scholarly journals SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization

2021 ◽  
Author(s):  
D.E. MacNeil ◽  
P. Lambert-Lanteigne ◽  
J. Qin ◽  
F. McManus ◽  
E. Bonneil ◽  
...  
Keyword(s):  
Nuclear Accumulation ◽  
Nucleolar Localization ◽  
Telomerase Rna ◽  
Dependent Manner ◽  
Complex Assembly ◽  
Fusion Construct ◽  
Subnuclear Localization ◽  
Complex Component ◽  
Nucleolar Localization Signal ◽  
Localization Signal

The nuclear and subnuclear compartmentalization of the telomerase-associated protein and H/ACA ribonucleoprotein component dyskerin is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein.

scholarly journals SUMOylation- and GAR1-dependent regulation of dyskerin nuclear and subnuclear localization

2020 ◽  
Author(s):  
D.E. MacNeil ◽  
P. Lambert-Lanteigne ◽  
J. Qin ◽  
F. McManus ◽  
E. Bonneil ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Fusion Construct ◽  
Subnuclear Localization ◽  
Complex Component ◽  
Nucleolar Localization Signal ◽  
Localization Signal ◽  
Human Telomerase

SummaryDyskerin, a telomerase-associated protein and H/ACA ribonucleoprotein complex component plays an essential role in human telomerase assembly and activity. The nuclear and subnuclear compartmentalization of dyskerin and the H/ACA complex is an important though incompletely understood aspect of H/ACA ribonucleoprotein function. The posttranslational modification, SUMOylation, targets a wide variety of proteins, including numerous RNA-binding proteins, and most identified targets reported to date localize to the nucleus. Four SUMOylation sites were previously identified in the C-terminal Nuclear/Nucleolar Localization Signal (N/NoLS) of dyskerin, each located within one of two lysine-rich clusters. We found that a cytoplasmic localized C-terminal truncation variant of dyskerin lacking most of the C-terminal N/NoLS and both lysine-rich clusters represents an under-SUMOylated variant of dyskerin compared to wildtype dyskerin. We demonstrate that mimicking constitutive SUMOylation of dyskerin using a SUMO3-fusion construct can drive nuclear accumulation of this variant, and that the SUMO site K467 in this N/NoLS is particularly important for the subnuclear localization of dyskerin to the nucleolus in a mature H/ACA complex assembly- and SUMO-dependent manner. We also characterize a novel SUMO-interacting motif in the mature H/ACA complex component GAR1 that mediates the interaction between dyskerin and GAR1. Mislocalization of dyskerin, either in the cytoplasm or excluded from the nucleolus, disrupts dyskerin function and leads to reduced interaction of dyskerin with the telomerase RNA. These data indicate a role for dyskerin C-terminal N/NoLS SUMOylation in regulating the nuclear and subnuclear localization of dyskerin, which is essential for dyskerin function as both a telomerase-associated protein and as an H/ACA ribonucleoprotein involved in rRNA and snRNA biogenesis.

scholarly journals NOA36 Protein Contains a Highly Conserved Nucleolar Localization Signal Capable of Directing Functional Proteins to the Nucleolus, in Mammalian Cells

PLoS ONE ◽  
2013 ◽  
Vol 8 (3) ◽  
pp. e59065 ◽  
Author(s):  
Ivan S. de Melo ◽  
Maria D. Jimenez-Nuñez ◽  
Concepción Iglesias ◽  
Antonio Campos-Caro ◽  
David Moreno-Sanchez ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Nucleolar Localization ◽  
Nucleolar Localization Signal ◽  
Localization Signal

scholarly journals Nonstructural Protein NSs of Schmallenberg Virus Is Targeted to the Nucleolus and Induces Nucleolar Disorganization

2016 ◽  
Vol 91 (1) ◽  
Author(s):  
Julie Gouzil ◽  
Aurore Fablet ◽  
Estelle Lara ◽  
Grégory Caignard ◽  
Marielle Cochet ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Virulence Factor ◽  
Nonstructural Protein ◽  
Schmallenberg Virus ◽  
Nucleolar Localization ◽  
Pol Ii ◽  
Nucleolar Localization Signal ◽  
Localization Signal ◽  
Infected Cells ◽  
Cellular Transcription

