Nucleoli Of Human Cells Research Articles

Homeotic DUX4 Genes Shape Dynamic Inter-Chromosomal Contacts with Nucleoli in Human Cells.

Nucleoli form interchromosomal contacts with genes controlling differentiation and carcinogenesis. DUX4 genes specify transcription factor possessing two homeodomains. Previously, using Circular Chromosome Conformation Capture (4С) approach on population of cells, it was demonstrated that DUX4 gene clusters form frequent contacts with nucleoli. It was found also that these contacts are almost completely abolished after heat shock treatment. 4C approach as all ligation-mediated methods is capable to detect rather close interactions between chromatin loops in nuclei. In order to independently confirm the formation and the frequency of the contacts in single cells we used FISH approach. Here, we show that DUX genes in single cells form stable contacts in all tested HEK293T cells. During heat shock, DUX4 genes reversibly move 1-3 µm away from the nuclei. We conclude that interchromosomal contacts formed by nucleoli are strong, dynamic, and reversible, providing both the initiation and maintenance of a differentiated state.

Anomalous coarsening of coalescing nucleoli in human cells

Coarsening is a ubiquitous phenomenon in droplet systems near thermodynamic equilibrium—as an increase in droplet size lowers the system’s free energy—however, coarsening of droplets in nonequilibrium systems, such as the cell nucleus, is far from understood. Liquid condensates in the cell nucleus, like nucleoli, form by liquid-liquid phase separation and play a key role in the nuclear organization. In human cells, nucleolar droplets are nucleated at the beginning of the cell cycle and coarsen with time by coalescing with each other. Upon coarsening, human nucleoli exhibit an anomalous volume distribution P(V)∼V−1, which cannot be explained by any existing theory. In this work, we investigate physical mechanisms behind the anomalous coarsening of human nucleoli. Using spinning disk confocal microscopy, we simultaneously record dynamic behavior of nucleoli and their surrounding chromatin before their coalescence in live human cells. We find that nucleolar anomalous coarsening persists during the entire cell cycle. We measure chromatin flows and density between and around nucleoli, as well as relative motion of two nucleoli before they coalesce. We find that, before nucleolar coalescence, chromatin concentration decreases in the space between nucleoli and the nucleoli move faster toward each other, resembling an effective depletion attraction between the coalescing nucleoli. Indeed, our computational simulations of nucleolar dynamics show that short-ranged attraction is sufficient to explain the observed anomalous volume distribution of human nucleoli. Overall, our results reveal a potential physical mechanism contributing to coarsening of human nucleoli. Such knowledge expands our picture of the physical behavior of liquid condensates inside the cell nucleus and our understanding of the dynamic nuclear organization.

The Novel Role of hnRNP UL1 in Human Cell Nucleoli.

hnRNP UL1 plays an important role in cell nuclei, where it is recruited to DNA damage sites and is involved in the repair of DNA double-strand breaks. Furthermore, this protein is known as a transcriptional repressor of RNA polymerase II genes. In the present study, we have shown that hnRNP UL1 is also localized in the nucleoli of human cells. Upon investigating its function, we found that hnRNP UL1 stimulates ribosomal DNA (rDNA) gene transcription. Moreover, we observed that cells with hnRNP UL1 silencing exhibited increased sensitivity to DNA damage. We also showed that hnRNP UL1 interacts with γH2A.X, RPA32, XRCC1, and Chk1 in cell nucleoli, suggesting its involvement in the repair of rDNA damage.

Nucleolar TRF2 attenuated nucleolus stress-induced HCC cell-cycle arrest by altering rRNA synthesis

The nucleolus is an important organelle that is responsible for the biogenesis of ribosome RNA (rRNA) and ribosomal subunits assembly. It is also deemed to be the center of metabolic control, considering the critical role of ribosomes in protein translation. Perturbations of rRNA synthesis are closely related to cell proliferation and tumor progression. Telomeric repeat-binding factor 2 (TRF2) is a member of shelterin complex that is responsible for telomere DNA protection. Interestingly, it was recently reported to localize in the nucleolus of human cells in a cell-cycle-dependent manner, while the underlying mechanism and its role on the nucleolus remained unclear. In this study, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1), a nucleolar protein that is responsible for the nucleolus construction and rRNA synthesis, interacted with TRF2 and mediated the shuttle of TRF2 between the nucleolus and nucleus. Abating the expression of NOLC1 decreased the nucleolar-resident TRF2. Besides, the nucleolar TRF2 could bind rDNA and promoted rRNA transcription. Furthermore, in hepatocellular carcinoma (HCC) cell lines HepG2 and SMMC7721, TRF2 overexpression participated in the nucleolus stress-induced rRNA inhibition and cell-cycle arrest.

