Showing papers on "Nucleolus published in 2006"
TL;DR: Recent progress is discussed in understanding of how both ribosome biosynthesis and some non-ribosomal functions relate to observable nucleolar structure.
221 citations
TL;DR: The nucleolus appears at the center of several trafficking pathways in the nucleus, suggesting a steady state favoring residence of processing factors that are then released in a control- and time-dependent manner.
Abstract: The nucleolus, a large nuclear domain, is the ribosome factory of the cells. Ribosomal RNAs are synthesized, processed and assembled with ribosomal proteins in the nucleolus, and the ribosome subunits are then transported to the cytoplasm. In this review, the structural organization of the nucleolus and the dynamics of the nucleolar proteins are discussed in an attempt to link both information. By electron microscopy, three main nucleolar components corresponding to different steps of ribosome biogenesis are identified and the nucleolar organization reflects its activity. Time-lapse videomicroscopy and fluorescent recovery after photobleaching (FRAP) demonstrate that mobility of GFP-tagged nucleolar proteins is slower in the nucleolus than in the nucleoplasm. Fluorescent recovery rates change with inhibition of transcription, decreased temperature and depletion of ATP, indicating that recovery is correlated with cell activity. At the exit of mitosis, the nucleolar processing machinery is first concentrated in prenucleolar bodies (PNBs). The dynamics of the PNBs suggests a steady state favoring residence of processing factors that are then released in a control- and time-dependent manner. Time-lapse analysis of fluorescence resonance energy transfer demonstrates that processing complexes are formed in PNBs. Finally, the nucleolus appears at the center of several trafficking pathways in the nucleus.
189 citations
TL;DR: The authors' analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces Naf1 to yield mature H/ ACA RNPs in Cajal bodies and nucleoli.
Abstract: Mammalian H/ACA RNPs are essential for ribosome biogenesis, premessenger RNA splicing, and telomere maintenance. These RNPs consist of four core proteins and one RNA, but it is not known how they assemble. By interrogating the site of H/ACA RNA transcription, we dissected their biogenesis in single cells and delineated the role of the non-core protein NAF1 in the process. NAF1 and all of the core proteins except GAR1 are recruited to the site of transcription. NAF1 binds one of the core proteins, NAP57, and shuttles between nucleus and cytoplasm. Both proteins are essential for stable H/ACA RNA accumulation. NAF1 and GAR1 bind NAP57 competitively, suggesting a sequential interaction. Our analyses indicate that NAF1 binds NAP57 and escorts it to the nascent H/ACA RNA and that GAR1 then replaces NAF1 to yield mature H/ACA RNPs in Cajal bodies and nucleoli.
175 citations
TL;DR: RNA-selective dyes with better imaging properties relative to commercially available SYTORNASelect dye were identified, which could help to image the DNA distribution in relation to RNA distribution in live cells and therefore reveal different patterns of RNA-DNA colocalization.
172 citations
TL;DR: Results suggest that miR-206 may associate both with nascent ribosome in the nucleolus and with exported, functional ribosomes in the cytoplasm, which is in contrast to previous findings in muscle tissue in vivo.
Abstract: MicroRNAs are small, ≈21- to 24-nt RNAs that have been found to regulate gene expression. miR-206 is a microRNA that is expressed at high levels in Drosophila, zebrafish, and mouse skeletal muscle and is thought to be involved in the attainment and/or maintenance of the differentiated state. We used locked nucleic acid probes for in situ hybridization analysis of the intracellular localization of miR-206 during differentiation of rat myogenic cells. Like most microRNAs, which are presumed to suppress translation of target mRNAs, we found that miR-206 occupies a cytoplasmic location in cultured myoblasts and differentiated myotubes and that its level increases in myotubes over the course of differentiation, consistent with previous findings in muscle tissue in vivo. However, to our surprise, we also observed miR-206 to be concentrated in nucleoli. A probe designed to be complementary to the precursor forms of miR-206 gave no nucleolar signal. We characterized the intracellular localization of miR-206 at higher spatial resolution and found that a substantial fraction colocalizes with 28S rRNA in both the cytoplasm and the nucleolus. miR-206 is not concentrated in either the fibrillar centers of the nucleolus or the dense fibrillar component, where ribosomal RNA transcription and early processing occur, but rather is localized in the granular component, the region of the nucleolus where final ribosome assembly takes place. These results suggest that miR-206 may associate both with nascent ribosomes in the nucleolus and with exported, functional ribosomes in the cytoplasm.