ABSTRACT Schmallenberg virus (SBV) was discovered in Germany in late 2011 and then spread rapidly to many European countries. SBV is an orthobunyavirus that causes abortion and congenital abnormalities in ruminants. A virus-encoded nonstructural protein, termed NSs, is a major virulence factor of SBV, and it is known to promote the degradation of Rpb1, a subunit of the RNA polymerase II (Pol II) complex, and therefore hampers global cellular transcription. In this study, we found that NSs is mainly localized in the nucleus of infected cells and specifically appears to target the nucleolus through a nucleolar localization signal (NoLS) localized between residues 33 and 51 of the protein. NSs colocalizes with nucleolar markers such as B23 (nucleophosmin) and fibrillarin. We observed that in SBV-infected cells, B23 undergoes a nucleolus-to-nucleoplasm redistribution, evocative of virus-induced nucleolar disruption. In contrast, the nucleolar pattern of B23 was unchanged upon infection with an SBV recombinant mutant with NSs lacking the NoLS motif (SBVΔNoLS). Interestingly, unlike wild-type SBV, the inhibitory activity of SBVΔNoLS toward RNA Pol II transcription is impaired. Overall, our results suggest that a putative link exists between NSs-induced nucleolar disruption and its inhibitory function on cellular transcription, which consequently precludes the cellular antiviral response and/or induces cell death. IMPORTANCE Schmallenberg virus (SBV) is an emerging arbovirus of ruminants that spread in Europe between 2011 and 2013. SBV induces fetal abnormalities during gestation, with the central nervous system being one of the most affected organs. The virus-encoded NSs protein acts as a virulence factor by impairing host cell transcription. Here, we show that NSs contains a nucleolar localization signal (NoLS) and induces disorganization of the nucleolus. The NoLS motif in the SBV NSs is absolutely necessary for virus-induced inhibition of cellular transcription. To our knowledge, this is the first report of nucleolar functions for NSs within the Bunyaviridae family.

scholarly journals Internal Modification of U2 Small Nuclear (Snrna) Occurs in Nucleoli of Xenopus Oocytes

2001 ◽  
Vol 152 (6) ◽  
pp. 1279-1288 ◽  
Author(s):  
Yi-Tao Yu ◽  
Mei-Di Shu ◽  
Aarthi Narayanan ◽  
Rebecca M. Terns ◽  
Michael P. Terns ◽  
...  
Keyword(s):  
Binding Site ◽  
Xenopus Oocytes ◽  
Cajal Bodies ◽  
Nucleolar Localization ◽  
Small Nucleolar Rnas ◽  
Modified Nucleotides ◽  
Nucleolar Localization Signal ◽  
Localization Analysis ◽  
Localization Signal ◽  
Nucleolar Rnas

U2 small nuclear (sn)RNA contains a large number of posttranscriptionally modified nucleotides, including a 5′ trimethylated guanosine cap, 13 pseudouridines, and 10 2′-O-methylated residues. Using Xenopus oocytes, we demonstrated previously that at least some of these modified nucleotides are essential for biogenesis of a functional snRNP. Here we address the subcellular site of U2 internal modification. Upon injection into the cytoplasm of oocytes, G-capped U2 that is transported to the nucleus becomes modified, whereas A-capped U2 that remains in the cytoplasm is not modified. Furthermore, by injecting U2 RNA into isolated nuclei or enucleated oocytes, we observe that U2 internal modifications occur exclusively in the nucleus. Analysis of the intranuclear localization of fluorescently labeled RNAs shows that injected wild-type U2 becomes localized to nucleoli and Cajal bodies. Both internal modification and nucleolar localization of U2 are dependent on the Sm binding site. An Sm-mutant U2 is targeted only to Cajal bodies. The Sm binding site can be replaced by a nucleolar localization signal derived from small nucleolar RNAs (the box C/D motif), resulting in rescue of internal modification as well as nucleolar localization. Analysis of additional chimeric U2 RNAs reveals a correlation between internal modification and nucleolar localization. Together, our results suggest that U2 internal modification occurs within the nucleolus.