Probing the Liquid-Like Nature of Human Nucleoli and their Interaction with the Interphase Chromatin

The nucleolus is a membraneless organelle embedded in chromatin solution inside the cell nucleus. It has recently been reported that nucleoli in frog oocytes and C. elegans embryos form by liquid-liquid phase separation [1,2]. While fusion of nucleoli in human cells has been observed, it is unclear if they are liquid-like or solid-like in nature [3,4]. Moreover, the role of the chromatin polymer in the facilitation of nucleolar fusion is completely unknown. In this work, we investigate the kinetics of the nucleolar fusion in order to determine its physical nature, i.e. aggregation of solid-like particles vs. coalescence of liquid-like droplets. We monitor and analyze the nucleolar shape during fusion events and find it consistent with a coalescence of liquid droplets. Further, we analyzed nucleolar fusion events and found that after fusion, the eccentricity of the new nucleolus decreases, indicating the nucleolus undergoes a structural change and behaves as a liquid-like structure. Historically, the nucleolar assembly in mammalian cells was believed to complete within two hours after mitosis, when the final number of nucleoli forms via de novo assembly as well as fusion events [3]. However, we found that fusion events can also occur later in the cell cycle. To explore the interaction of nucleoli with chromatin, we altered chromatin state using biochemical perturbations. We found that chromatin is indeed involved in the positioning, shape and alignment of nucleoli inside the cell nucleus. 1. Brangwynne, CP et al., Proc. Natl. Acad. Sci., 4434, 2011 2. Berry, J, et. al. Proc. Natl. Acad. Sci., E5237, 2015 3. Savino, TM, et al. J. Cell Biol.,1097, 2001 4. Farley, KI, et al. Chromosoma, 323, 2015

Nucleolar and coiled-body phosphoprotein 1 (NOLC1) regulates the nucleolar retention of TRF2

Telomeric repeat-binding factor 2 (TRF2) was reported to localize in the nucleolus of human cells in a cell cycle-dependent manner; however, the underlying mechanism remains unclear. Here, we found that nucleolar and coiled-body phosphoprotein 1 (NOLC1) interacted with TRF2 and mediated the shuttling of TRF2 between the nucleolus and nucleus in human 293T and HepG2 cells. Ablation of NOLC1 expression increased the number of nuclear TRF2 foci and decreased the nucleolar level of TRF2. Conversely, NOLC1 overexpression promoted the nucleolar accumulation of TRF2. NOLC1 overexpression also increased the number of 53BP1 foci and induced the DNA damage response. In addition, co-expression of TRF2 rescued NOLC1 overexpression-induced cell cycle arrest and apoptosis.

Progesterone Receptor Membrane Component-1 (PGRMC1) nucleolar localization in bovine granulosa cells and its putative interaction with nucleolin.

PGRMC1 is a multifunctional protein that is found in multiple subcellular compartments, suggesting a specific function at each site. Among the several subcellular sites of expression, PGRMC1 was found in the nucleolus of human cells (Ahmad et al. 2009) and bovine zygotes (Luciano et al. 2010). However, the role at this site is not clear. Therefore, the aim of this study is to assess whether PGRMC1 modulates nucleolar function. Immunofluorescence experiments confirmed nucleolar localization in rat spontaneously immortalized granulosa cells, bovine granulosa cells (bGC) and bovine oocytes. Moreover, in bGC PGRMC1 co-localizes with nucleolin, a well-known nucleolar marker exerting important functions within the nucleolus. Additionally, siRNA mediated gene knockdown experiments showed that when PGRMC1 expression is silenced, nucleolin localization shifts from the nucleolus to the nucleoplasm, suggesting a PGRMC1/nucleolin functional association. However, in situ proximity ligation assay did not detect a direct interaction between these two proteins, suggesting the involvement of additional molecules that could mediate PGRMC1/nucleolin interaction. In conclusion, these studies suggest a function for PGRMC1 in nucleolar activity and set the stage for further investigations aimed at dissecting PGRMC1’s molecular mechanisms of action in the nuclear compartment.

