The nucleolus: an old factory with unexpected capabilities

doi:10.1016/S0962-8924(00)01738-4

Trends in Cell Biology

Volume 10, Issue 5, 1 May 2000, Pages 189-196

https://doi.org/10.1016/S0962-8924(00)01738-4 Get rights and content

Abstract

The function of the nucleolus as a factory for assembling ribosomal subunits is well established, but many unrelated activities have been discovered over the past decade. Our understanding of the dynamics of nucleolar structure and its reassembly at the end of mitosis has recently advanced and the small nucleolar RNAs have been shown to be major players in the processing and modification of preribosomal RNA. Unexpectedly, the nucleolus also seems to play a role in nuclear export, sequestering regulatory molecules, modifying small RNAs, assembling ribonucleoprotein (RNP) and controlling aging.

Section snippets

The variability of nucleolar structure

The structure of the interphase nucleolus is organized around the tandemly repeated genes for preribosomal RNA (these genes are called rDNA; see Box 1). Transcription of these genes generates two structures that are found in all nucleoli: the dense fibrillar component (DFC) and the granular component² (GC). The first of these contains newly synthesized preribosomal RNA (pre-rRNA) and a collection of proteins. The second is made up of nearly completed preribosomal particles destined for the

After massive mitotic destruction, how is the factory reassembled?

One of the most remarkable features of the cell cycle is the disassembly of the nucleolus during mitosis and its subsequent reassembly as the daughter cells proceed into interphase. The arrest of rDNA transcription² and the suppression of pre-rRNA processing¹⁰ accompany the structural disassembly of the nucleolus. The reactivation of these processes late in mitosis provides a unique system for studying nucleologenesis.

A curious feature of mitosis is the separation of certain nucleolar

Many components participate in ribosome assembly

The close coupling of pre-rRNA transcription and early events in processing is suggested by the presence of electron-dense ‘terminal balls’ near the leading ends of nascent pre-rRNA transcripts¹⁸. The terminal balls contain U3 small nucleolar RNA (snoRNA) and a number of unidentified proteins; these structures might be equivalent to the ‘processomes’, which are complexes of processing proteins and snoRNAs¹⁹. Although there is evidence that the first processing event occurs before the pre-rRNA

The nucleolus as a subnuclear storage depot and export clearinghouse

One of the more surprising findings of the past decade is the discovery of a variety of macromolecules in the nucleolus with no apparent function in ribosome assembly. Of these, the HIV-1 Rev protein initially attracted the most attention³⁴, with the obvious question of what the nucleolus is doing in Rev function. Although Rev can regulate HIV-1 mRNA splicing without ever visiting the nucleolus, the possibility that nucleolar location is important for optimal Rev function cannot be excluded

Activities not related to ribosome biogenesis occur within the nucleolus

A handful of cellular processes not involved in conventional nucleolar functions seem to take place in the nucleolus⁴³. The first of these is the maturation of signal recognition particle (SRP) RNA. When SRP RNA is injected into the nuclei of mammalian cells, it initially and rapidly localizes to nucleoli, after which there is a gradual decline in nucleolar signal and an increase in its presence in the cytoplasm⁴³. In addition, three of the SRP proteins have been found in the nucleolus⁴⁵. These

The enigmatic relationship between the nucleolus and the coiled body

The coiled body (CBs), more recently named the Cajal body, was originally called the nucleolar accessory body, implying that the CB and the nucleolus are in some way related⁴⁹. The juxtaposition of the CB and the nucleolus is illustrated in Fig. 1. A functional interaction between them is suggested by the finding that expressing mutant forms of coilin can disrupt the nucleolar structure; conversely, mutants of the nucleolar protein Nopp140 affect CB structure⁴⁹. However, other studies indicate

Is the nucleolus a control valve on the fountain of youth?

One of the most fascinating findings in recent years is the possible link between aging and the nucleolus⁵⁵. The initial finding was that a mutation in the SIR4 gene extended the life span of yeast. Subsequent studies revealed that the silencing proteins Sir3p and Sir4p relocate from telomeres to nucleoli in this mutant strain. This relocation is dependent on another gene, UTH4, which extends life span. Even more interestingly, the Sir complex also relocates from telomeres to nucleoli in aging

Concluding remarks

The past few years have brought remarkable progress in our understanding of nucleolar structure and dynamics and of traditional and non traditional nucleolar functions. At the same time, the recent discoveries raise intriguing questions. Is a well-organized nucleolus necessary for ribosome biogenesis? Is it possible that the real reason for the nucleolar structure is for the newly uncovered, nontraditional roles? Some of the new activities might need an immobile platform, thereby taking