145 citations
TL;DR: A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis, which is an intriguing observation since the role of these pre-rRNAs is presently unknown.
Abstract: Nucleoli are the prominent contrasted structures of the cell nucleus. In the nucleolus, ribosomal RNAs (rRNAs) are synthesized, processed and assembled with ribosomal proteins. The size and organization of the nucleolus are directly related to ribosome production. The organization of the nucleolus reveals the functional compartmentation of the nucleolar machineries that depends on nucleolar activity. When this activity is blocked, disrupted or impossible, the nucleolar proteins have the capacity to interact independently of the processing activity. In addition, nucleoli are dynamic structures in which nucleolar proteins rapidly associate and dissociate with nucleolar components in continuous exchanges with the nucleoplasm. At the time of nucleolar assembly, the processing machineries are recruited in a regulated manner in time and space, controlled by different kinases and form intermediate structures, the prenucleolar bodies. The participation of stable pre-rRNAs in nucleolar assembly was demonstrated after mitosis and during development but this is an intriguing observation since the role of these pre-rRNAs is presently unknown. A brief report on the nucleolus and diseases is proposed as well as of nucleolar functions different from ribosome biogenesis.
143 citations
TL;DR: It is concluded that the nuclear‐restricted pre‐ribosomes are polyadenylated by TRAMP and degraded by the exosome.
Abstract: We previously hypothesized that HEAT-repeat (Huntington, elongation A subunit, TOR) ribosome synthesis factors function in ribosome export. We report that the HEAT-repeat protein Sda1p is a component of late 60S pre-ribosomes and is required for nuclear export of both ribosomal subunits. In strains carrying the ts-lethal sda1–2 mutation, pre-60S particles were rapidly degraded following transfer to 37°C. Polyadenylated forms of the 27S pre-rRNA and the 25S rRNA were detected, suggesting the involvement of the Trf4p/Air/Mtr4p polyadenylation complex (TRAMP). The absence of Trf4p suppressed polyadenylation and stabilized the pre-rRNA and rRNA. The absence of the nuclear exosome component Rrp6p also conferred RNA stabilization, with some hyperadenylation. We conclude that the nuclear-restricted pre-ribosomes are polyadenylated by TRAMP and degraded by the exosome. In sda1–2 strains at 37°C, pre-40S and pre-60S ribosomes initially accumulated in the nucleoplasm, but then strongly concentrated in a subnucleolar focus, together with exosome and TRAMP components. Localization of pre-ribosomes to this focus was lost in sda1–2 strains lacking Trf4p or Rrp6p. We designate this nucleolar focus the No-body and propose that it represents a site of pre-ribosome surveillance.
137 citations
TL;DR: Nuclear tau associated with AT-rich α-satellite DNA sequences is discussed as a potential molecular link between trisomy 21 and AD and a functional role for nuclear tau is proposed in relation to the nucleolar organization and/or heterochromatinization of a portion of RNA genes.
Abstract: The microtubule-associated tau protein participates in the organization and integrity of the neuronal cytoskeleton. A nuclear form of tau has been described in neuronal and non-neuronal cells, which displays a nucleolar localization during interphase but is associated with nucleolar-organizing regions in mitotic cells. In the present study, based on immunofluorescence, immuno-FISH and confocal microscopy, we show that nuclear tau is mainly present at the internal periphery of nucleoli, partially colocalizing with the nucleolar protein nucleolin and human AT-rich alpha-satellite DNA sequences organized as constitutive heterochromatin. By using gel retardation, we demonstrate that tau not only colocalizes with, but also specifically binds to, AT-rich satellite DNA sequences apparently through the recognition of AT-rich DNA stretches. Here we propose a functional role for nuclear tau in relation to the nucleolar organization and/or heterochromatinization of a portion of RNA genes. Since nuclear tau has also been found in neurons from patients with Alzheimer's disease (AD), aberrant nuclear tau could affect the nucleolar organization during the course of AD. We discuss nucleolar tau associated with AT-rich alpha-satellite DNA sequences as a potential molecular link between trisomy 21 and AD.
136 citations
TL;DR: This work is the first description of a higher eukaryotic organism with a disrupted nucleolin-like gene and defines a new role for nucleolin in nucleolus structure and rDNA chromatin organization.
