA and facilitate transport of singly spliced and unspliced RNA to the cytoplasm, it is likely that the nucleolus plays a critical role in HIV-1 RNA export. As a test for trafficking of HIV-1 RNAs into the nucleolus, a hammerhead ribozyme that specifically cleaves HIV-1 RNA was inserted into the body of the U16 small nucleolar RNA, resulting in accumulation of the ribozyme within the nucleoli of human cells. HeLa CD4(+) and T cells expressing this nucleolar localized ribozyme exhibit dramatically suppressed HIV-1 replication. The results presented here suggest a trafficking of HIV-1 RNA through the nucleoli of human cells, thus posing a different paradigm for lentiviral RNA processing.

Nucleolar localization of human methionyl-tRNA synthetase and its role in ribosomal RNA synthesis

Abstract: Human aminoacyl–tRNA synthetases (ARSs) are normally located in cytoplasm and are involved in protein synthesis. In the present work, we found that human methionyl–tRNA synthetase (MRS) was translocated to nucleolus in proliferative cells, but disappeared in quiescent cells. The nucleolar localization of MRS was triggered by various growth factors such as insulin, PDGF, and EGF. The presence of MRS in nucleoli depended on the integrity of RNA and the activity of RNA polymerase I in the nucleolus. The ribosomal RNA synthesis was specifically decreased by the treatment of anti-MRS antibody as determined by nuclear run-on assay and immunostaining with anti-Br antibody after incorporating Br-UTP into nascent RNA. Thus, human MRS plays a role in the biogenesis of rRNA in nucleoli, while it is catalytically involved in protein synthesis in cytoplasm.

The nucleolus and the four ribonucleoproteins of translation.

Abstract: The classical view of the nucleolus as solely committed to ribosome biosynthesis has been modified by recent studies pointing to additional roles for this nuclear domain. These newly recognized features include the nucleolar presence of several nonribosomal RNAs transcribed by RNA polymerase III, as

Nucleolar dominance: uniparental gene silencing on a multi-megabase scale in genetic hybrids.

Abstract: Nucleolar dominance is a phenomenon in hybrids or allopolyploids in which nucleoli form on chromosomes inherited from only one of the two parents. The molecular basis for nucleolar dominance is the transcription by RNA polymerase I of only one parental set of ribosomal RNA genes (rRNA genes). These rRNA genes are clustered by the hundreds, or thousands, of copies, often spanning tens of millions of basepairs of chromosomal DNA at loci known as nucleolus organizer regions (NORs). Enforcement of nucleolar dominance appears to be accomplished by selectively silencing one set of rRNA genes via chemical modifications of chromatin. However, the mechanisms responsible for initially discriminating among the parental sets of rRNA genes and establishing nucleolar dominance remain unclear. Possibilities include mechanisms that act on each rRNA gene or mechanisms that affect whole NORs or even larger chromosomal domains. This review provides a historical perspective of nucleolar dominance research, explores the most popular hypotheses and their shortcomings, and offers some speculations concerning alternative hypotheses to be considered.

Transient nucleolar localization Of U6 small nuclear RNA in Xenopus Laevis oocytes.

Abstract: Recent studies on the 2'-O-methylation and pseudouridylation of U6 small nuclear RNA (snRNA) hypothesize that these posttranscriptional modifications might occur in the nucleolus. In this report, we present direct evidence for the nucleolar localization of U6 snRNA and analyze the kinetics of U6 nucleolar localization after injection of in vitro transcribed fluorescein-labeled transcripts into Xenopus laevis oocytes. In contrast to U3 small nucleolar RNA (snoRNA) which developed strong nucleolar labeling over 4 h and maintained strong nucleolar signals through 24 h, U6 snRNA localized to nucleoli immediately after injection, but nucleolar staining decreased after 4 h. By 24 h after injection of U6 snRNA, only weak nucleolar signals were observed. Unlike the time-dependent profile of strong nucleolar localization of U6 snRNA or U3 snoRNA, injection of fluorescein-labeled U2 snRNA gave weak nucleolar staining at all times throughout a 24-h period; U2 snRNA modifications are believed to occur outside of the nucleolus. The notion that the decrease of U6 signals over time was due to its trafficking out of nucleoli and not to transcript degradation was supported by the demonstration of U6 snRNA stability over time. Therefore, in contrast to snoRNAs like U3, U6 snRNA transiently passes through nucleoli.

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.

Abstract: In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4–90 min at 37°C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br- labeled RNA is a useful technique for high resolution studies of structure–function associations in the nucleolus.

Plant nuclei can contain extensive grooves and invaginations.

