Nucleolar Targeting Research Articles

Nucleolar targeting of coilin is regulated by its hypomethylation state

Coilin, a molecular marker for Cajal bodies (CBs), is a phosphoprotein that contains a cryptic nucleolar localization signal and multiple interacting domains, such as the RG-box. Post-translational symmetrical dimethylation of arginines on the coilin RG-box is required for the recruitment of the survival motor neuron (SMN) protein and splicing small ribonucleoproteins (snRNPs) to CBs. Here, we analyze the role of the methylation state of coilin in the regulation of its localization to the nucleolus. We use the MCF7 MTAP(-/-) cell line, which lacks the gene encoding 5'-methylthioadenosine phosphorylase (MTAP). This is a key enzyme of the methionine salvage pathway. The reduction of the levels of coilin methylation causes disruption of the canonical CBs and coilin redistribution to nucleoplasmic microfoci and to the nucleolus. Intranucleolar coilin is unmethylated and appears restricted to the dense fibrillar component. Interestingly, intranucleolar coilin is not associated with SMN or snRNPs, and does not interfere with global transcriptional activity. Overexpression of wild-type MTAP reverts the intranucleolar localization of coilin and the disruption of CBs to the normal coilin phenotype. Our results suggest the existence of a dynamic flux of coilin between CBs, nucleoplasm and nucleolus, and indicate that coilin methylation plays a key role in this process.

Abstract 3062: Identification and functional analysis of NOL7 tumor suppressor gene nuclear and nucleolar localization signals

Abstract NOL7 is candidate tumor suppressor found within the nucleus and nucleolus. However, the mechanisms regulating its subcellular localization have not been elucidated. An in vitro import assay demonstrated that NOL7 requires cytosolic machinery for active nuclear transport. Using NOL7 deletion constructs and cytoplasmic pyruvate kinase (PK) fusion proteins, three nuclear localization signals (NLSs) were identified. Site-directed mutagenesis of PK fusions and full-length NOL7 defined the functional regions within each NLS. Further characterization revealed that NLS2 and NLS3 were critical for both the rate and efficiency of nuclear targeting. In addition, four basic clusters within NLS2 and NLS3 were independently capable of nucleolar targeting. The dynamics of nucleolar localization were investigated by FRAP and specific nucleic acid depletion. NOL7 is relatively immobile within the nucleolus, but shuttles rapidly between the nucleolus and nucleoplasm. In addition, targeting to the nucleolar compartment was dependent on the presence of RNA, as depletion of total RNA or rRNA resulted in a nucleoplasmic shift of NOL7. Take together, these results identify the minimal sequences required for the regulation of NOL7 subcellular localization, characterizes the contribution of each sequence to NOL7 nuclear and nucleolar dynamics and suggests a functional role for NOL7 in both compartments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3062.

Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity

The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.

Nucleolar Targeting of RelA(p65) Is Regulated by COMMD1-Dependent Ubiquitination

Stimulation of the NF-kappaB pathway can have proapoptotic or antiapoptotic consequences, and one mechanism that determines the outcome is the nuclear distribution of RelA. Certain stress stimuli induce nucleolar accumulation of RelA thereby mediating apoptosis, whereas others induce nucleoplasmic accumulation and inhibition of apoptosis. Here we investigated the mechanisms that regulate the nuclear distribution of RelA, specifically, the role of the ubiquitin/proteasome system. We found that stress-induced nucleolar translocation of RelA is preceded by ubiquitination of the protein. We also found that chemical proteasome inhibitors induce the ubiquitination and nucleolar translocation of RelA and that this is required for the apoptotic response to these agents. We show that the RelA nucleolar localization signal (amino acids 27-30) is a critical domain for ubiquitination of the protein but that the lysine residue within this motif is not a direct target. We show that RelA binds COMMD1, the rate-limiting component of the RelA ubiquitin ligase complex, in response to stress. Furthermore, we show that overexpression of COMMD1 promotes stress-mediated nucleolar targeting of RelA, whereas knockdown of COMMD1 blocks this effect, causing RelA to remain in the nucleoplasm. These data identify a new role for COMMD1 in regulating the nuclear/nucleolar distribution of RelA and suggest that ubiquitination acts as a signal for transport of RelA to the nucleolus. These findings have relevance to the design of chemopreventative/anticancer agents that act by targeting RelA to the nucleolar compartment.