scholarly journals The Nucleolar Localization Signal of Porcine Circovirus Type 4 Capsid Protein Is Essential for Interaction With Serine-48 Residue of Nucleolar Phosphoprotein Nucleophosmin-1

2021 ◽  
Vol 12 ◽  
Author(s):  
Jianwei Zhou ◽  
Yonghui Qiu ◽  
Ning Zhu ◽  
Linyi Zhou ◽  
Beining Dai ◽  
...  
Keyword(s):  
Porcine Circovirus Type ◽  
Cellular Proteins ◽  
Porcine Circovirus ◽  
Nucleolar Localization ◽  
Serine Residue ◽  
Nucleolar Localization Signal ◽  
Localization Signal ◽  
Charge Property ◽  
First Time ◽  
Oligomerization Domain

Porcine circovirus type 4 (PCV4) is an emerging etiological agent which was first detected in 2019. The nucleolar localization signal (NoLS) of PCV4 Cap protein and its binding host cellular proteins are still not elucidated. In the present study, we discovered a distinct novel NoLS of PCV4 Cap, which bound to the nucleolar phosphoprotein nucleophosmin-1 (NPM1). The NoLS of PCV4 Cap and serine-48 residue at the N-terminal oligomerization domain of NPM1 were necessary for PCV4 Cap/NPM1 interaction. Furthermore, the charge property of serine residue at position 48 of the NPM1 was crucial for its oligomerization and interaction with PCV4 Cap. In summary, our findings show for the first time that the PCV4 Cap NoLS and the NPM1 oligomerization determine the interaction of Cap/NPM1.

scholarly journals ARF Function Does Not Require p53 Stabilization or Mdm2 Relocalization

2002 ◽  
Vol 22 (1) ◽  
pp. 196-206 ◽  
Author(s):  
Chandrashekhar Korgaonkar ◽  
Lili Zhao ◽  
Modestos Modestou ◽  
Dawn E. Quelle
Keyword(s):  
Transcriptional Activation ◽  
Growth Arrest ◽  
Nucleolar Localization ◽  
Wild Type ◽  
Growth Inhibitory ◽  
Nucleolar Localization Signal ◽  
Localization Signal ◽  
Reporter Assays ◽  
Dependent Growth ◽  
Block Growth

ABSTRACT It is generally accepted that the ARF tumor suppressor induces p53-dependent growth arrest by sequestering the p53 antagonist Mdm2 in the nucleolus. Previous mutagenic studies of murine ARF suggested that residues 1 through 14 and 26 through 37 were critical for Mdm2 binding, while the latter domain also governed ARF nucleolar localization. We show that mouse ARF residues 6 to 10 and 21 to 25 are required for ARF-induced growth arrest whereas residues 1 to 5 and 29 to 34 are dispensable. Deletion of the putative nucleolar localization signal 31RRPR34 did not prevent nucleolar localization. Surprisingly, unlike wild-type ARF, growth-inhibitory mutants D1-5 and D29-34 failed to stabilize p53 yet induced its transcriptional activation in reporter assays. This suggests that p53 stabilization is not essential for ARF-mediated activation of p53. Like wild-type ARF, both mutants also exhibited p53-independent function since they were able to arrest p53/Mdm2-null cells. Notably, other mutants lacking conserved residues 6 to 10 or 21 to 25 were unable to suppress growth in p53-positive cells despite nucleolar localization and the ability to import Mdm2. Those observations stood in apparent contrast to the ability of wild-type ARF to block growth in some cells without relocalizing endogenous Mdm2 to nucleoli. Together, these data show a lack of correlation between ARF activity and Mdm2 relocalization, suggesting that additional events other than Mdm2 import are required for ARF function.

