scholarly journals Bud23 Methylates G1575 of 18S rRNA and Is Required for Efficient Nuclear Export of Pre-40S Subunits

2008 ◽  
Vol 28 (10) ◽  
pp. 3151-3161 ◽  
Author(s):  
Joshua White ◽  
Zhihua Li ◽  
Richa Sardana ◽  
Janusz M. Bujnicki ◽  
Edward M. Marcotte ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Fluorescent Protein ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Nuclear Accumulation ◽  
Methyltransferase Activity ◽  
Late Step ◽  
Green Fluorescent

ABSTRACT BUD23 was identified from a bioinformatics analysis of Saccharomyces cerevisiae genes involved in ribosome biogenesis. Deletion of BUD23 leads to severely impaired growth, reduced levels of the small (40S) ribosomal subunit, and a block in processing 20S rRNA to 18S rRNA, a late step in 40S maturation. Bud23 belongs to the S-adenosylmethionine-dependent Rossmann-fold methyltransferase superfamily and is related to small-molecule methyltransferases. Nevertheless, we considered that Bud23 methylates rRNA. Methylation of G1575 is the only mapped modification for which the methylase has not been assigned. Here, we show that this modification is lost in bud23 mutants. The nuclear accumulation of the small-subunit reporters Rps2-green fluorescent protein (GFP) and Rps3-GFP, as well as the rRNA processing intermediate, the 5′ internal transcribed spacer 1, indicate that bud23 mutants are defective for small-subunit export. Mutations in Bud23 that inactivated its methyltransferase activity complemented a bud23Δ mutant. In addition, mutant ribosomes in which G1575 was changed to adenosine supported growth comparable to that of cells with wild-type ribosomes. Thus, Bud23 protein, but not its methyltransferase activity, is important for biogenesis and export of the 40S subunit in yeast.

Requirements for the nuclear export of the small ribosomal subunit

2002 ◽  
Vol 115 (14) ◽  
pp. 2985-2995 ◽  
Author(s):  
Terence I. Moy ◽  
Pamela A. Silver
Keyword(s):  
Nuclear Export ◽  
Ribosome Biogenesis ◽  
Large Scale ◽  
Ribosomal Subunit ◽  
Small Subunit ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Small Ribosomal Subunit ◽  
Factors Affecting ◽  
The Stability

Eukaryotic ribosome biogenesis requires multiple steps of nuclear transport because ribosomes are assembled in the nucleus while protein synthesis occurs in the cytoplasm. Using an in situ RNA localization assay in the yeast Saccharomyces cerevisiae, we determined that efficient nuclear export of the small ribosomal subunit requires Yrb2, a factor involved in Crm1-mediated export. Furthermore, in cells lacking YRB2, the stability and abundance of the small ribosomal subunit is decreased in comparison with the large ribosomal subunit. To identify additional factors affecting small subunit export, we performed a large-scale screen of temperature-sensitive mutants. We isolated new alleles of several nucleoporins and Ran-GTPase regulators. Together with further analysis of existing mutants,we show that nucleoporins previously shown to be defective in ribosomal assembly are also defective in export of the small ribosomal subunit.

scholarly journals Nip7p Interacts with Nop8p, an Essential Nucleolar Protein Required for 60S Ribosome Biogenesis, and the Exosome Subunit Rrp43p

1999 ◽  
Vol 19 (2) ◽  
pp. 1518-1525 ◽  
Author(s):  
Nilson I. T. Zanchin ◽  
David S. Goldfarb
Keyword(s):  
Ribosome Biogenesis ◽  
18S Rrna ◽  
Fluorescent Protein ◽  
Protein A ◽  
Nucleolar Protein ◽  
Mrna Turnover ◽  
5.8S Rrna ◽  
Multistep Process ◽  
Green Fluorescent ◽  
Two Hybrid

