Nucleolar Localization Sequences Research Articles

Proteins of the Nucleolus of Dictyostelium discoideum: Nucleolar Compartmentalization, Targeting Sequences, Protein Translocations and Binding Partners.

The nucleoli of Dictyostelium discoideum have a comparatively unique, non-canonical, localization adjacent to the inner nuclear membrane. The verified nucleolar proteins of this eukaryotic microbe are detailed while other potential proteins are introduced. Heat shock protein 32 (Hsp32), eukaryotic translation initiation factor 6 (eIF6), and tumour necrosis factor receptor-associated protein 1 (TRAP1) are essential for cell survival. NumA1, a breast cancer type 1 susceptibility protein-C Terminus domain-containing protein linked to cell cycle, functions in the regulation of nuclear number. The cell cycle checkpoint kinase 2 homologue forkhead-associated kinase A (FhkA) and BRG1-associated factor 60a homologue Snf12 are also discussed. While nucleoli appear homogeneous ultrastructurally, evidence for nucleolar subcompartments exists. Nucleolar localization sequences (NoLS) have been defined that target proteins to either the general nucleolar area or to a specific intranucleolar domain. Protein translocations during mitosis are protein-specific and support the multiple functions of the Dictyostelium nucleolus. To enrich the picture, binding partners of NumA1, the most well-characterized nucleolar protein, are examined: nucleolar Ca2+-binding protein 4a (CBP4a), nuclear puromycin-sensitive aminopeptidase A (PsaA) and Snf12. The role of Dictyostelium as a model for understanding the contribution of nucleolar proteins to various diseases and cellular stress is discussed throughout the review.

Mapping and Engineering Functional Domains of the Assembly-Activating Protein of Adeno-associated Viruses.

Adeno-associated viruses (AAVs) encode a unique assembly-activating protein (AAP) within their genomes that is essential for capsid assembly. Studies to date have focused on establishing the role of AAP as a chaperone that mediates the stability, nucleolar transport, and assembly of AAV capsid proteins. Here, we map structure-function correlates of AAP using secondary structure analysis, followed by deletion and substitutional mutagenesis of specific domains, namely, the N-terminal hydrophobic region (HR), conserved core (CC), proline-rich region (PRR), threonine/serine-rich region (T/S), and basic region (BR). First, we establish that the centrally located PRR and T/S are flexible linker domains that can either be deleted completely or replaced by heterologous functional domains that enable ancillary functions such as fluorescent imaging or increased AAP stability. We also demonstrate that the C-terminal BR domains can be substituted with heterologous nuclear or nucleolar localization sequences that display various abilities to support AAV capsid assembly. Further, by replacing the BR domain with immunoglobulin (IgG) Fc domains, we assessed AAP complexation with AAV capsid subunits and demonstrate that the hydrophobic region (HR) and the conserved core (CC) in the AAP N terminus are the sole determinants for viral protein (VP) recognition. However, VP recognition alone is not sufficient for capsid assembly. Our study sheds light on the modular structure-function correlates of AAP and provides multiple approaches to engineer AAP that might prove useful toward understanding and controlling AAV capsid assembly.IMPORTANCE Adeno-associated viruses (AAVs) encode a unique assembly-activating protein (AAP) within their genomes that is essential for capsid assembly. Understanding how AAP acts as a chaperone for viral assembly could help improve efficiency and potentially control this process. Our studies reveal that AAP has a modular architecture, with each module playing a distinct role and can be engineered for carrying out new functions.

