Intact Nucleoli Research Articles

The nucleolar phase of signal recognition particle assembly.

The signal recognition particle is essential for targeting transmembrane and secreted proteins to the endoplasmic reticulum. Remarkably, because they work together in the cytoplasm, the SRP and ribosomes are assembled in the same biomolecular condensate: the nucleolus. How important is the nucleolus for SRP assembly is not known. Using quantitative proteomics, we have investigated the interactomes of SRP components. We reveal that SRP proteins are associated with scores of nucleolar proteins important for ribosome biogenesis and nucleolar structure. Having monitored the subcellular distribution of SRP proteins upon controlled nucleolar disruption, we conclude that an intact organelle is required for their proper localization. Lastly, we have detected two SRP proteins in Cajal bodies, which indicates that previously undocumented steps of SRP assembly may occur in these bodies. This work highlights the importance of a structurally and functionally intact nucleolus for efficient SRP production and suggests that the biogenesis of SRP and ribosomes may be coordinated in the nucleolus by common assembly factors.

In situ cryo-electron tomography reveals gradient organization of ribosome biogenesis in intact nucleoli

Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.

Ribosomal 18 S RNA Processing by the IGF-I-responsive WDR3 Protein Is Integrated with p53 Function in Cancer Cell Proliferation

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G(1) phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

Nucleolar disruption impairs Kaposi’s sarcoma-associated herpesvirus ORF57-mediated nuclear export of intronless viral mRNAs

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 plays a pivotal role in mediating the nuclear export of KSHV intronless transcripts. We demonstrate that ORF57 localises and dynamically trafficks through the nucleolus. To assess if nucleolar shuttling of ORF57 is important for the export of intronless viral mRNA, the ability of ORF57 to export mRNA was assessed in cells where the nucleolus was disrupted using Actinomycin D or DRB. We show that rapid disorganisation of the nucleolus was accompanied by a reduction in intronless virus mRNA export, suggesting that an intact nucleolus is essential for efficient KSHV ORF57-mediated intronless mRNA export. Structured summaryMINT-7291377: Aly (uniprotkb:Q86V81) physically interacts (MI:0915) with ORF57 (uniprotkb:Q2HR75) by pull down (MI:0096)MINT-7291330, MINT-7291320: Nucleolin (uniprotkb:P19338) and ORF57 (uniprotkb:Q2HR75) colocalise (MI:0403) by fluorescence microscopy (MI:0416)MINT-7291715: ORF57 (uniprotkb:Q2HR75) and Aly (uniprotkb:Q86V81) colocalise (MI:0403) by fluorescence microscopy (MI:0416)

Critical Role of Nucleostemin in Pre-rRNA Processing

Nucleostemin is a nucleolar protein widely expressed in proliferating cells. Nucleostemin is involved in the regulation of cell proliferation, and both depletion and overexpression of nucleostemin induce cell cycle arrest through the p53 signaling pathway. Although the presence of p53-independent functions of nucleostemin has been previously suggested, the identities of these additional functions remained to be investigated. Here, we show that nucleostemin has a novel role as an integrated component of ribosome biogenesis, particularly pre-rRNA processing. Nucleostemin forms a large protein complex (>700 kDa) that co-fractionates with the pre-60 S ribosomal subunit in a sucrose gradient. This complex contains proteins related to pre-rRNA processing, such as Pes1, DDX21, and EBP2, in addition to several ribosomal proteins. We show that the nucleolar retention of DDX21 and EBP2 is dependent on the presence of nucleostemin in the nucleolus. Furthermore, the knockdown of nucleostemin delays the processing of 32 S pre-rRNA into 28 S rRNA. This is accompanied by a substantial decrease of protein synthesis as well as the levels of rRNAs and some mRNAs. In addition, overexpressed nucleostemin significantly promotes the processing of 32 S pre-rRNA. Collectively, these biochemical and functional studies demonstrate a novel role of nucleostemin in ribosome biogenesis. This is a key aspect of the role of nucleostemin in regulating cell proliferation.