Perturbation of nucleo-cytoplasmic transport affects size of nucleus and nucleolus in human cells.

Size regulation of human cell nucleus and nucleolus are poorly understood subjects. 3D reconstruction of live image shows that the karyoplasmic ratio (KR) increases by 30-80% in transformed cell lines compared to their immortalized counterpart. The attenuation of nucleo-cytoplasmic transport causes the KR value to increase by 30-50% in immortalized cell lines. Nucleolus volumes are significantly increased in transformed cell lines and the attenuation of nucleo-cytoplasmic transport causes a significant increase in the nucleolus volume of immortalized cell lines. A cytosol and nuclear fraction swapping experiment emphasizes the potential role of unknown cytosolic factors in nuclear and nucleolar size regulation.

A novel fragile X syndrome mutation reveals a conserved role for the carboxy-terminus in FMRP localization and function.

Loss of function of the FMR1 gene leads to fragile X syndrome (FXS), the most common form of intellectual disability. The loss of FMR1 function is usually caused by epigenetic silencing of the FMR1 promoter leading to expansion and subsequent methylation of a CGG repeat in the 5′ untranslated region. Very few coding sequence variations have been experimentally characterized and shown to be causal to the disease. Here, we describe a novel FMR1 mutation and reveal an unexpected nuclear export function for the C-terminus of FMRP. We screened a cohort of patients with typical FXS symptoms who tested negative for CGG repeat expansion in the FMR1 locus. In one patient, we identified a guanine insertion in FMR1 exon 15. This mutation alters the open reading frame creating a short novel C-terminal sequence, followed by a stop codon. We find that this novel peptide encodes a functional nuclear localization signal (NLS) targeting the patient FMRP to the nucleolus in human cells. We also reveal an evolutionarily conserved nuclear export function associated with the endogenous C-terminus of FMRP. In vivo analyses in Drosophila demonstrate that a patient-mimetic mutation alters the localization and function of Dfmrp in neurons, leading to neomorphic neuronal phenotypes.

Driving nucleolar assembly

In this issue of Genes & Development, Grob and colleagues (pp. 220-230) identify the minimal molecular requirements to assemble a fully functional nucleolus in human cells and demonstrate the importance of the nucleolar transcription factor upstream binding factor (UBF) as a mitotic bookmark at the ribosomal DNA (rDNA).

Construction of synthetic nucleoli in human cells reveals how a major functional nuclear domain is formed and propagated through cell division.

Human cell nuclei are functionally organized into structurally stable yet dynamic bodies whose cell cycle inheritance is poorly understood. Here, we investigate the biogenesis and propagation of nucleoli, sites of ribosome biogenesis and key regulators of cellular growth. Nucleolar and cell cycles are intimately connected. Nucleoli disappear during mitosis, reforming around prominent uncharacterized chromosomal features, nucleolar organizer regions (NORs). By examining the effects of UBF depletion on both endogenous NORs and synthetic pseudo-NORs, we reveal its essential role in maintaining competency and establishing a bookmark on mitotic NORs. Furthermore, we demonstrate that neo-NORs, UBF-binding site arrays coupled with rDNA transcription units, direct the de novo biogenesis of functional compartmentalized neonucleoli irrespective of their site of chromosomal integration. For the first time, we establish the sequence requirements for nucleolar biogenesis and provide proof that this is a staged process where UBF-dependent mitotic bookmarking precedes function-dependent nucleolar assembly.

Abstract 835: hRrp15, a ribosome RNA processing protein, has profound function on nucleoli construction, ribosome biogenesis and cell proliferation.