Contribution of the NS1 Gene of H7 Avian Influenza Virus Strains to Pathogenicity in Chickens

Using reverse genetics (rg), we generated two reassortant viruses carrying the NS1 gene of two closely related HPAIV and LPAIV H7N1 variants (designated rgH7N7 HP(HPNS1) and rgH7N7 HP(LPNS1), respectively) in the backbone of the HP H7N7 strain A/Chicken/Netherlands/621557/03 (rgH7N7 HP). Comparison of these reassortants allowed us to determine the effect of amino acid differences in the nuclear export and nucleolar localization sequences of NS1 on pathogenesis in chickens. Compared to rgH7N7 HP(LPNS1), a delay in weight gain and an increase in mortality were observed for rgH7N7 HP(HPNS1). Furthermore, an increase in viral load in brains, lungs, and cloacal swabs, as well as an increased induction of mRNA for type I interferons and pro-inflammatory cytokines in brains, were observed for rgH7N7 HP(HPNS1). Comparison of rgH7N7 HP(LPNS1) with the backbone strain rgH7N7 HP allowed us to examine differences in pathogenesis due to differences in NS1 alleles. rgH7N7 HP, which contained allele A of NS1 showed a higher in vitro replication rate and proved to be more virulent than the isogenic virus carrying allele B of NS1(rgH7N7 HP(LPNS1)). In addition, higher virus accumulation in the lungs and brains, and an increased induction of host gene responses, especially in the brains, were found for rgH7N7 HP compared to rgH7N7 HP(LPNS1). No large differences were observed in type I interferon expression in the lungs of chickens infected with any of the viruses, suggesting that differences in virulence due to differences in NS1 could be related to differences in the induction of pro-inflammatory cytokines in vital organs such as the brains.

72. Identification of Nucleolar Localization Sequences in the C-Terminal Region of Assembly-Activating Protein

72. Identification of Nucleolar Localization Sequences in the C-Terminal Region of Assembly-Activating Protein

Nucleolar Localization of GLTSCR2/PICT-1 Is Mediated by Multiple Unique Nucleolar Localization Sequences

The human glioma tumor suppressor candidate region 2 gene product, GLTSCR2, also called ‘protein interacting with carboxyl terminus 1’ (PICT-1), has been implicated in the regulation of two major tumor suppressor proteins, PTEN and p53, and reported to bind the membrane-cytoskeleton regulator of cell signaling, Merlin. PICT-1 is a nucleolar protein, conserved among eukaryotes, and its yeast homolog has been functionally associated with ribosomal RNA processing. By means of confocal microscopy of EGFP and myc-tagged PICT-1 fusion proteins, we delineate that the nucleolar localization of PICT-1 is mediated by two independent nucleolar localization sequences (NoLS). Unlike most NoLSs, these NoLSs are relatively long with flexible boundaries and contain arginine and leucine clusters. In addition, we show that PICT-1 exhibits a nucleolar distribution similar to proteins involved in ribosomal RNA processing, yet does not colocalize precisely with either UBF1 or Fibrillarin under normal or stressed conditions. Identification of the precise location of PICT-1 and the signals that mediate its nucleolar localization is an important step towards advancing our understanding of the demonstrated influence of this protein on cell fate and tumorigenesis.

NoD: a Nucleolar localization sequence detector for eukaryotic and viral proteins

BackgroundNucleolar localization sequences (NoLSs) are short targeting sequences responsible for the localization of proteins to the nucleolus. Given the large number of proteins experimentally detected in the nucleolus and the central role of this subnuclear compartment in the cell, NoLSs are likely to be important regulatory elements controlling cellular traffic. Although many proteins have been reported to contain NoLSs, the systematic characterization of this group of targeting motifs has only recently been carried out.ResultsHere, we describe NoD, a web server and a command line program that predicts the presence of NoLSs in proteins. Using the web server, users can submit protein sequences through the NoD input form and are provided with a graphical output of the NoLS score as a function of protein position. While the web server is most convenient for making prediction for just a few proteins, the command line version of NoD can return predictions for complete proteomes. NoD is based on our recently described human-trained artificial neural network predictor. Through stringent independent testing of the predictor using available experimentally validated NoLS-containing eukaryotic and viral proteins, the NoD sensitivity and positive predictive value were estimated to be 71% and 79% respectively.ConclusionsNoD is the first tool to provide predictions of nucleolar localization sequences in diverse eukaryotes and viruses. NoD can be run interactively online at http://www.compbio.dundee.ac.uk/nod or downloaded to use locally.

STK35L1 associates with nuclear actin and regulates cell cycle and migration of endothelial cells.