Abstract: Nucleolin is one of the most abundant protein in the nucleolus and is a multifunctional protein involved in different steps of ribosome biogenesis. In contrast to animals and yeast, the genome of the model plant Arabidopsis thaliana encodes two nucleolin-like proteins, AtNUC-L1 and AtNUC-L2. However, only the AtNUC-L1 gene is ubiquitously expressed in normal growth conditions. Disruption of this AtNUC-L1 gene leads to severe plant growth and development defects. AtNUC-L1 is localized in the nucleolus, mainly in the dense fibrillar component. Absence of this protein in Atnuc-L1 plants induces nucleolar disorganization, nucleolus organizer region decondensation, and affects the accumulation levels of pre-rRNA precursors. Remarkably, in Atnuc-L1 plants the AtNUC-L2 gene is activated, suggesting that AtNUC-L2 might rescue, at least partially, the loss of AtNUC-L1. This work is the first description of a higher eukaryotic organism with a disrupted nucleolin-like gene and defines a new role for nucleolin in nucleolus structure and rDNA chromatin organization.
122 citations
TL;DR: It is demonstrated that affinity-purified Prp43p-associated material includes the expected spliceosomal components; however, it is also identified several preribosomal complexes that are specifically purified with Prp 43p.
Abstract: The known function of the DEXH/D-box protein Prp43p is the removal of the U2, U5, and U6 snRNPs from the postsplicing lariat-intron ribonucleoprotein complex. We demonstrate that affinity-purified Prp43p-associated material includes the expected spliceosomal components; however, we also identify several preribosomal complexes that are specifically purified with Prp43p. Conditional prp43 mutant alleles confer a 35S pre-rRNA processing defect, with subsequent depletion of 27S and 20S precursors. Upon a shift to a nonpermissive temperature, both large and small-ribosomal-subunit proteins accumulate in the nucleolus of prp43 mutants. Pulse-chase analysis demonstrates delayed kinetics of 35S, 27S, and 20S pre-rRNA processing with turnover of these intermediates. Microarray analysis of pre-mRNA splicing defects in prp43 mutants shows a very mild effect, similar to that of nonessential pre-mRNA splicing factors. Prp43p is the first DEXH/D-box protein shown to function in both RNA polymerase I and polymerase II transcript metabolism. Its essential function is in its newly characterized role in ribosome biogenesis of both ribosomal subunits, positioning Prp43p to regulate both pre-mRNA splicing and ribosome biogenesis.
108 citations
TL;DR: The results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.
Abstract: We have investigated the possible involvement of the ubiquitin-proteasome system (UPS) in ribosome biogenesis. We find by immunofluorescence that ubiquitin is present within nucleoli and also demonstrate by immunoprecipitation that complexes associated with pre-rRNA processing factors are ubiquitinated. Using short proteasome inhibition treatments, we show by fluorescence microscopy that nucleolar morphology is disrupted for some but not all factors involved in ribosome biogenesis. Interference with proteasome degradation also induces the accumulation of 90S preribosomes, alters the dynamic properties of a number of processing factors, slows the release of mature rRNA from the nucleolus, and leads to the depletion of 18S and 28S rRNAs. Together, these results suggest that the UPS is probably involved at many steps during ribosome biogenesis, including the maturation of the 90S preribosome.
TL;DR: The role that chromatin plays in determining the activity status of the genes that encode ribosomal RNAs (rRNAs) is elucidated, including the appearance of NORs as secondary constrictions on metaphase chromosomes (McClintock 1934).