Abstract Nucleolus is the sub-organelle that controls ribosome biogenesis in cells, and ribosome biogenesis is a highly regulated process that ensures cell growth (increase in biomass) coordinated with cell proliferation. In the nucleolus, ribosomal RNA precursor (47S rRNA) is initially transcribed from rDNA and then subsequently processes into 5.8S, 18S and 28S rRNAs, together with numerous ribosome proteins and nonribosomal factors, to form 60S and 40S pre-ribosomal particles before they are exported to the cytoplasm. A large body of evidence demonstrates that perturbation of rRNA synthesis, process and/or pre-ribosomal factor function impairs ribosome biogenesis and induces cell cycle arrest. This is attributed to nucleolar disruption and diffusion of ribosomal proteins, such as Rpl11 and Rpl5, into the nucleoplasm in which these ribosomal proteins bind to and inhibit MDM2 function, resulting in p53 induction and activation. In this study, we described the identification and characterization of a human protein, hRrp15, that has sequence homology with yeast pre-ribosomal factor, Rrp15p. Previous studies showed that Rrp15p is a ribosome RNA processing protein required for 60S pre-ribosomal particle formation and/or maturation in yeast. Using immunofluorescence and sucrose gradient centrifugation analyses, we demonstrated that hRrp15 was mainly localized at nucleoli in human cells. Depletion of hRrp15 expression disrupted nucleolar structure, inducing diffusion of nucleolar protein, nucleolin, in the nucleus. In contrast to Rrp15p, hRrp15 mainly associated with 60S pre-ribosomal particle but it also presented in 40S pre-ribosomal particle. Consistently, inhibition of hRrp15 expression perturbed nucleolar formation and impaired ribosomal biogenesis in both 60S and 40S pre-ribosomal subunits. In addition, depletion of hRrp15 in human non-transformed cells, RPE1, resulted in the induction of p53 and p21 proteins and cell cycle arrest at G1-G1/S whereas depletion of hRrp15 in human tumor cells, HeLa and MCF7, caused DNA damage response with detectable γ-H2AX and apoptosis. These results indicated that hRrp15 plays a critical role in regulating formation of both 60S and 40S pre-ribosomal particles, ribosome biogenesis, construction of nucleoli, cell growth and cell proliferation in human cells. Hence, hRrp15 may serve as a potential target for cancer therapy. Citation Format: Zhixiong Dong, Changjun Zhu, Wei Jiang. hRrp15, a ribosome RNA processing protein, has profound function on nucleoli construction, ribosome biogenesis and cell proliferation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 835. doi:10.1158/1538-7445.AM2013-835

The Human Homolog of Escherichia coli Endonuclease V Is a Nucleolar Protein with Affinity for Branched DNA Structures

Loss of amino groups from adenines in DNA results in the formation of hypoxanthine (Hx) bases with miscoding properties. The primary enzyme in Escherichia coli for DNA repair initiation at deaminated adenine is endonuclease V (endoV), encoded by the nfi gene, which cleaves the second phosphodiester bond 3′ of an Hx lesion. Endonuclease V orthologs are widespread in nature and belong to a family of highly conserved proteins. Whereas prokaryotic endoV enzymes are well characterized, the function of the eukaryotic homologs remains obscure. Here we describe the human endoV ortholog and show with bioinformatics and experimental analysis that a large number of transcript variants exist for the human endonuclease V gene (ENDOV), many of which are unlikely to be translated into functional protein. Full-length ENDOV is encoded by 8 evolutionary conserved exons covering the core region of the enzyme, in addition to one or more 3′-exons encoding an unstructured and poorly conserved C-terminus. In contrast to the E. coli enzyme, we find recombinant ENDOV neither to incise nor bind Hx-containing DNA. While both enzymes have strong affinity for several branched DNA substrates, cleavage is observed only with E. coli endoV. We find that ENDOV is localized in the cytoplasm and nucleoli of human cells. As nucleoli harbor the rRNA genes, this may suggest a role for the protein in rRNA gene transactions such as DNA replication or RNA transcription.