Ischemia Leads to Apoptosis—and Necrosis‐like Neuron Death in the Ischemic Rat Hippocampus

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis-related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho-c-Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis-like cell death and necrosis-like cell death, which were confirmed by electron microscopy. Activated caspase-3 was present in the vast majority of cells with apoptosis-like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase-3, revealed a profound loss of nucleoli in cells with apoptosis-like morphology, whereas cells with necrosis-like morphology showed intact nucleoli. Overall, cells with apoptosis-like morphology and/or caspase-3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis-like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c-Jun N-terminal kinase (JNK) pathway activation, which then either follows an apoptosis-like cell death pathway or leads to secondary necrosis.

Single ribosomal transcription units are linear, compacted Christmas trees in plant nucleoli.

The rDNA transcription units are enormous macromolecular structures located in the nucleolus and containing 50-100 RNA polymerases together with the nascent pre-rRNA attached to the rDNA. It has not previously been possible to visualize nucleolar transcription units directly in intact nucleoli, although highly spread preparations in the electron microscope have been imaged as "Christmas trees" 2-3 microm long. Here we determine the relative conformation of individual transcription units in Pisum sativum plant nucleoli using a novel labelling technique. Nascent transcripts were detected by a highly sensitive silver-enhanced 1 nm gold procedure, followed by 3D electron microscopy of entire nucleoli. Individual transcription units are seen as conical, elongated clusters approximately 300 nm in length and 130 nm in width at the thickest end. We further show that there were approximately 300 active ribosomal genes in the nucleoli examined. The underlying chromatin structure of the transcribing rDNA was directly visualized by applying a novel limited extraction procedure to fixed specimens in order to wash out the proteins and RNA, thus specifically revealing DNA strands after uranyl acetate staining. Using this technique, followed by post-embedding in situ hybridization, we observed that the nucleolar rDNA fibres are not extended but show a coiled, thread-like appearance. Our results show for the first time that native rDNA transcription units are linear, compacted Christmas trees.

Reassembly of Functional Nucleoli Followingin SituUnraveling by Low-Ionic-Strength Treatment of Cultured Mammalian Cells

In order to determine the most persistent components of the nucleolus that might serve as “core” nucleolar elements, we studied the reactivity of nucleoli in living mammalian cells subjected to hypotonic buffer saline followed by the incubation of the cells in an isotonic medium. To document as precisely as possible the fine structural changes which occurred, the cells were examined by video-enhanced optical microscopy, fluorescence confocal laser scanning microscopy, and electron microscopy combined with cytochemistry. Light microscopic autoradiography was used to demonstrate the transcriptional characteristics of the reassembled nucleoli. It was shown that all the major compartments of the intact nucleolus could be substantially affected by reduction of the osmolarity of the environmental media. The dynamic events of the nucleolar unraveling in low-salt buffers occurred in the following order: dispersion of the nucleolar pars granulosa, disassociation of the fibrillar complexes into discrete fibrillar centers (FCs) and the dense fibrillar component (DFC), and the almost complete unraveling of the DFC and FCs. At the terminal stages of nucleolar dispersion, the nuclear interior was mainly composed of a loose filamentous meshwork, and none of the typically discerned nucleolar constituents was recognized. Nevertheless, when hypotonically treated cells were returned to isotonic conditions, the nucleolar bodies rapidly began to reassemble. Within 1–2 h of cell incubation under isotonicity, the nucleoli not only became clearly visible, but also reconstituted to their initial size, shape, and position within the nucleus. The ultrastructure and functional activity of the reassembled nucleoli were also found to be fully comparable to those of the untreated controls. These data indicate that the architectural composition of the interphase nucleolus is strictly controlled by the cell. As far as could be determined, none of the usual substructures of the intact nucleolus that could be substituted by complete reassembly of the nucleolar bodies in normotonic conditions, including FCs and the DFC, remained clearly preserved in the terminal stage of nucleolar unraveling. We concluded that the integrity of the nucleolus was mainly preserved by the nuclear or nucleolar matrix system rather than by any other nucleolar structural domains.