Abstract: Research over the last decade has demonstrated that the regulated packaging of DNA into chromatin is fundamental to keeping genes in an active/open or a more condensed/inactive conformation. Nucleosomes, the basic repeating unit of chromatin, contain two molecules each of canonical histones, H2A, H2B, H3, and H4. These are organized as an H3–H4 tetramer associated with two H2A–H2B dimers. The NH2 termini of histones protrude from the nucleosome, and are the target for a wide array of covalent modifications including acetylation, methylation, and phosphorylation. These modifications are applied and removed in a highly specific manner to generate what has been described as the histone code (Jenuwein and Allis 2001). This code is, in turn, read by chromatin-associated factors. For example, individual acetylated lysine residues are recognized by factors with bromodomains and methylated lysines by chromodomain-containing proteins. Additionally, the action of ATP-dependent chromatin remodeling activities can displace nucleosomes altering the accessibility of DNA within chromatin templates (Flaus and OwenHughes 2001). While most of this work has focused on protein-encoding genes transcribed by RNA polymerase II, more recent studies, including one by Earley et al. (2006) in this issue of Genes & Development, are aimed at elucidating the role that chromatin plays in determining the activity status of the genes that encode ribosomal RNAs (rRNAs). Eukaryotic genomes contain many rRNA gene copies, ranging from hundreds to thousands in some plants, organized in tandem arrays. rRNA genes are transcribed by RNA polymerase I (Pol I) into a precursor RNA (prerRNA) that encodes the three largest RNA components of ribosomes. Pre-rRNA coding sequences are separated by intergenic spacers (IGS) that can range in size from 3 kb in yeast to 30 kb in mammals. Pre-rRNA synthesis is regulated by elements including promoters, transcriptional enhancers, and terminators that are located within the IGS (Grummt 1999). Transcription of rRNA gene arrays results in formation of a nucleolus; consequently, they are termed nucleolar organizer regions (NORs). Importantly, not all NORs are transcriptionally active. Species with multiple NORs can vary the active proportion. Pre-rRNA is matured into 18S, 5.8S, and 28S rRNA by a precisely ordered series of events that includes both cleavages and targeted base modifications. These are required to ensure correct folding of rRNAs for ribosome assembly and function. These multiple steps in ribosome biogenesis require a bewildering array of proteins and small nucleolar RNAs that converge on transcriptionally active NORs, forming such a high concentration of ribonucleoprotein complexes that nucleoli are the most prominent feature of the eukaryotic nucleus. The electron micrographs of Miller and Beatty (1969) demonstrated that the coding regions of active repeats are fully loaded with Pol I. rRNA gene transcription accounts for ∼50% of nascent RNA synthesis in a cell. This high density of transcription and the recent demonstration that it is tightly coupled to pre-rRNA processing (Granneman and Baserga 2004) argues that rRNA gene chromatin needs to be especially accessible. As cells approach metaphase, Pol I transcription is actively shut down and the nucleolus disappears. It is at this point that the specialized nature of active rRNA gene chromatin is most dramatically revealed by the appearance of NORs as secondary constrictions on metaphase chromosomes (McClintock 1934).
TL;DR: Improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering the understanding of the role of nucleoli in different physiological and pathological cell states.
Abstract: Nucleoli are plurifunctional nuclear domains involved in the regulation of several major cellular processes such as ribosome biogenesis, the biogenesis of non-ribosomal ribonucleoprotein complexes, cell cycle, and cellular aging. Until recently, the protein content of nucleoli was poorly described. Several proteomic analyses have been undertaken to discover the molecular bases of the biological roles fulfilled by nucleoli. These studies have led to the identification of more than 700 proteins. Extensive bibliographic and bioinformatic analyses allowed the classification of the identified proteins into functional groups and suggested potential functions of 150 human proteins previously uncharacterized. The combination of improvements in mass spectrometry technologies, the characterization of protein complexes, and data mining will assist in furthering our understanding of the role of nucleoli in different physiological and pathological cell states.
TL;DR: Dyskerin gene silencing in the MCF‐7 human breast carcinoma cell line reduced telomerase activity and rRNA pseudo‐uridylation, and patients with low dyskerin expression were characterized by a better clinical outcome than those with a high Dyskerin level.
Abstract: Dyskerin is a nucleolar protein, altered in dyskeratosis congenita, which carries out two separate functions, both fundamental for proliferating cells. One function is the pseudo-uridylation of ribosomal RNA (rRNA) molecules, necessary for their processing, and the other is the stabilization of the telomerase RNA component, necessary for telomerase activity. A significant feature of dyskeratosis congenita is an increased susceptibility to cancer; so far, however, no data have been reported on dyskerin changes in human tumours. Therefore, in this study, the distribution of dyskerin in a large series of human tumours from the lung, breast, and colon, as well as from B-cell lymphomas, was analysed by immunohistochemistry. Dyskerin proved never to be lost or delocalized outside the nucleolus. A quantitative analysis of dyskerin mRNA expression was then performed in 70 breast carcinomas together with the evaluation of telomerase RNA component levels and rRNA pseudo-uridylation. Dyskerin mRNA levels were highly variable and directly associated with both telomerase RNA component levels and rRNA pseudo-uridylation. Dyskerin gene silencing in the MCF-7 human breast carcinoma cell line reduced telomerase activity and rRNA pseudo-uridylation. Significantly, patients with low dyskerin expression were characterized by a better clinical outcome than those with a high dyskerin level. These data indicate that dyskerin is not lost in human cancers and that the levels of its expression and function are associated with tumour progression.