GLTSCR2/PICT-1, a Putative Tumor Suppressor Gene Product, Induces the Nucleolar Targeting of the Kaposi's Sarcoma-Associated Herpesvirus KS-Bcl-2 Protein

KS-Bcl-2, encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), is a structural and functional homologue of the Bcl-2 family of apoptosis regulators. Like several other Bcl-2 family members, KS-Bcl-2 protects cells from apoptosis and autophagy. Using a yeast two-hybrid screen and coimmunoprecipitation assays, we identified a novel KS-Bcl-2-interacting protein, referred to as protein interacting with carboxyl terminus 1 (PICT-1), encoded by a candidate tumor suppressor gene, GLTSCR2. Confocal laser scanning microscopy revealed nucleolar localization of PICT-1, whereas KS-Bcl-2 was located mostly at the mitochondrial membranes with a small fraction in the nucleoli. Ectopic expression of PICT-1 resulted in a large increase in the nucleolar fraction of KS-Bcl-2, and only a minor fraction remained in the cytoplasm. Furthermore, knockdown of endogenous PICT-1 abolished the nucleolar localization of KS-Bcl-2. However, ectopically expressed PICT-1 did not alter the cellular distribution of human Bcl-2. Subsequent analysis mapped the crucial amino acid sequences of both KS-Bcl-2 and PICT-1 required for their interaction and for KS-Bcl-2 targeting to the nucleolus. Functional studies suggest a correlation between nucleolar targeting of KS-Bcl-2 by PICT-1 and reduction of the antiapoptotic activity of KS-Bcl-2. Thus, these studies demonstrate a cellular mechanism to sequester KS-Bcl-2 from the mitochondria and to downregulate its virally encoded antiapoptotic activity. Additional characterization of the interaction of KS-Bcl-2 and PICT-1 is likely to shed light on the functions of both proteins.

Proteomics Analysis of Nucleolar SUMO-1 Target Proteins upon Proteasome Inhibition

Many cellular processes are regulated by the coordination of several post-translational modifications that allow a very fine modulation of substrates. Recently it has been reported that there is a relationship between sumoylation and ubiquitination. Here we propose that the nucleolus is the key organelle in which SUMO-1 conjugates accumulate in response to proteasome inhibition. We demonstrated that, upon proteasome inhibition, the SUMO-1 nuclear dot localization is redirected to nucleolar structures. To better understand this process we investigated, by quantitative proteomics, the effect of proteasome activity on endogenous nucleolar SUMO-1 targets. 193 potential SUMO-1 substrates were identified, and interestingly in several purified SUMO-1 conjugates ubiquitin chains were found to be present, confirming the coordination of these two modifications. 23 SUMO-1 targets were confirmed by an in vitro sumoylation reaction performed on nuclear substrates. They belong to protein families such as small nuclear ribonucleoproteins, heterogeneous nuclear ribonucleoproteins, ribosomal proteins, histones, RNA-binding proteins, and transcription factor regulators. Among these, histone H1, histone H3, and p160 Myb-binding protein 1A were further characterized as novel SUMO-1 substrates. The analysis of the nature of the SUMO-1 targets identified in this study strongly indicates that sumoylation, acting in coordination with the ubiquitin-proteasome system, regulates the maintenance of nucleolar integrity.

Nucleolar binding of an anti‐NMDA receptor antibody in hydra: a non‐canonical role for an NMDA receptor protein?