BackgroundMigration and proliferation of vascular endothelial cells are essential for repair of injured endothelium and angiogenesis. Cyclins, cyclin-dependent kinases (CDKs), and cyclin-dependent kinase inhibitors play an important role in vascular tissue injury and wound healing. Previous studies suggest a link between the cell cycle and cell migration: cells present in the G1 phase have the highest potential to migrate. The molecular mechanism linking these two processes is not understood.Methodology/Principal FindingsIn this study, we explored the function of STK35L1, a novel Ser/Thr kinase, localized in the nucleus and nucleolus of endothelial cells. Molecular biological analysis identified a bipartite nuclear localization signal, and nucleolar localization sequences in the N-terminal part of STK35L1. Nuclear actin was identified as a novel binding partner of STK35L1. A class III PDZ binding domains motif was identified in STK35L1 that mediated its interaction with actin. Depletion of STK35L1 by siRNA lead to an accelerated G1 to S phase transition after serum-stimulation of endothelial cells indicating an inhibitory role of the kinase in G1 to S phase progression. Cell cycle specific genes array analysis revealed that one gene was prominently downregulated (8.8 fold) in STK35L1 silenced cells: CDKN2A alpha transcript, which codes for p16INK4a leading to G1 arrest by inhibition of CDK4/6. Moreover in endothelial cells seeded on Matrigel, STK35L1 expression was rapidly upregulated, and silencing of STK35L1 drastically inhibited endothelial sprouting that is required for angiogenesis. Furthermore, STK35L1 depletion profoundly impaired endothelial cell migration in two wound healing assays.Conclusion/SignificanceThe results indicate that by regulating CDKN2A and inhibiting G1- to S-phase transition STK35L1 may act as a central kinase linking the cell cycle and migration of endothelial cells. The interaction of STK35L1 with nuclear actin might be critical in the regulation of these fundamental endothelial functions.

Characterization and prediction of protein nucleolar localization sequences

Although the nucleolar localization of proteins is often believed to be mediated primarily by non-specific retention to core nucleolar components, many examples of short nucleolar targeting sequences have been reported in recent years. In this article, 46 human nucleolar localization sequences (NoLSs) were collated from the literature and subjected to statistical analysis. Of the residues in these NoLSs 48% are basic, whereas 99% of the residues are predicted to be solvent-accessible with 42% in α-helix and 57% in coil. The sequence and predicted protein secondary structure of the 46 NoLSs were used to train an artificial neural network to identify NoLSs. At a true positive rate of 54%, the predictor’s overall false positive rate (FPR) is estimated to be 1.52%, which can be broken down to FPRs of 0.26% for randomly chosen cytoplasmic sequences, 0.80% for randomly chosen nucleoplasmic sequences and 12% for nuclear localization signals. The predictor was used to predict NoLSs in the complete human proteome and 10 of the highest scoring previously unknown NoLSs were experimentally confirmed. NoLSs are a prevalent type of targeting motif that is distinct from nuclear localization signals and that can be computationally predicted.

Ribosomal stress induces processing of Mybbp1a and its translocation from the nucleolus to the nucleoplasm

Myb-binding protein 1a (Mybbp1a) was originally identified as a c-myb proto-oncogene product (c-Myb)-interacting protein, and also binds to various other transcription factors. The 160-kDa Mybbp1a protein (p160(MBP)) is ubiquitously expressed and is post-translationally processed in some types of cells to generate an amino-terminal 67 kDa fragment (p67(MBP)). Despite its interaction with various transcription factors, Mybbp1a is localized predominantly, but not exclusively, in nucleoli. Here, we have purified the two Mybbp1a-containing complexes. The smaller complex contained p67(MBP) and p140(MBP), which lacked the C-terminal region of p160(MBP) containing the nucleolar localization sequences. The larger complex contained the intact p160(MBP) and various ribosomal subunits. Treatment of cells with actinomycin D (ActD), cisplatin or UV, all of which inhibit ribosome biogenesis, induced processing of p160(MBP) into p140(MBP) and p67(MBP). ActD, cisplatin and UV also induced a translocation of Mybbp1a from the nucleolus to the nucleoplasm. Both small and large Mybbp1a complexes contained nucleophosmin and nucleolin. In contrast, nucleostemin was detected only in the large complex, while the cell cycle-regulated protein EBP1 was only in the small complex. These results suggest that Mybbp1a may connect the ribosome biogenesis and the Myb-dependent transcription, which controls cell cycle progression and proliferation.