HIV-1 Rev Is Capable of Shuttling between the Nucleus and Cytoplasm

The mechanism by which Rev facilitates the export, and consequently, the translation of the structural protein mRNAs of the human immunodeficiency virus type 1 remains undefined. Previous immunolocalization has determined that Rev is predominately in the nucleus with significant accumulation in the nucleolus, a localization consistent with the assumed site of Rev action. To determine whether the subcellular distribution is more dynamic than what was indicated by the original studies, the capacity of Rev to shuttle between the nucleus and cytoplasm was examined. It was observed that treatment of cells with DRB or actinomycin D resulted in a dramatic alteration in Rev distribution, the majority of the protein being found in the cytoplasm. Removal of the drug resulted in a rapid accumulation of Rev in the nucleus indicating that the block to nuclear import was reversible. Subsequent studies indicated that the movement of Rev into the cytoplasm was a passive process while its accumulation in the nucleus was an active one, given that only the latter displayed sensitivity to temperature. Finally, it was demonstrated that, while extensive redistribution of Rev could be attained by inhibition of RNA polymerase I alone, Rev was still capable of inducing expression of HIV structural gene expression under these conditions. Consequently, Rev activity does not appear to be dependent on either an intact nucleolus or the accumulation of the protein in the nucleus.

Cytotoxic Activity of rev Protein of Human Immunodeficiency Virus Type 1 by Nucleolar Dysfunction

The rev protein (Rev) of the human immunodeficiency virus type 1 (HIV-1) is known as a post-transcriptional regulator of viral gene expression. It is located in the cell nucleolus. Transiently expressed Rev caused nucleolar ballooning and deformity with aberrant accumulation of rRNAs, and de novo synthesis of rRNAs decreased dramatically in these cells. However, similarly expressed rex protein (Rex) of the human T-cell leukemia virus type I, which is a functional homologue to Rev, did not affect nucleolar structure and function. Rev expression resulted in cell death with nucleolar destruction in an inducible cell line. Analysis of Rev routants revealed that both the nucleolar targeting signal of Rev and the multimerization domain are prerequisites to the nucleolar disintegration by Rev. Human T-cells acutely infected with HIV-1 contained nucleoli which were deformed and filled with Rev, but chronically infected cells had intact nucleoli. Involvement of Rev in cytopathic effects in HIV-1 infection is discussed.

Interpreting nucleolar structure: where are the transcribing genes?

ABSTRACT The nucleolus, the site of ribosomal RNA synthesis, provides us with one of the best opportunities for unravelling the relationships between transcription and the spatial organization of chromatin. Some of the best visualizations of the transcription process have come from nucleolar spread preparations (Miller, 1981) but this procedure introduces major structural reorganizations that deny us a molecular interpretation of the intact nucleolus.

Proteins and RNA in mouse L cell core nucleoli and nucleolar matrix

When intact nucleoli were prepared in the presence of enough leupeptin and phenylmethanesulfonyl fluoride to inhibit protease action, electrophoretic patterns of their constituent proteins were reproducible and very similar for L, HeLa, CHO, and rat hepatoma cells. "Core nucleoli", defined as that nucleolar fraction which remains after extensive DNase I action, had a protein composition similar to that of crude intact nucleoli, but were enriched for snRNA U3. Core nucleolar proteins included all of the histones, ribosomal proteins, and phosphorylated proteins with mobilities corresponding to 110 (protein C23) and 160 kilodaltons (kDa). The presence of protein C23 and of lamins A and C in nucleoli and core nucleoli was further verified by reaction with specific antibodies after one- or two-dimensional electrophoresis. A class of higher molecular weight proteins, ranging from 70 to greater than 200 kDa by mobility, was observed. It included at least 25 specific proteins, almost all of them highly acidic (pI less than 3.5). Treatment of core nucleoli with ethylenediaminetetraacetic acid/hypotonic buffer solubilized 30-35% of the small and large molecular weight proteins. In contrast, washing core nucleoli with 2 M NaCl selectively released U3 snRNA, 95% of the ribosomal RNA, and about half of the proteins, including C23 and most of the histones, ribosomal proteins, and other lower molecular weight proteins. The fraction remaining insoluble, "nucleolar matrix", was enriched for proteins of 34 and 57 kDa, lamins A and C, and most higher molecular weight proteins, as well as a portion of ribosomal spacer DNA.

Isolation of an MSP1-derived transcriptionally active nucleolar particle

A discrete macromolecular structure with inherent transcription activity has been isolated from Novikoff nucleoli. The particle (350 Å diameter) was released from the intact nucleoli by digestion with restriction endonuclease (Msp1) and is enriched in repetitive DNA sequences both 5′ and 3′ to the ribosomal genes. In vitro transcription analyses suggest that the particle contains the rDNA polymerase template, and that its 12–15 major proteins are responsible for transcriptional activity without added soluble protein factors.