Acknowledgements

We thank the following individuals for providing results prior to publication and for stimulating discussions: F. Amalric, J. Aris, S. Baserga, H. Busch, P. Chan, K. Collins, E. Egyhazi, D. Engelke, I. Grummt, D. Hernandez-Verdun, K. Hingorani, A. Hopper, A. Lamond, F. Medina, T. Misteli, W. Mosgoeller, T. Pederson, I. Ras̆ka, U. Scheer, M. Schmidt-Zachmann, C. Schoefer, P. Silver, A. Tartakoff and M. Terns. We thank Romie Brown for assistance in typing the manuscript. We apologize to those

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Considered the brain of the nucleus, alterations in the structure and function of the nucleolus are linked to numerous cellular functions and, consequently, contribute to several diseases. The identification of nucleolar morphology and activity via novel biomarkers presents new avenues for the development of therapeutic approaches for a variety of human diseases, including cancer, neurodegeneration, and aging. Therefore, specific detection of the nucleolus with fluorescence probes is of critical importance for clinical applications. In the present study, a series of benzoxazole-N,N-dialkylphenylamines derivative compounds were designed and synthesized based on the benzothiazole-based fluorescence probe Thioflavin T (ThT). Among the compounds, BX-3 and BX-16, which carry electron-withdrawing substituents in the benzoxazole ring, were observed to have higher fluorescence emission at wavelengths of 470 and 465 nM, respectively. The general morphology and divisions of the cells were examined under inverted and light microscopes, respectively, and the fluorescence potentials of selected compounds were determined using immunofluorescence microscopy. The fluorescence intensity of molecules and 3D interactive surface plot images of cells were analyzed using ImageJ software. Cell imaging analyses showed that BX-6 and BX-13, like ThT, specifically stain the cell nucleolus. Moreover, molecular docking studies showed that the compounds could identify the RNA-rich nucleolus by binding with high affinity to the guanine region in the RNA structure. The results suggest that the compounds may be an initial route in developing specific biosensor compounds for nucleolus imaging.
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The nucleolus was one of the earliest intracellular structures (and the first within the nucleus) to be observed by microscopy, around 1860. Throughout the early twentieth century, cytologists were struck by the consistent presence of nucleoli inside the nuclei of virtually all cells examined, although the number and shape of nucleoli were quite variable (Figure 1). In the 1930s, it was discovered that the formation of the nucleolus is determined by a distinct genetic locus. This genetic element, termed the nucleolus organizer, was found in the 1960s to consist of repeated genes for ribosomal RNA (rRNA). It is an interesting historical fact that the rRNA genes were not only the first ones from eukaryotes to be observed at high resolution in the electron microscope, but also the first genes to be physically isolated, well before the advent of recombinant DNA techniques. Although a considerable amount of cytochemical and ultrastructural research had been done on nucleoli up through the 1950s, a major advance occurred when they were first isolated by cell fractionation in the mid-1960s. This opened the door to the investigation of their protein and RNA components, and accelerated the study of rRNA synthesis and processing.
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Nucleolus is a sub-cellular nuclear structure identified with an optical microscope. Surprisingly, the fluorescent labelling tools for nucleolus remains very scarce compared to other cellular organelles. In recent years, the biorelevance of nucleolus in various diseases receive intensity scrutiny, and therefore high-performance fluorescence stains for nucleolus are warranted. The proximal pH of the cell nucleolus is slightly lower compared to the other nucleoplasmic region. However, differentiation of a minor pH variation with a traditional Henderson-Hasselbalch type pH probe is difficult due to its inferior sensitivity. Harnessing a molecular mechanism for pH-sensing with positive cooperativity, we were able to design Hill-type small-molecular pH probes with a profile. They exhibit a reduced acid-base transition range and better differentiate biorelevant small pH changes. We showcase that such Hill-type pH probes are suitable for selective nucleolus staining. It exhibits fast membrane permeability and applies to live, fixed, and permeated cells. PHN is expected to find applications in nucleolar chemical biological studies.
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The nucleocapsid (N) protein of porcine epidemic diarrhea virus (PEDV), the most important pathogen causing severe diarrhea in piglets, is a highly conserved structural protein. In this study, 5 monoclonal antibodies (McAbs) against the PEDV N-protein were prepared and identified. Three new epitopes, ⁵⁶QIRWRMRRGERI⁶⁷, ³¹⁸GYAQIASLAPNVAALLFGGNVA VRE³⁴² and ³⁹⁸HEEAIYDDV⁴⁰⁶, were firstly identified in the viral N-protein, by using McAbs 3F10, 6A11, and 1C9. The epitope ³⁹⁸HEEAIYDDV⁴⁰⁶ was deleted in SH strain (isolated by our lab) and different between CV777 and YZ strain (isolated by our lab). To study the characters of this epitope, four peptides were synthesized according to the sequence of SH and CV777 and used in the study. The result showed that the 398^th amino acid maybe an important amino acid of the epitope. Biological information analysis showed that the three B cell linear epitopes are highly conserved among different PEDV isolates. In addition, McAb 1C9, which attached to the epitope ³⁹⁸HEEAIYDDV⁴⁰⁶, showed variant reactivity with PEDV CV777, SH, YZ and MS strains. McAb 1C9 reacted with PEDV strains CV777 and YZ, but not with SH which had a deletion from 399 to 410 amino acids in N-protein (No. MK841494). Among the three McAbs, 6A11, 3F10 and 1C9, only 6A11 reacted with porcine transmissible gastroenteritis virus (TGEV) in immunofluorescence assay, therefore the other two could be used to distinguish TGEV and PEDV. These mAbs and their defined epitopes may provide useful tool for the study of the PEDV N-protein structure and function, and facilitate the development of diagnostic methods for PEDV.
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Polyguanylic acid potassium salt (PolyG) has an anti-fibrotic G-quadruplex (G4) structure. It could inhibit the expression of nucleolin, a protein involved in cell proliferation and apoptosis. However, its role in regulating nucleolin in silicosis is still unknown. After instillation of 50 μl of crystalline silica suspension (50 mg/ml) into the trachea of C57BL/6 mice, we show that nucleolin expression is upregulated in mouse pulmonary tissue following the treatment with silica and that PolyG, which were injected 2.5 mg/kg body weight into mice by abdomen, could alleviate pulmonary fibrosis through inhibiting the expression of nucleolin. Further, we demonstrated that the expression of the DNA double-strand break (DSB) marker, γ-H2AX, increased in response to silica treatment. PolyG could efficiently reduce the protein expression of γ-H2AX and decreased the level of fibrosis-related genes, such as Col1a1 and Col3a1, as well as the levels of fibrosis-associated proteins α-SMA and vimentin in the lungs of silica-treated mice. These findings show that PolyG could regulate nucleolin and DNA damage repair to control fibrotic response in experimental silicosis and provide a new target for preventive intervention.
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Trends in Cell Biology