TL;DR: This work examines the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis and suggests that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A(1) and/or A(2.).
Abstract: Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A1 and/or A2.
TL;DR: This Survey and Summary analyzes the reports regarding the spatial organization of pol III genes and addresses the potential influence of this organization on transcriptional regulation.
Abstract: RNA polymerase III (pol III) transcribes many essential, small, noncoding RNAs, including the 5S rRNAs and tRNAs. While most pol III-transcribed genes are found scattered throughout the linear chromosome maps or in multiple linear clusters, there is increasing evidence that many of these genes prefer to be spatially clustered, often at or near the nucleolus. This association could create an environment that fosters the coregulation of transcription by pol III with transcription of the large ribosomal RNA repeats by RNA polymerase I (pol I) within the nucleolus. Given the high number of pol III-transcribed genes in all eukaryotic genomes, the spatial organization of these genes is likely to affect a large portion of the other genes in a genome. In this Survey and Summary we analyze the reports regarding the spatial organization of pol III genes and address the potential influence of this organization on transcriptional regulation.
TL;DR: It is suggested that sumoylation of preribosomal particles in the nucleus and subsequent desumoylation at the NPC is necessary for efficient ribosome biogenesis and export in eukaryotes.
Abstract: Ribosomal precursor particles are initially assembled in the nucleolus prior to their transfer to the nucleoplasm and export to the cytoplasm. In a screen to identify thermosensitive (ts) mutants defective in the export of pre-60S ribosomal subunit, we isolated the rix16-1 mutant. In this strain, nucleolar accumulation of the Rpl25-eGFP reporter was complemented by UBA2 (a subunit of the E1 sumoylation enzyme). Mutations in UBC9 (E2 enzyme), ULP1 [small-ubiquitin-related modifier (SUMO) isopeptidase] and SMT3 (SUMO-1) caused 60S export defects. A directed analysis of the SUMO proteome revealed that many ribosome biogenesis factors are sumoylated. Importantly, preribosomal particles along both the 60S and the 40S synthesis pathways were decorated with SUMO, showing its direct involvement. Consistent with this, early 60S assembly factors were genetically linked to SUMO conjugation. Notably, the SUMO deconjugating enzyme Ulp1, which localizes to the nuclear pore complex (NPC), was functionally linked to the 60S export factor Mtr2. Together our data suggest that sumoylation of preribosomal particles in the nucleus and subsequent desumoylation at the NPC is necessary for efficient ribosome biogenesis and export in eukaryotes.
TL;DR: The data show that nucleolar localization of CTCF is associated with growth arrest and it is demonstrated that the central zinc-finger domain of C TCF is the region responsible for nucleolar targeting.
Abstract: Multiple functions have been reported for the transcription factor and candidate tumour suppressor, CTCF. Among others, they include regulation of cell growth, differentiation and apoptosis, enhancer-blocking activity and control of imprinted genes. CTCF is usually localized in the nucleus and its subcellular distribution during the cell cycle is dynamic; CTCF was found associated with mitotic chromosomes and the midbody, suggesting different roles for CTCF at different stages of the cell cycle. Here we report the nucleolar localization of CTCF in several experimental model systems. Translocation of CTCF from nucleoplasm to the nucleolus was observed after differentiation of K562 myeloid cells and induction of apoptosis in MCF7 breast cancer cells. CTCF was also found in the nucleoli in terminally differentiated rat trigeminal ganglion neurons. Thus our data show that nucleolar localization of CTCF is associated with growth arrest. Interestingly, the 180 kDa poly(ADP-ribosyl)ated isoform of CTCF was predominantly found in the nucleoli fractions. By transfecting different CTCF deletion constructs into cell lines of different origin we demonstrate that the central zinc-finger domain of CTCF is the region responsible for nucleolar targeting. Analysis of subnucleolar localization of CTCF revealed that it is distributed homogeneously in both dense fibrillar and granular components of the nucleolus, but is not associated with fibrillar centres. RNA polymerase I transcription and protein synthesis were required to sustain nucleolar localization of CTCF. Notably, the labelling of active transcription sites by in situ run-on assays demonstrated that CTCF inhibits nucleolar transcription through a poly(ADP-ribosyl)ation-dependent mechanism.