Effect of human placental ribonuclease inhibitor in cell-free ribosomal RNA synthesis

Exposure to purified placental ribonuclease inhibitor caused a marked reduction in ribonuclease activity associated with intact nucleoli. Addition of the placental RNase inhibitor to a nucleolar RNA synthesizing system, prior to the addition of ribonucleoside triphosphates, resulted in a significant increase in the size but did not alter the yield of the RNA products. The effect of the RNase inhibitor on the size of distribution of in, vitro transcriptional products could be reversed by the addition of N-ethylmaleimide after transcription was terminated. The properties of the ribonnclease(s) affected by the inhibitor were indicated by its resistance to N-ethylmaleimide and EDTA inhibition and by its limited endonucleolytic cleavage of the in, vitro transcript.

Termination of transcription in nucleoli isolated from Tetrahymena.

Correct termination of transcription of the rRNA gene in nucleoli isolated from Tetrahymena is dependent on a protein factor with a molecular weight between 50,000 and 100,000. At low ionic strength the endogenous RNA polymerase synthesizes a transcript identical in size to the precursor rRNA extracted from cells. In the presence of ammonium sulfate, however, the RNA polymerase reads through the normal termination point as demonstrated by size and hybridization studies of the transcript. After ammonium sulfate treatment, rDNA associated with chromosomal proteins (r-chromatin) can be separated from the termination factor by differential centrifugation. The endogenous RNA polymerase on the salt-treated r-chromatin is no longer able to recognize the normal terminator even at low ionic strength. Normal termination properties can be reconstituted by complementation with intact nucleoli or with a protein factor extracted from nucleoli.

Studies on the specificity of preribosomal RNA transcription in nucleoli after selective deproteinization.

Fidelity of preribosomal RNA transcription in vitro was studied after selective deproteinization of nucleoli using either sequential salt extraction or sodium deoxycholate treatment. Homochromatography fingerprinting and identification of marker oligonucleotides from a T1 ribonuclease digest of the transcripts were used to evaluate the RNA products. These studies indicated that: (1) nucleoli retained their endogenous RNA polymerase I activity and the specificity of transcription up to 0.6 M NaCl extraction; (2) exogenous RNA polymerase I transcribed nucleolar chromatin only after 1.0 M NaCl extraction and the transcription pattern, like that of totally deproteinized DNA, was completely random; (3) extraction of nucleoli with deoxycholate resulted in a DNP complex in which the endogenous RNA polymerase I transcribed pre-rRNA specifically; however, it also initiated random transcription, producing a "mixed" fingerprint pattern on the homochromatogram. The random transcription was selectively inhibited either by deoxycholate or rifampicin AF/013. These studies indicate that the selectivity of pre-rRNA transcription is due both to the endogenous RNA polymerase I molecules that were involved in transcription in vivo and are tightly bound to the template and to factors in intact nucleoli which prevent random transcription by the released RNA polymerase I molecules.

Effect of Sarkosyl on the fidelity of preribosomal RNA synthesis in isolated nucleoli

When nucleoli isolated from Novikoff hepatoma ascites cells were treated with 2% Sarkosyl, 95% of the nucleolar proteins were extracted. In addition to RNA polymerase I, a heterogeneous group of proteins remained bound to DNA. The homochromatography fingerprinting analysis of the transcripts of the Sarkosyl pellet had virtually all of the marker oligonucleotides that are found in transcripts from whole nucleoli. In addition, seven discrete spots were present which were not found either in transcripts from intact nucleoli or in 45S pre-rRNA labeled in vivo . These results suggest that the endogenous RNA polymerase I present in the Sarkosyl pellet may have transcribed sequences juxtaposed to pre-rRNA coding region in addition to faithfully transcribing the ribosomal genes. It is possible that Sarkosyl extracted factor(s) necessary for proper termination or processing of pre-rRNA.

Polyamine stimulation of phosphorylation of nonhistone acidic protein in nuclei and nucleoli from Physarum polycephalum.