scholarly journals Nuclear Trafficking of La Protein Depends on a Newly Identified Nucleolar Localization Signal and the Ability to Bind RNA

2004 ◽  
Vol 279 (25) ◽  
pp. 26563-26570 ◽  
Author(s):  
Sven Horke ◽  
Kerstin Reumann ◽  
Michaela Schweizer ◽  
Hans Will ◽  
Tilman Heise
Keyword(s):  
Nucleolar Localization ◽  
Nuclear Trafficking ◽  
La Protein ◽  
Nucleolar Localization Signal ◽  
Localization Signal

scholarly journals Identification and Characterization of the Nucleolar Localization Signal of Autographa californica Multiple Nucleopolyhedrovirus LEF5

2019 ◽  
Vol 94 (4) ◽  
Author(s):  
Guoqing Chen ◽  
Qing Yan ◽  
Haoran Wang ◽  
Shufen Chao ◽  
Lijuan Wu ◽  
...  
Keyword(s):  
Autographa Californica ◽  
Nucleolar Localization ◽  
Sf9 Cells ◽  
Multiple Nucleopolyhedrovirus ◽  
Nucleolar Localization Signal ◽  
Viral Progeny ◽  
Localization Signal ◽  
Infected Cells ◽  
Autographa Californica Multiple Nucleopolyhedrovirus ◽  
Late Expression

ABSTRACT Autographa californica multiple nucleopolyhedrovirus (AcMNPV) late expression factor 5 (LEF5) is highly conserved in all sequenced baculovirus genomes and plays an important role in production of infectious viral progeny. In this study, nucleolar localization of AcMNPV LEF5 was characterized. Through transcriptome analysis, we identified two putative nucleolar proteins, Spodoptera frugiperda nucleostemin (SfNS) and fibrillarin (SfFBL), from Sf9 cells. Immunofluorescence analysis demonstrated that SfNS and SfFBL were localized to the nucleolus. AcMNPV infection resulted in reorganization of the nucleoli of infected cells. Colocalization of LEF5 and SfNS showed that AcMNPV LEF5 was localized to the nucleolus in Sf9 cells. Bioinformatic analysis revealed that basic amino acids of LEF5 are enriched at residues 184 to 213 and may contain a nucleolar localization signal (NoLS). Green fluorescent protein (GFP) fused to NoLS of AcMNPV LEF5 localized to the nucleoli of transfected cells. Multiple-point mutation analysis demonstrated that amino acid residues 197 to 204 are important for nucleolar localization of LEF5. To identify whether the NoLS in AcMNPV LEF5 is important for production of viral progeny, a lef5-null AcMNPV bacmid was constructed; several NoLS-mutated LEF5 proteins were reinserted into the lef5-null AcMNPV bacmid with a GFP reporter. The constructs containing point mutations at residues 185 to 189 or 197 to 204 in AcMNPV LEF5 resulted in reduction in production of infectious viral progeny and occlusion body yield in bacmid-transfected cells. Together, these data suggested that AcMNPV LEF5 contains an NoLS, which is important for nucleolar localization of LEF5, progeny production, and occlusion body production. IMPORTANCE Many viruses, including human and plant viruses, target nucleolar functions as part of their infection strategy. However, nucleolar localization for baculovirus proteins has not yet been characterized. In this study, two nucleolar proteins, SfNS and SfFBL, were identified in Sf9 cells. Our results showed that Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infection resulted in redistribution of the nucleoli of infected cells. We demonstrated that AcMNPV late expression factor 5 (LEF5) could localize to the nucleolus and contains a nucleolar localization signal (NoLS), which is important for nucleolar localization of AcMNPV LEF5 and for production of viral progeny and yield of occlusion bodies.

scholarly journals The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal.

1995 ◽  
Vol 130 (2) ◽  
pp. 255-263 ◽  
Author(s):  
T Tagawa ◽  
T Kuroki ◽  
P K Vogt ◽  
K Chida
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Kinase Inhibitors ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Localization Signal ◽  
Cycle Dependent

Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.

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