Two emerging concepts in cell biology are the back-and-forth trafficking of receptor proteins and nuclear transcription factors between the nucleus and the cell membrane, and the alternative splicing of messenger RNA to produce similar proteins targeted to different cell sites. Recent evidence suggests that the nucleolus is a dynamic structure whose components may be involved in both types of trafficking. In the nervous system of higher animals, the N-methyl-D-aspartate (NMDA)-specific glutamate receptor has various roles in development and cell communication. It is involved in learning, memory, axonal guidance and nerve regeneration. We have reported earlier that the NR1 subunit of the NMDA receptor is present in the cell periphery and the nucleus of stem cells, neurons and epitheliomuscular cells of the early-evolved cnidarian, Hydra vulgaris (Scappaticci et al., 2004. Cell Tissue Res 316:263-270); it is involved in coordinating hydra's neuroeffector systems (Kass-Simon and Scappaticci, 2003. Hydrobiologia 530/531:67-71; Pierobon et al., 2004. Eur J Neurosci 20:2598-2604; Scappaticci and Kass-Simon, 2008. Comp Biochem Physiol A 150:415-422; Kay and Kass-Simon, 2009. Bio Bull 216:113-129). Here we report immunocytochemical experiments, using a mouse monoclonal antibody raised against the mammalian NR1 receptor subunit, and an in silico genomic and gene expression analysis identifying the homologues in hydra of mammalian NR1 and fibrillarin (FBL) genes, and their expressed proteins. The experiments reveal that the NR1 antibody specifically labels the nucleoli of large and small interstitial cells (stem cells), nematoblasts, neuroblasts, and epitheliomusclar cells; antibody labeling of the nucleolar marker, FBL, confirms the nucleolar localization of NR1 antibody labeling. Genomic analysis reveals that NR1 and FBL genes are conserved in hydra, and suggests that there are at least two NR1 splice variants, one of which contains both nuclear and nucleolar targeting signals. The finding that an NR1 receptor subunit (or a portion of it) appears in nucleoli of hydra cells is unique, and has not been reported for any other organism. Its presence in nucleoli of hydra may signal the existence of a yet-undescribed shuttle mechanism between the cell surface and the nucleous, or the alternative deployment of NR1 splice variants to different cell sites.

Avian reovirus sigmaA localizes to the nucleolus and enters the nucleus by a nonclassical energy- and carrier-independent pathway.

Avian reovirus sigmaA is a double-stranded RNA (dsRNA)-binding protein that has been shown to stabilize viral core particles and to protect the virus against the antiviral action of interferon. To continue with the characterization of this viral protein, we have investigated its intracellular distribution in avian cells. Most sigmaA accumulates into cytoplasmic viral factories of infected cells, and yet a significant fraction was detected in the nucleolus. The protein also localizes in the nucleolus of transfected cells, suggesting that nucleolar targeting is not facilitated by the viral infection or by viral factors. Assays performed in both intact cells and digitonin-permeabilized cells demonstrate that sigmaA is able to enter the nucleus via a nucleoporin-dependent nondiffusional mechanism that does not require added cytosolic factors or energy input. These results indicate that sigmaA by itself is able to penetrate into the nucleus using a process that is mechanistically different from the classical nuclear localization signal/importin pathway. On the other hand, two sigmaA arginines that are necessary for dsRNA binding are also required for nucleolar localization, suggesting that dsRNA-binding and nucleolar targeting are intimately linked properties of the viral protein.

Hormones and osteoporosis update. Histological aspects on the action of parathyroid hormone (PTH) and PTH-related peptide (PTHrP) on bone and cartilage

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) , which were shown to bind the common receptor, PTH/PTHrP receptor, affect fetal chondrogenesis and adult bone turnover, respectively. PTH appears to predominantly stimulate proliferation of osteoblastic precursors, and consequently affect osteoblastic differentiation mediating coupling with osteoclasts. A recent study has reported the biological function of C-terminal region of PTHrP including nucleolar targeting signal. We will review recent reports for the action of PTH and PTHrP on bone and cartilage.

Nucleophosmin protein expression level, but not threonine 198 phosphorylation, is essential in growth and proliferation

Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM's phosphorylation by cyclin E-cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell's presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM's described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM's capacity to drive cell cycle progression and proliferation.