Abstract: Plant cells can exhibit highly complex nuclear organization. Through dye-labeling experiments in untransformed onion epidermal and tobacco culture cells and through the expression of green fluorescent protein targeted to either the nucleus or the lumen of the endoplasmic reticulum/nuclear envelope in these cells, we have visualized deep grooves and invaginations into the large nuclei of these cells. In onion, these structures, which are similar to invaginations seen in some animal cells, form tubular or planelike infoldings of the nuclear envelope. Both grooves and invaginations are stable structures, and both have cytoplasmic cores containing actin bundles that can support cytoplasmic streaming. In dividing tobacco cells, invaginations seem to form during cell division, possibly from strands of the endoplasmic reticulum trapped in the reforming nucleus. The substantial increase in nuclear surface area resulting from these grooves and invaginations, their apparent preference for association with nucleoli, and the presence in them of actin bundles that support vesicle motility suggest that the structures might function both in mRNA export from the nucleus and in protein import from the cytoplasm to the nucleus.

Import of proteins into the trypanosome nucleus and their distribution at karyokinesis.

Abstract: In all eukaryotic organisms proteins are targeted to the nucleus via a receptor-mediated mechanism that requires a specific nuclear localization sequence (NLS) in the protein. Little is known about this process in trypanosomatid protozoa that are considered amongst the earliest divergent eukaryotes. We have used the green fluorescent protein (gfp) and beta-galactosidase reporters to identify the NLS of two trypanosomal proteins, namely the Trypanosoma brucei La protein homologue and histone H2B of T. cruzi. A monopartite NLS was demonstrated at the C terminus of the La protein, whereas a bipartite NLS was identified within the first 40 amino acids of histone H2B. Treatment of live trypanosomes with poisons of ATP synthesis resulted in exit of the La NLS-gfp fusion from the nucleus. Interestingly, this fusion protein accumulated at several discrete sites in the cytoplasm, rather than equilibrating between the nucleus and the cytoplasm. When ATP levels returned to normal, the protein reentered the nucleus, demonstrating that the process was energy dependent. Finally, using fusion proteins that localize to the nucleoplasm or the nucleolus, we identified a subpopulation of mitotic cells in which the chromosomes have segregated but the daughter nuclei remain connected by a thin thread-like structure. We propose that cells containing this structure represent a late stage in nuclear division that can be placed after chromosome segregation, but before completion of karyokinesis.

RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest.

Abstract: When 3T6 cells are confluent, they withdraw from the cell cycle. Concomitant with cell cycle arrest a significant reduction in RNA polymerase I transcription (80% decrease at 100% confluence) is observed. In the present study, we examined mechanism(s) through which transcription of the ribosomal genes is coupled to cell cycle arrest induced by cell density. Interestingly with an increase in cell density (from 3 - 43% confluence), a significant accumulation in the cellular content of hyperphosphorylated Rb was observed. As cell density increased further, the hypophosphorylated form of Rb became predominant and accumulated in the nucleoli. Co-immunoprecipitation experiments demonstrated there was also a significant rise in the amount of hypophosphorylated Rb associated with the rDNA transcription factor UBF. This increased interaction between Rb and UBF correlated with the reduced rate of rDNA transcription. Furthermore, overexpression of recombinant Rb inhibited UBF-dependent activation of transcription from a cotransfected rDNA reporter in either confluent or exponential cells. The amounts or activities of the rDNA transcription components we examined did not significantly change with cell cycle arrest. Although the content of PAF53, a polymerase associated factor, was altered marginally (decreased 38%), the time course and magnitude of the decrease did not correlate with the reduced rate of rDNA transcription. The results presented support a model wherein regulation of the binding of UBF to Rb and, perhaps the cellular content of PAF53, are components of the mechanism through which cell cycle and rDNA transcription are linked. Oncogene (2000) 19, 3487 - 3497

Locus specificity determinants in the multifunctional yeast silencing protein Sir2.

Abstract: Yeast SIR2, the founding member of a conserved gene family, acts to modulate chromatin structure in three different contexts: silent (HM) mating-type loci, telomeres and rDNA. At HM loci and telomeres, Sir2p forms a complex with Sir3p and Sir4p. However, Sir2p's role in rDNA silencing is Sir3/4 independent, requiring instead an essential nucleolar protein, Net1p. We describe two novel classes of SIR2 mutations specific to either HM/telomere or rDNA silencing. Despite their opposite effects, both classes of mutations cluster in the same two regions of Sir2p, each of which borders on a conserved core domain. A surprising number of these mutations are dominant. Several rDNA silencing mutants display a Sir2p nucleolar localization defect that correlates with reduced Net1p binding. Although the molecular defect in HM/telomere-specific mutants is unclear, they mimic an age-related phenotype where Sir3p and Sir4p relocalize to the nucleolus. Artificial targeting can circumvent the silencing defect in a subset of mutants from both classes. These results define distinct functional domains of Sir2p and provide evidence for additional Sir2p-interacting factors with locus-specific silencing functions.