Centromere RNA is a key component for the assembly of nucleoproteins at the nucleolus and centromere.

The centromere is a complex structure, the components and assembly pathway of which remain inadequately defined. Here, we demonstrate that centromeric alpha-satellite RNA and proteins CENPC1 and INCENP accumulate in the human interphase nucleolus in an RNA polymerase I-dependent manner. The nucleolar targeting of CENPC1 and INCENP requires alpha-satellite RNA, as evident from the delocalization of both proteins from the nucleolus in RNase-treated cells, and the nucleolar relocalization of these proteins following alpha-satellite RNA replenishment in these cells. Using protein truncation and in vitro mutagenesis, we have identified the nucleolar localization sequences on CENPC1 and INCENP. We present evidence that CENPC1 is an RNA-associating protein that binds alpha-satellite RNA by an in vitro binding assay. Using chromatin immunoprecipitation, RNase treatment, and "RNA replenishment" experiments, we show that alpha-satellite RNA is a key component in the assembly of CENPC1, INCENP, and survivin (an INCENP-interacting protein) at the metaphase centromere. Our data suggest that centromere satellite RNA directly facilitates the accumulation and assembly of centromere-specific nucleoprotein components at the nucleolus and mitotic centromere, and that the sequestration of these components in the interphase nucleolus provides a regulatory mechanism for their timely release into the nucleoplasm for kinetochore assembly at the onset of mitosis.

Mapping nucleolar localization sequences of 1A6/DRIM

Human 1A6/downregulated in metastasis (DRIM) is a nucleolar protein with multiple HEAT-repeat motifs ( Huntington, elongation factor 3, a subunit of protein phosphatase 2A, target of rapamycin). The yeast homologue to 1A6/DRIM, Utp20, is part of the small subunit processome and functions in 18S RNA processing. In the present study, we utilized the green fluorescent protein as the fusion protein marker to investigate the sequence responsible for 1A6/DRIM accumulation in nucleolus. Deletion sequence analysis demonstrated that a single region located between amino acids 2744 and 2761 at the C-terminus of 1A6/DRIM is capable of nucleolar accumulation. Two basic amino acid clusters within this region are essential for nucleolar accumulation. The sequences required for nucleolar accumulation overlaps the putative nuclear localization signal of 1A6/DRIM.

Mapping nucleolar and spliceosome localization sequences of neuregulin1-β3

Mitogenic growth factors are generally cell surface associated or secreted proteins, which produce effects by binding to cell surface receptor tyrosine kinases. More recently, it has become clear that some of these proteins can accumulate in the nucleus, where they are proposed to have transcriptional activity. We show here that neuregulin1 (NRG1-β), an EGF-like growth factor, localizes to the cell nuclei of a human breast cancer. We also show that a nonsecreted isoform of this family of ligands, neuregulin1-β3, localizes to two distinct compartments within the nucleus, nucleoli, and SC35-positive speckles. Importantly, localization of NRG-β3 to either structure is receptor-independent, as it occurs in cells lacking its cognate receptors, erbB-3 and erbB-4, and is unaffected by removal of the receptor-binding domain. A panel of deletion mutants was used to demonstrate that the first 21 amino acids of the N-terminus are essential for nucleolar localization, while targeting to nuclear speckles requires residues 49–79 of the 241 amino acid protein. These observations support the idea that secretion and subsequent cell surface receptor binding of mitogenic growth factors are not a prerequisite for nuclear localization and that nonsecreted ligands may have highly specific functions in defined nuclear compartments.