Nucleolar targeting: the hub of the matter

The nucleolus is a dynamic structure that has roles in various processes, from ribosome biogenesis to regulation of the cell cycle and the cellular stress response. Such functions are frequently mediated by the sequestration or release of nucleolar proteins. Our understanding of protein targeting to the nucleolus is much less complete than our knowledge of membrane-spanning translocation systems--such as those involved in nuclear targeting--and the experimental evidence reveals that few parallels exist with these better-characterized systems. Here, we discuss the current understanding of nucleolar targeting, explore the types of sequence that control the localization of a protein to the nucleolus, and speculate that certain subsets of nucleolar proteins might act as hub proteins that are able to bind to multiple protein targets. In parallel to other subnuclear structures, such as PML bodies, the proteins that are involved in the formation and maintenance of the nucleolus are inexorably linked to nucleolar trafficking.

Two wobble-splicing events affect ING4 protein subnuclear localization and degradation

ING4 (inhibitor of growth 4) is a candidate tumor suppressor gene that is implicated as a repressor of cell growth, angiogenesis, cell spreading and cell migration and can suppress loss of contact inhibition in vitro. Another group and we identified four wobble-splicing isoforms of ING4 generated by alternative splicing at two tandem splice sites, GC(N) 7GT and NAGNAG, which caused canonical (GT-AG) and non-canonical (GC-AG) splice site wobbling selection. Expression of the four ING4 wobble-splicing isoforms did not vary significantly in any of the cell lines examined. Here we show that ING4_v1 is translocated to the nucleolus, indicating that ING4 contains an intrinsic nucleolar localization signal. We further demonstrate that the subcellular localization of ING4 is modulated by two wobble-splicing events at the exon 4–5 boundary, causing displacement from the nucleolus to the nucleus. We also observed that ING4 is degraded through the ubiquitin–proteasome pathway and that it is subjected to N-terminal ubiquitination. We demonstrate that nucleolar accumulation of ING4 prolongs its half-life, but lack of nucleolar targeting potentially increases ING4 degradation. Taken together, our data suggest that the two wobble-splicing events at the exon 4–5 boundary influence subnuclear localization and degradation of ING4.

Structural Consequences of Nucleophosmin Mutations in Acute Myeloid Leukemia

Mutations affecting NPM1 (nucleophosmin) are the most common genetic lesions found in acute myeloid leukemia (AML). NPM1 is one of the most abundant proteins found in the nucleolus and has links to the MDM2/p53 tumor suppressor pathway. A distinctive feature of NPM1 mutants in AML is their aberrant localization to the cytoplasm of leukemic cells. This mutant phenotype is the result of the substitution of several C-terminal residues, including one or two conserved tryptophan residues, with a leucine-rich nuclear export signal. The exact molecular mechanism underlying the loss of nucleolar retention, and the role of the tryptophans, remains unknown. In this study we have determined the structure of an independently folded globular domain in the C terminus of NPM1 using NMR spectroscopy, and we report that the conserved tryptophans are critical for structure. This domain is necessary for the nucleolar targeting of NPM1 and is disrupted by mutations in AML with cytoplasmic NPM1. Furthermore, we identify conserved surface-exposed lysine residues that are functionally rather than structurally important for nucleolar localization. This study provides new focus for efforts to understand the pathogenesis of AML with cytoplasmic NPM1 and may be used to aid the design of small molecules that target the C-terminal domain of NPM1 to act as novel anti-proliferative and anti-leukemia therapeutics.