ReviewThe nucleolus: an old factory with unexpected capabilities

Abstract

Section snippets

The variability of nucleolar structure

After massive mitotic destruction, how is the factory reassembled?

Many components participate in ribosome assembly

The nucleolus as a subnuclear storage depot and export clearinghouse

Activities not related to ribosome biogenesis occur within the nucleolus

The enigmatic relationship between the nucleolus and the coiled body

Is the nucleolus a control valve on the fountain of youth?

Concluding remarks

Acknowledgements

Curr. Opin. Cell Biol.

Histochem. Cell Biol.

J. Struct. Biol.

J. Cell Biol.

Prog. Nucleic Acids Res. Mol. Biol.

Curr. Opin. Cell Biol.

Curr. Opin. Cell Biol.

J. Mol. Biol.

J. Gen. Virol.

Proc. Natl. Acad. Sci. U. S. A.

Cell

Proc. Natl. Acad. Sci. U. S. A.

J. Biol. Chem.

Nature

The Nucleolus and Ribosome Biogenesis

The organization of replication and transcription

Science

The nucleolus: an organelle formed by the act of building a ribosome

Curr. Opin. Cell Biol.

3-D organization of ribosomal transcription units after DRB inhibition of RNA polymerase II transcription

J. Cell Sci.

Mutational analysis of the structure and localization of the nucleolus in the yeast Saccharomyces cerevisiae

J. Cell Biol.

Does Saccharomyces need an organized nucleolus?

Chromosoma

Partially processed pre-rRNA is preserved in association with processing components in nucleolus-derived foci during mitosis

Mol. Biol. Cell

Association of nonribosomal nucleolar proteins in ribonucleoprotein complexes during interphase and mitosis

Mol. Biol. Cell

Cell cycle-dependent regulation of RNA polymerase I transcription: the nucleolar transcription factor UBF is inactive in mitosis and early G1

Proc. Natl. Acad. Sci. U. S. A.

Phosphorylation by G1-specific cdk–cyclin complexes activates the nucleolar transcription factor UBF

EMBO J.

In vivo release of mitotic silencing of ribosomal gene transcription does not give rise to precursor ribosomal RNA processing

J. Cell Biol.

Regulation of ribosomal DNA transcription by insulin

Am. J. Physiol.

Ribosomal RNA processing in eukaryotes

The small nucleolar RNAs

Annu. Rev. Biochem.

Analysis of nucleolar transcription and processing domains and pre-rRNA movements by in situ hybridization

Chromosoma

Review
The nucleolus: an old factory with unexpected capabilities

Cell cycle-dependent regulation of RNA polymerase I transcription: the nucleolar transcription factor UBF is inactive in mitosis and early G₁

Phosphorylation by G₁-specific cdk–cyclin complexes activates the nucleolar transcription factor UBF