Intact isolated nuclei and nucleoli from the slime mold Physarum polycephalum incorporated inorganic [32P]phosphate into the phenol-soluble acidic nuclear proteins when incubated for 1 h in a phosphorylation mixture consisting of [32P]phosphate (pH 6.8), glucose, Mg2+, and ATP. The polyamines spermidine and spermine, or equimolar combinations of these compounds with putrescine, at 1 mM total amine concentration, stimulated phosphorylation of these nuclear proteins compared to control experiments lacking these compounds. The magnitude of enhanced phosphorylation ranged from 6-fold to 30-fold depending on the combination of polyamines employed and the given nuclear preparation. Magnesium ion could not substitute for the polyamines in effecting this stimulation. Fractionation of the phosphorylated proteins by polyacrylamide gel electrophoresis in sodium dodecylsulfate revealed that different polypeptides among them were phosphorylated by nuclei in the presence, versus the absence, of the polyamines. In the presence of all three polyamines, each at 0.33 mM, enhanced phosphorylation was primarily due to [32P]-phosphate incorporation into four major nuclear proteins. These proteins had molecular weights of 14000, 27000, 52000, and 70000, respectively. In the absence of the polyamines, three major proteins were phosphorylated with molecular weights of 17500, 21200, and 25000, respectively. The polypeptides of 52000 and 70000 molecular weight were also selectively phosphorylated in, and isolated from, intact nucleoli after incubation in phosphorylation mixtures supplemented with 1 mM total polyamines. The [32P]phospho-protein linkage of the 32P-containing substances in polyacrylamide gels was established by the capacity of pronase to eliminate these protein bands and by their insensitivity to nuclease digestion. Polyamine stimulation of the phosphorylation of these nuclear proteins was unaltered by 2-propynylamine, an irreversible inhibitor of amine oxidase. Moreover, H2O2 did not stimulate phosphorylation of these proteins by intact nuclei in which amine oxidase was inhibited by 2-propynylamine.

A light- and electron-microscope study of nuclear structure throughout the cell cycle in the euglenoid Astasia longa (Jahn).

The structure of nuclei of Astasia longa in synchronized cultures was examined at the light- and electron-microscope levels. Three types of nuclei, differing mainly in chromatin conformation, were observed during interphase and were tentatively classed in the G1, S and G2-periods. The fibrillar nucleolar regions exhibited a most complex organization and appeared to consist of convoluted, coarse filaments or nucleolonemata approximately 0.15 micrometer in diameter. Chromosome condensation was evidenced first by the longer, thicker profiles of chromatin observed in late prophase. Furthermore, the nucleolus, that persists throughout mitosis, began to elongate at late prophase. Furthermore, the nucleolus, that persists thorughout mitosis, began to elongate at this stage, simultaneously with the appearance of short, unoriented profiles of intranuclear microtubules. Chromosome condensation was complete by mid-metaphase and the nucleolus was elongated into a cylindrical shape with irregular extremities. Microtubule profiles were longer than in prophase; they were now oriented parallel to the nucleolus and frequently lay closely appressed to its sides. In anaphase, the chromosomes segregated into 2 groups, one towards each extremity of the dumb-bell-shaped nucleolus. The telophase chromosomes assumed a random orientation with respect to the still intact nucleolus. Throughout the division stages the persiting nucleolus maintained its ultrastructural organization and consisted partly of conspicuous nucleolonemal profiles which tended to be oriented along the major axis of this organelle. Nucleolar separation into 2 fragments occurred late in telophase and was followed by a reformation of daughter nuclei and initiation of cell fission during cytokinesis.

Increased RNA polymerase activity in isolated liver nucleoli from thyroidectomized rats treated with triiodothyronine.

In isolated liver nucleoli from thyroidectomized rats the activity of the two RNA polymerase I populations, one of which is active and the other inactive towards the endogenous chromatin template, is greatly enhanced 10 and 24h after a single ip injection triiodothyronine (T3). When the nucleolar enzyme is solubilized and assayed with exogenous DNA as template, it retains, after T3 treatment, the same increase in activity as observed in intact nucleoli. On the contrary, the template availability, as judged by the binding capacity of isolated nucleoli for [3H]actinomycin D, does not appear to be modified by the hormone. These observations support the conclusion that the enhanced nucleolar RNA synthesis following T3 administration is due to an increased activity of the RNA polymerase I itself rather than to a greater availability of ribosomal RNA cistrons. The hormonal stimulation of both nucleolar RNA polymerase activities depends on continuous protein synthesis since it is almost completely abolished by the administration of cycloheximide.