Journal Article•
TL;DR: It is concluded that the dominant-negative effect of the M1 and M5 mutants is mediated by the impaired function of the PeBoW-complex.
Abstract: The nucleolar PeBoW-complex, consisting of Pes1, Bop1 and WDR12, is essential for cell proliferation and processing of ribosomal RNA in mammalian cells. Here we have analysed the physical and functional interactions of Pes1 deletion mutants with the PeBoW-complex. Pes1 mutants M1 and M5, with N- and C-terminal truncations, respectively, displayed a dominant-negative phenotype. Both mutants showed nucleolar localization, blocked processing of the 36S/32S precursors to mature 28S rRNA, inhibited cell proliferation, and induced high p53 levels in proliferating, but not in resting cells. Mutant M1 and M5 proteins associated with large pre-ribosomal complexes and co-immunoprecipitated Bop1 and WDR12 proteins indicating their proper incorporation into the PeBoW-complex. We conclude that the dominant-negative effect of the M1 and M5 mutants is mediated by the impaired function of the PeBoW-complex.
TL;DR: Nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export.
Abstract: The nucleolus is the largest subnuclear structure and is plurifunctional in nature. Here, we demonstrate that nucleolar localization of a key herpesvirus regulatory protein is essential for its role in virus mRNA nuclear export. The herpesvirus saimiri ORF57 protein is a nucleocytoplasmic shuttle protein that is conserved in all herpesviruses and orchestrates the nuclear export of viral intronless mRNAs. We demonstrate that expression of the ORF57 protein induces nucleolar redistribution of human TREX (transcription/export) proteins that are involved in mRNA nuclear export. Moreover, we describe a previously unidentified nucleolar localization signal within ORF57 that is composed of two distinct nuclear localization signals. Intriguingly, point mutations that ablate ORF57 nucleolar localization lead to a failure of ORF57-mediated viral mRNA nuclear export. Furthermore, nucleolar retargeting of the ORF57 mutant was achieved by the incorporation of the HIV-1 Rev nucleolar localization signal, and analysis demonstrated that this modification was sufficient to restore viral mRNA nuclear export. This finding represents a unique and fundamental role for the nucleolus in nuclear export of viral mRNA.
TL;DR: It is shown that Cid14 is a Trf4/5 functional homolog in the distantly related fission yeast Schizosaccharomyces pombe, and surprisingly, both fully processed 25S rRNA and rRNA processing intermediates appear to be channeled into this pathway.
Abstract: Polyadenylation in eukaryotes is conventionally associated with increased nuclear export, translation, and stability of mRNAs In contrast, recent studies suggest that the Trf4 and Trf5 proteins, members of a widespread family of noncanonical poly(A) polymerases, share an essential function in Saccharomyces cerevisiae that involves polyadenylation of nuclear RNAs as part of a pathway of exosome-mediated RNA turnover Substrates for this pathway include aberrantly modified tRNAs and precursors of snoRNAs and rRNAs Here we show that Cid14 is a Trf4/5 functional homolog in the distantly related fission yeast Schizosaccharomyces pombe Unlike trf4 trf5 double mutants, cells lacking Cid14 are viable, though they suffer an increased frequency of chromosome missegregation The Cid14 protein is constitutively nucleolar and is required for normal nucleolar structure A minor population of polyadenylated rRNAs was identified These RNAs accumulated in an exosome mutant, and their presence was largely dependent on Cid14, in line with a role for Cid14 in rRNA degradation Surprisingly, both fully processed 25S rRNA and rRNA processing intermediates appear to be channeled into this pathway Our data suggest that additional substrates may include the mRNAs of genes involved in meiotic regulation Polyadenylation-assisted nuclear RNA turnover is therefore likely to be a common eukaryotic mechanism affecting diverse biological processes
TL;DR: The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell.