Nucleolar-cytoplasmic shuttling of PRRSV nucleocapsid protein: a simple case of molecular mimicry or the complex regulation by nuclear import, nucleolar localization and nuclear export signal sequences

The order Nidovirales, which includes the arteriviruses and coronaviruses, incorporate a cytoplasmic replication scheme; however, the nucleocapsid (N) protein of several members of this group localizes to the nucleolus suggesting that viral proteins influence nuclear processes during replication. The relatively small, 123 amino acid, N protein of porcine reproductive and respiratory syndrome virus (PRRSV), an arterivirus, presents an ideal model system for investigating the properties and mechanism of N protein nucleolar localization. The PRRSV N protein is found in both cytoplasmic and nucleolar compartments during infection and after transfection of gene constructs that express N-enhanced green fluorescent protein (EGFP) fusion proteins. Experiments using oligopeptides, truncated polypeptides and amino acid-substituted proteins have identified several domains within PRRSV N protein that participate in nucleo-cytoplasmic shuttling, including a cryptic nuclear localization signal (NLS) called NLS-1, a functional NLS (NLS-2), a nucleolar localization sequence (NoLS), as well as a possible nuclear export signal (NES). The purpose of this paper is to review our current understanding of PRRSV N protein shuttling and propose a shuttling scheme regulated by RNA binding and post-translational modification.

Basic domains target protein subunits of the RNase MRP complex to the nucleolus independently of complex association.

The RNase MRP and RNase P ribonucleoprotein particles both function as endoribonucleases, have a similar RNA component, and share several protein subunits. RNase MRP has been implicated in pre-rRNA processing and mitochondrial DNA replication, whereas RNase P functions in pre-tRNA processing. Both RNase MRP and RNase P accumulate in the nucleolus of eukaryotic cells. In this report we show that for three protein subunits of the RNase MRP complex (hPop1, hPop4, and Rpp38) basic domains are responsible for their nucleolar accumulation and that they are able to accumulate in the nucleolus independently of their association with the RNase MRP and RNase P complexes. We also show that certain mutants of hPop4 accumulate in the Cajal bodies, suggesting that hPop4 traverses through these bodies to the nucleolus. Furthermore, we characterized a deletion mutant of Rpp38 that preferentially associates with the RNase MRP complex, giving a first clue about the difference in protein composition of the human RNase MRP and RNase P complexes. On the basis of all available data on nucleolar localization sequences, we hypothesize that nucleolar accumulation of proteins containing basic domains proceeds by diffusion and retention rather than by an active transport process. The existence of nucleolar localization sequences is discussed.

Conserved boxes C and D are essential nucleolar localization elements of U14 and U8 snoRNAs.

Sequences necessary for nucleolar targeting were identified in Box C/D small nucleolar RNAs (snoRNAs) by fluorescence microscopy. Nucleolar preparations were examined after injecting fluorescein-labelled wild-type and mutated U14 or U8 snoRNA into Xenopus oocyte nuclei. Regions in U14 snoRNA that are complementary to 18S rRNA and necessary for rRNA processing and methylation are not required for nucleolar localization. Truncated U14 molecules containing Boxes C and D with or without the terminal stem localized efficiently. Nucleolar localization was abolished upon mutating just one or two nucleotides within Boxes C and D. Moreover, the spatial position of Boxes C or D in the molecule is essential. Mutations in Box C/D of U8 snoRNA also impaired nucleolar localization, suggesting the general importance of Boxes C and D as nucleolar localization sequences for Box C/D snoRNAs. U14 snoRNA is shown to be required for 18S rRNA production in vertebrates.

Occurrence of beta-turn potentials around nuclear and nucleolar localization sequences.

The biological significance of turn structures is now of great topical interest. By using the protein conformational prediction method of Chou and Fasman, the present work predicts that 17 nuclear localization signals and a nucleolar localization signal reported so far contain turn potentials. Two nuclear localization signals, human lamin A and c-myc protein (peptide M1), however, cannot be predicted as containing beta-turns by the prediction method. To date, no physical characterization of any nuclear or nucleolar location signal by X-ray crystallography and nuclear magnetic resonance spectroscopy has been performed. Employing conformation prediction methods, therefore, would be useful for elucidating structural features of nuclear and nucleolar location signals.