An MBoC Favorite: High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli

Molecular Biology of the CellVol. 23, No. 11 MBoC 20th Anniversary FavoritesFree AccessAn MBoC Favorite: High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoliSui HuangSui HuangSearch for more papers by this authorPublished Online:13 Oct 2017https://doi.org/10.1091/mbc.e12-02-0155AboutSectionsView PDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmail In celebration of MBoC's first 20 years, members of the Editorial Board, members of the ASCB Council, and others comment on their favorite MBoC papers from the past two decades.This article is my favorite MBoC paper, because I believe that it broke ground for a new field—the study of the role of nucleoli in genome organization. Van Koningsbruggen et al. (2010) demonstrate that parts of all chromosomes are associated with nucleoli in human cells. The association is biased toward more transcriptionally repressive and AT-rich regions of the genome. The patterns of nucleolar-associated chromatins partially overlap with nuclear lamina–associated chromatin domains and are heritable through cell cycles. Nucleoli and nuclear lamina have long been known to nucleate heterochromatin. However, little attention has been directed to exploring possible roles of nucleoli in global genome organization. This article, together with a study published in the same period by Nemeth et al. (2010), provided the first molecular evidence of the extensive interactions between nucleoli and the genome. These findings raise the possibility that nucleoli play a key role in anchoring and organizing the mammalian genome.FOOTNOTESDOI: 10.1091/mbc.E12-02-0155Molecular Biology of the Cell Volume 23 Page 2016.REFERENCES Nemeth A, Conesa A, Santoyo-Lopez J, Medina I, Montaner D, Peterfia B, Solovei I, Cremer T, Dopazo J, Langst G (2010). Initial genomics of the human nucleolus. PLoS Genet 6, e1000889. Crossref, Medline, Google Scholar van Koningsbruggen S, Gierlinski M, Schofield P, Martin D, Barton GJ, Ariyurek Y, den Dunnen JT, Lamond AI (2010). High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli. Mol Biol Cell 21, 3735-3748. Link, Google ScholarFiguresReferencesRelatedDetailsCited byCalreticulin promotes EGF-induced EMT in pancreatic cancer cells via Integrin/EGFR-ERK/MAPK signaling pathway26 October 2017 | Cell Death & Disease, Vol. 8, No. 10 Vol. 23, No. 11 June 01, 20122015-2234 Metrics Downloads & Citations Downloads: 27Citations: 1 History Information© 2012 Huang. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).PDF download

Air1 Zinc Knuckles 4 and 5 and a Conserved IWRXY Motif Are Critical for the Function and Integrity of the Trf4/5-Air1/2-Mtr4 Polyadenylation (TRAMP) RNA Quality Control Complex

In Saccharomyces cerevisiae, non-coding RNAs, including cryptic unstable transcripts (CUTs), are subject to degradation by the exosome. The Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex in S. cerevisiae is a nuclear exosome cofactor that recruits the exosome to degrade RNAs. Trf4/5 are poly(A) polymerases, Mtr4 is an RNA helicase, and Air1/2 are putative RNA-binding proteins that contain five CCHC zinc knuckles (ZnKs). One central question is how the TRAMP complex, especially the Air1/2 protein, recognizes its RNA substrates. To characterize the function of the Air1/2 protein, we used random mutagenesis of the AIR1/2 gene to identify residues critical for Air protein function. We identified air1-C178R and air2-C167R alleles encoding air1/2 mutant proteins with a substitution in the second cysteine of ZnK5. Mutagenesis of the second cysteine in AIR1/2 ZnK1-5 reveals that Air1/2 ZnK4 and -5 are critical for Air protein function in vivo. In addition, we find that the level of CUT, NEL025c, in air1 ZnK1-5 mutants is stabilized, particularly in air1 ZnK4, suggesting a role for Air1 ZnK4 in the degradation of CUTs. We also find that Air1/2 ZnK4 and -5 are critical for Trf4 interaction and that the Air1-Trf4 interaction and Air1 level are critical for TRAMP complex integrity. We identify a conserved IWRXY motif in the Air1 ZnK4-5 linker that is important for Trf4 interaction. We also find that hZCCHC7, a putative human orthologue of Air1 that contains the IWRXY motif, localizes to the nucleolus in human cells and interacts with both mammalian Trf4 orthologues, PAPD5 and PAPD7 (PAP-associated domain containing 5 and 7), suggesting that hZCCHC7 is the Air component of a human TRAMP complex.