Abstract: Data on the in situ structural-functional organization of ribosomal genes in the mammalian cell nucleolus are reviewed here. Major findings on chromatin structure in situ come from investigations carried out using the Feulgen-like osmium ammine reaction as a highly specific electron-opaque DNA tracer. Intranucleolar chromatin shows three different levels of organization: compact clumps, fibers ranging from 11 to 30 nm, and loose agglomerates of extended DNA filaments. Both clumps and fibers of chromatin exhibit a nucleosomal organization that is lacking in the loose agglomerates of extended DNA filaments. In fact, these filaments constantly show a thickness of 2-3 nm, the same as a DNA double-helix molecule. The loose agglomerates of DNA filaments are located in the fibrillar centers, the interphase counterpart of metaphase NORs, therefore being constituted by ribosomal DNA. The extended, non-nucleosomal configuration of this rDNA has been shown to be independent of transcriptional activity and characterizes ribosome genes that are either transcribed or transcriptionally silent. Data reviewed are consistent with a model of control for ribosome gene activity that is not mediated by changes in chromatin structure. The presence of rDNA in mammalian cells always structurally ready for transcription might facilitate a more rapid adjustment of the ribosome production in response to the metabolic needs of the cell.
TL;DR: The isolation of the WTM (WD40-containing transcriptional modulator) proteins are reported as regulators of Rnr2/Rnr4 localization and increased hydroxyurea (HU)-resistance in mec1 mutants.
Abstract: The control of deoxyribonucleotide levels is essential for DNA synthesis and repair. This control is exerted through regulation of ribonucleotide reductase (RNR). One mode of RNR regulation is differential localization of its subunits. In Saccharomyces cerevisiae, the catalytic subunit hererodimer, Rnr2/Rnr4, is localized to the nucleus while its regulatory subunit, Rnr1, is cytoplasmic. During S phase and in response to DNA damage, Rnr2-Rnr4 enters the cytoplasm, where it presumably combines with Rnr1 to form an active complex. The mechanism of its nuclear localization is not understood. Here, we report the isolation of the WTM (WD40-containing transcriptional modulator) proteins as regulators of Rnr2/Rnr4 localization. Overproduction of Wtm2 increased Rnr2/Rnr4. Deletion of WTM1, a homolog of WTM2, leads to the cytoplasmic localization of Rnr2/Rnr4, and increased hydroxyurea (HU)-resistance in mec1 mutants. Wtm1 binds Rnr2/4 complexes and release them to the cytoplasm in response to DNA damage. Forced localization of Wtm1 to the nucleolus causes Rnr2/Rnr4 complexes to relocalize to the nucleolus. Thus, Wtm1 acts as a nuclear anchor to maintain nuclear localization of Rnr2/4 complexes outside of S phase. In the presence of DNA damage this association is disrupted and Rnr2/Rnr4 become cytoplasmic, where they join with Rnr1 to form an intact complex.
TL;DR: The dynamic localization of the Arabidopsis thaliana SR protein RSZp22 was investigated and it was confirmed that RSp31, a plant-specific SR protein, is dynamically distributed in nucleolar cap-like structures upon phosphorylation inhibition.
Abstract: Serine/arginine-rich (SR) proteins are splicing regulators that share a modular structure consisting of one or two N-terminal RNA recognition motif domains and a C-terminal RS-rich domain. We investigated the dynamic localization of the Arabidopsis thaliana SR protein RSZp22, which, as we showed previously, distributes in predominant speckle-like structures and in the nucleolus. To determine the role of RSZp22 diverse domains in its nucleolar distribution, we investigated the subnuclear localization of domain-deleted mutant proteins. Our results suggest that the nucleolar localization of RSZp22 does not depend on a single targeting signal but likely involves different domains/motifs. Photobleaching experiments demonstrated the unrestricted dynamics of RSZp22 between nuclear compartments. Selective inhibitor experiments of ongoing cellular phosphorylation influenced the rates of exchange of RSZp22 between the different nuclear territories, indicating that SR protein mobility is dependent on the phosphorylation state of the cell. Furthermore, based on a leptomycin B- and fluorescence loss in photobleaching-based sensitive assay, we suggest that RSZp22 is a nucleocytoplasmic shuttling protein. Finally, with electron microscopy, we confirmed that RSp31, a plant-specific SR protein, is dynamically distributed in nucleolar cap-like structures upon phosphorylation inhibition. Our findings emphasize the high mobility of Arabidopsis SR splicing factors and provide insights into the dynamic relationships between the different nuclear compartments.
TL;DR: A brief history of nucleolus research and new concepts and unresolved questions is presented and hepatitis D virus is introduced for studying the communication between theucleolus and other subnuclear compartments, and Caenorhabditis elegans for the role of nucleoli in the development and the epistatic control of nucleologenesis are introduced.