The HIV Tat protein affects processing of ribosomal RNA precursor

BackgroundInside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown.ResultsTo clarify the significance of the Tat nucleolar localization, we induced the expression of the protein during oogenesis in Drosophila melanogaster strain transgenic for HIV-tat gene. Here we show that Tat localizes in the nucleoli of Drosophila oocyte nurse cells, where it specifically co-localizes with fibrillarin. Tat expression is accompanied by a significant decrease of cytoplasmic ribosomes, which is apparently related to an impairment of ribosomal rRNA precursor processing. Such an event is accounted for by the interaction of Tat with fibrillarin and U3 snoRNA, which are both required for pre-rRNA maturation.ConclusionOur data contribute to understanding the function of Tat in the nucleolus, where ribosomal RNA synthesis and cell cycle control take place. The impairment of nucleolar pre-rRNA maturation through the interaction of Tat with fibrillarin-U3snoRNA complex suggests a process by which the virus modulates host response, thus contributing to apoptosis and protein shut-off in HIV-uninfected cells.

Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence 916NFNHIHKRIRRVADKYLSG 934 comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin α/β mechanism employing importins α1 and α3 but not α5. This transport was inhibited by wheat germ agglutinin and GTPγS. The sequence 1414SKKTNRGSQLHKYYMKRRTL 1433, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166–1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin α1/β or α3/β complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

Mycophenolic Acid Activation of p53 Requires Ribosomal Proteins L5 and L11

Mycophenolate mofetil (MMF), a prodrug of mycophenolic acid (MPA), is widely used as an immunosuppressive agent. MPA selectively inhibits inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme for the de novo synthesis of guanine nucleotides, leading to depletion of the guanine nucleotide pool. Its chemotherapeutic effects have been attributed to its ability to induce cell cycle arrest and apoptosis. MPA treatment has also been shown to induce and activate p53. However, the mechanism underlying the p53 activation pathway is still unclear. Here, we show that MPA treatment results in inhibition of pre-rRNA synthesis and disruption of the nucleolus. This treatment enhances the interaction of MDM2 with L5 and L11. Interestingly, knockdown of endogenous L5 or L11 markedly impairs the induction of p53 and G(1) cell cycle arrest induced by MPA. These results suggest that MPA may trigger a nucleolar stress that induces p53 activation via inhibition of MDM2 by ribosomal proteins L5 and L11.

Nucleolus and c-Myc: potential targets of cardenolide-mediated antitumor activity

The use of cardenolides like ouabain, digitoxin, or oleandrin has been reported previously many times as a means of potentially combating human refractory prostate cancer by inducing apoptosis through an increase in intracellular calcium concentrations. The aims of the current study were to investigate if part of the antitumor effects mediated by cardenolides concerned disorganization of nucleolar structure and whether this was further associated with a marked decrease in c-Myc expression. Accordingly, the antitumor activity of a novel hemisynthetic cardenolide [1R,3aS,3bR,5aS,6aR,7aS,9R,12aR,13aR,15aR]-3a,11a-dihydroxy-13a-(hydroxymethyl)-9,15a-dimethyl-1-(5-oxo-2,5-dihydrofuran-3-yl)icosahydro-1H,4'H-spiro[cyclopenta [7,8]phenanthro[2,3-b]pyrano[3,2-e][1,4]dioxine-11,2'-[1,3]thiazolidin]-4'-one (UNBS1450)] was compared with that of classic cardenolides and reference anticancer agents in prostate cancer cell lines in vitro and in vivo following s.c. and orthotopic prostate cancer cell grafting into mice. The present study indicates that UNBS1450 markedly decreases the in vitro viability/proliferation of human prostate cancer cell lines but not of normal cells. The induced effects are not linked to an increase in intracellular calcium concentrations and subsequent induction of apoptosis. Rather, they appear to relate to the compound's capacity to disorganize nucleolar structure and function (through an impairment of cyclin-dependent kinase and c-Myc expression and related signaling pathways; paralleled by the disorganization of cancer cell-specific perinucleolar bodies as revealed by disruption of Sam68). This nonapoptotic cancer cell death mediated by severe nucleolar targeting and down-regulation of c-Myc expression is a completely new cardenolide-induced mechanism of antitumor action.