Nucleolar Targeting of the Chaperone Hsc70 Is Regulated by Stress, Cell Signaling, and a Composite Targeting Signal Which Is Controlled by Autoinhibition

Hsc70s are constitutively synthesized members of the 70-kDa chaperone family; they are essential for viability and conserved among all organisms. When eukaryotic cells recover from stress, hsc70s accumulate in nucleoli by an unknown mechanism. Our studies were undertaken to characterize the signaling events and the targeting sequence required to concentrate hsc70 in the nucleoli of human cells. Here, we show that pharmacological inhibitors of phosphatidylinositol (PI) 3-kinase and MEK kinases as well as protein-tyrosine phosphatases abolished the stress-dependent nucleolar accumulation of hsc70. Furthermore, to identify the hsc70 nucleolar targeting sequence, green fluorescent protein-tagged fusion proteins with defined segments of hsc70 were generated and their subcellular distribution was analyzed in growing cells. These studies demonstrated that residues 225 to 297 serve as a heat-inducible nucleolar targeting signal. This segment directs green fluorescent protein to nucleoli in response to stress, but fails to do so under nonstress conditions. Fine mapping of the nucleolar targeting signal revealed that it has two separable functions. First, residues 225 to 262 direct reporter proteins constitutively to nucleoli, even without stress. Second, segment 263 to 287 functions as an autoinhibitory element that prevents hsc70 from concentrating in nucleoli when cells are not stressed. Taken together, PI 3-kinase and MEK kinase signaling as well as tyrosine dephosphorylation are essential for the accumulation of hsc70 in nucleoli of stressed cells. This process relies on a stress-dependent composite targeting signal that combines multiple functions.

Plk1-mediated mitotic phosphorylation of PinX1 regulates its stability

PinX1 was originally identified as a Pin2/TRF1-interacting protein that suppresses telomerase activity via its telomerase inhibitor domain (TID) and regulates the nucleolar localization of TRF1 in telomerase-positive cells. In addition to its telomeric localization, PinX1 can be found in the nucleoli of human cells. Our recent studies have shown that PinX1 localizes to the chromosome periphery and kinetochores in mitosis. Depletion of PinX1 results in lagging chromosomes in mitosis and micronuclei in interphase. However, less is known about the post-translational modification of PinX1 in mitosis. Here, we show that Polo-like kinase 1 (Plk1) is a novel interacting protein of PinX1. Plk1 interacts with and phosphorylates PinX1 in vivo and in vitro. Overexpression of Plk1 promotes protein turnover of PinX1, a process that depends on ubiquitin-associated proteasomal degradation. Depletion of Plk1 using siRNA increases the stability of PinX1 at protein level in mitosis. Moreover, Plk1-mediated phosphorylation of PinX1 at five phosphorylation sites is essential for its Plk1-induced degradation. These findings suggest that Plk1 may negatively regulate the stability of PinX1 by mitotic phosphorylation.

Changes in the status of nucleolus during long-term culturing of human HeLa cells

Changes in the immunocytochemical status of the nucleoli during long-term (6-8 months) in vitro culturing of HeLa (carcinoma of the cervix uteri) cells were described using new A3 monoclonal antibodies selectively reacting with human cell nucleoli. The appearance of cells with abnormal location of A3 antigen was paralleled by a significant increase of culture sensitivity to some external factors (protein synthesis inhibition and oxidative stress). The data indicate that location of one of the nucleolar antigens is an indicator of the qualitative status of HeLa cells in the culture.

Reduction of phase thickness is a characteristic reaction of the nucleoli to toxicity as shown by coherent phase microscopy

Optical parameters of human cell nucleoli (HCT116 colorectal cancer cells) in depolymerization of microtubules and depletion of intracellular ATP pool were studied by coherent phase microscopy. These influences were associated with a rapid (recorded within the first minutes) reduction of the phase thickness of the nucleoli. These changes are similar to the nucleolar response to direct inhibitors of transcription. Hence, quantitative parameters of coherent phase microscopy describe common reaction of the nucleolus to stress; reduction of optical thickness of the nucleolus is a component of this reaction.

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

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.