Abstract: The nucleolus is the most prominent compartment in the nucleus and known as the site for ribosome biogenesis in eucaryotes. In contrast, there is no such equivalent structure for ribosome synthesis in procaryotes. This raises two concerns that how does the nucleolus evolve and that whether the nucleolus remains playing a single role in ribosome biogenesis along the evolution. Increasing data support new nucleolus functions, including signal recognition particle assembly, small RNA modification, telomerase maturation, cell-cycle and aging control, and cell stress sensor. Multiple functions of the nucleolus possibly result from the plurifunctionality of nucleolar proteins, such as nucleolin and Nopp140. Proteomic analyses of human and Arabidopsis nucleolus lead a remarkable progress in understanding the evolution and new functions of nucleoli. In this review, we present a brief history of nucleolus research and new concepts and unresolved questions. Also, we introduce hepatitis D virus for studying the communication between the nucleolus and other subnuclear compartments, and Caenorhabditis elegans for the role of nucleolus in the development and the epistatic control of nucleologenesis.
TL;DR: Changes in the intracellular localization of RECQL4 in response to oxidative stress are demonstrated and an interaction betweenRECQL4 and PARP-1 is identified, a nuclear enzyme that promotes genomic integrity through its involvement in DNA repair and signaling pathways.
TL;DR: The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response.
Abstract: The p53/p14ARF/mdm2 stress response pathway plays a central role in mediating cellular responses to oncogene activation, genome instability, and therapy-induced DNA damage. Abrogation of the pathway occurs in most if not all cancers, and may be essential for tumor development. The high frequency with which the pathway is disabled in cancer and the fact that the pathway appears to be incompatible with tumor cell growth, has made it an important point of focus in cancer research and therapeutics development. Recently, Nucleophosmin (NPM, B23, NO38 and numatrin), a multifunctional nucleolar protein, has emerged as a p14ARF binding protein and regulator of p53. While complex formation between ARF and NPM retains ARF in the nucleolus and prevents ARF from activating p53, DNA damaging treatments promote a transient subnuclear redistribution of ARF to the nucleoplasm, where it interacts with mdm2 and promotes p53 activation. The results add support to a recently proposed model in which the nucleolus serves as a p53-uspstream sensor of stress, and where ARF links nucleolar stress signals to nucleoplasmic effectors of the stress response. A better understanding of ARF’s nucleolar interactions could further elucidate the regulation of the p53 pathway and suggest new therapeutic approaches to restore p53 function.
TL;DR: A nucleolar phosphoprotein B23 is identified as a protein binding with the core protein of JEV but not with that of dengue virus, and overexpression of dominant negatives of B23 reduced JEV replication.
Abstract: Japanese encephalitis virus (JEV) core protein is detected not only in the cytoplasm but also in the nucleoli of infected cells. We previously showed that a mutant JEV lacking the nucleolar localization of the core protein impaired viral replication in mammalian cells. In this study, we identified a nucleolar phosphoprotein B23 as a protein binding with the core protein of JEV but not with that of dengue virus. The region binding with JEV core protein was mapped to amino acid residues 38 to 77 of B23. Upon JEV infection, some fraction of B23 was translocated from the nucleoli to the cytoplasm, and cytoplasmic B23 was colocalized with the core protein of wild-type JEV but not with that of the mutant JEV. Furthermore, overexpression of dominant negatives of B23 reduced JEV replication. These results suggest that B23 plays an important role in the intracellular localization of the core protein and replication of JEV.
TL;DR: Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N‐protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization.
Abstract: Unlike nuclear localization signals, there is no obvious consensus sequence for the targeting of proteins to the nucleolus. The nucleolus is a dynamic subnuclear structure which is crucial to the normal operation of the eukaryotic cell. Studying nucleolar trafficking signals is problematic as many nucleolar retention signals (NoRSs) are part of classical nuclear localization signals (NLSs). In addition, there is no known consensus signal with which to inform a study. The avian infectious bronchitis virus (IBV), coronavirus nucleocapsid (N) protein, localizes to the cytoplasm and the nucleolus. Mutagenesis was used to delineate a novel eight amino acid motif that was necessary and sufficient for nucleolar retention of N protein and colocalize with nucleolin and fibrillarin. Additionally, a classical nuclear export signal (NES) functioned to direct N protein to the cytoplasm. Comparison of the coronavirus NoRSs with known cellular and other viral NoRSs revealed that these motifs have conserved arginine residues. Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N-protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization. We hypothesise that the N-protein uses these signals to traffic to and from the nucleolus and the cytoplasm.