Nucleolar targeting of proteins by the tandem array of basic amino acid stretches identified in the RNA polymerase I-associated factor PAF49

There is accumulating evidence to indicate that the regulation of subnuclear compartmentalization plays important roles in cellular processes. The RNA polymerase I-associated factor PAF49 has been shown to accumulate in the nucleolus in growing cells, but disperse into the nucleoplasm in growth-arrested cells. Serial deletion analysis revealed that amino acids 199–338 were necessary for the nucleolar localization of PAF49. Combinatorial point mutation analysis indicated that the individual basic amino acid stretches (BS) within the central (BS1-4) and the C-terminal (BS5 and 6) regions may cooperatively confer the nucleolar localization of PAF49. Addition of the basic stretches in tandem to a heterologous protein, such as the interferon regulatory factor-3, translocated the tagged protein into the nucleolus, even in the presence of an intrinsic nuclear export sequence. Thus, tandem array of the basic amino acid stretches identified here functions as a dominant nucleolar targeting sequence.

NOM1 Targets Protein Phosphatase I to the Nucleolus

Protein phosphatase I (PP1) is an essential eukaryotic serine/threonine phosphatase required for many cellular processes, including cell division, signaling, and metabolism. In mammalian cells there are three major isoforms of the PP1 catalytic subunit (PP1alpha, PP1beta, and PP1gamma) that are over 90% identical. Despite this high degree of identity, the PP1 catalytic subunits show distinct localization patterns in interphase cells; PP1alpha is primarily nuclear and largely excluded from nucleoli, whereas PP1gamma and to a lesser extent PP1beta concentrate in the nucleoli. The subcellular localization and the substrate specificity of PP1 catalytic subunits are determined by their interaction with targeting subunits, most of which bind PP1 through a so-called "RVXF" sequence. Although PP1 targeting subunits have been identified that direct PP1 to a number of subcellular locations and/or substrates, no targeting subunit has been identified that localizes PP1 to the nucleolus. Identification of nucleolar PP1 targeting subunit(s) is important because all three PP1 isoforms are included in the nucleolar proteome, enzymatically active PP1 is present in nucleoli, and PP1gamma is highly concentrated in nucleoli of interphase cells. In this study, we identify NOM1 (nucleolar protein with MIF4G domain 1) as a PP1-interacting protein and further identify the NOM1 RVXF motif required for its binding to PP1. We also define the NOM1 nucleolar localization sequence. Finally, we demonstrate that NOM1 can target PP1 to the nucleolus and show that a specific NOM1 RVXF motif and the NOM1 nucleolar localization sequence are required for this targeting activity. We therefore conclude that NOM1 is a PP1 nucleolar targeting subunit, the first identified in eukaryotic cells.

Nucleolar Targeting of Hepatitis Delta Antigen Abolishes Its Ability To Initiate Viral Antigenomic RNA Replication

Hepatitis delta virus (HDV) is a small RNA virus that contains one 1.7-kb single-stranded circular RNA of negative polarity. The HDV particle also contains two isoforms of hepatitis delta antigen (HDAg), small (SHDAg) and large HDAg. SHDAg is required for the replication of HDV, which is presumably carried out by host RNA-dependent RNA polymerases. The localization and the HDAg and host RNA polymerase responsible for HDV replication remain important issues to be addressed. In this study, using recombinant SHDAg fused with a heterologous nucleolar localization sequence (NoLS) to confine its subcellular localization in nucleoli, we aimed to study the effect of SHDAg subcellular localization on HDV RNA replication. The initiation of genomic RNA synthesis from antigenomic template was hardly detectable when SHDAg was fused with the NoLS motif and localized mainly in nucleoli. In contrast, the initiation of antigenomic RNA synthesis was not affected. Drug treatment to release a SHDAg-NoLS mutant from nucleoli could partially restore the replication of HDV genomic RNA from antigenomic RNA. This also recovered the cointeraction between SHDAg and RNA polymerase II. These data strongly suggest that nuclear polymerase (RNA polymerase II) is involved in the synthesis of genomic RNA and that the synthesis of antigenomic RNA can occur in nucleoli. Our results support the idea that the replication of HDV genomic RNA or antigenomic RNA is likely to be carried out by different machineries in different subcellular localizations.