ABSTRACT NIP7 encodes a conserved Saccharomyces cerevisiae nucleolar protein that is required for 60S subunit biogenesis (N. I. T. Zanchin, P. Roberts, A. DeSilva, F. Sherman, and D. S. Goldfarb, Mol. Cell. Biol. 17:5001–5015, 1997). Rrp43p and a second essential protein, Nop8p, were identified in a two-hybrid screen as Nip7p-interacting proteins. Biochemical evidence for an interaction was provided by the copurification on immunoglobulin G-Sepharose of Nip7p with protein A-tagged Rrp43p and Nop8p. Cells depleted of Nop8p contained reduced levels of free 60S ribosomes and polysomes and accumulated half-mer polysomes. Nop8p-depleted cells also accumulated 35S pre-rRNA and an aberrant 23S pre-rRNA. Nop8p-depleted cells failed to accumulate either 25S or 27S rRNA, although they did synthesize significant levels of 18S rRNA. These results indicate that 27S or 25S rRNA is degraded in Nop8p-depleted cells after the section containing 18S rRNA is removed. Nip7p-depleted cells exhibited the same defects as Nop8p-depleted cells, except that they accumulated 27S precursors. Rrp43p is a component of the exosome, a complex of 3′-to-5′ exonucleases whose subunits have been implicated in 5.8S rRNA processing and mRNA turnover. Whereas both green fluorescent protein (GFP)-Nop8p and GFP-Nip7p localized to nucleoli, GFP-Rrp43p localized throughout the nucleus and to a lesser extent in the cytoplasm. Distinct pools of Rrp43p may interact both with the exosome and with Nip7p, possibly both in the nucleus and in the cytoplasm, to catalyze analogous reactions in the multistep process of 60S ribosome biogenesis and mRNA turnover.

scholarly journals CRM1 Mediates the Export of ADAR1 through a Nuclear Export Signal within the Z-DNA Binding Domain

2001 ◽  
Vol 21 (22) ◽  
pp. 7862-7871 ◽  
Author(s):  
Hanne Poulsen ◽  
Jakob Nilsson ◽  
Christian K. Damgaard ◽  
Jan Egebjerg ◽  
Jørgen Kjems
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Protein A ◽  
Nuclear Export Signal ◽  
Nuclear Accumulation ◽  
Adenosine Deaminases ◽  
Editing Activity ◽  
Green Fluorescent ◽  
Export Signal

ABSTRACT RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

scholarly journals A Novel In Vivo Assay Reveals Inhibition of Ribosomal Nuclear Export in Ran-Cycle and Nucleoporin Mutants

1999 ◽  
Vol 144 (3) ◽  
pp. 389-401 ◽  
Author(s):  
Ed Hurt ◽  
Stefan Hannus ◽  
Birgit Schmelzl ◽  
Denise Lau ◽  
David Tollervey ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Nuclear Accumulation ◽  
Wild Type ◽  
Ribosomal Subunits ◽  
In Vivo Assay ◽  
Green Fluorescent ◽  
Transport Factors

To identify components involved in the nuclear export of ribosomes in yeast, we developed an in vivo assay exploiting a green fluorescent protein (GFP)-tagged version of ribosomal protein L25. After its import into the nucleolus, L25-GFP assembles with 60S ribosomal subunits that are subsequently exported into the cytoplasm. In wild-type cells, GFP-labeled ribosomes are only detected by fluorescence in the cytoplasm. However, thermosensitive rna1-1 (Ran-GAP), prp20-1 (Ran-GEF), and nucleoporin nup49 and nsp1 mutants are impaired in ribosomal export as revealed by nuclear accumulation of L25-GFP. Furthermore, overexpression of dominant-negative RanGTP (Gsp1-G21V) and the tRNA exportin Los1p inhibits ribosomal export. The pattern of subnuclear accumulation of L25-GFP observed in different mutants is not identical, suggesting that transport can be blocked at different steps. Thus, nuclear export of ribosomes requires the nuclear/cytoplasmic Ran-cycle and distinct nucleoporins. This assay can be used to identify soluble transport factors required for nuclear exit of ribosomes.

scholarly journals Kinetic Analysis of Smad Nucleocytoplasmic Shuttling Reveals a Mechanism for Transforming Growth Factor β-Dependent Nuclear Accumulation of Smads

2005 ◽  
Vol 25 (22) ◽  
pp. 9845-9858 ◽  
Author(s):  
Bernhard Schmierer ◽  
Caroline S. Hill
Keyword(s):  
Green Fluorescent Protein ◽  
Growth Factor ◽  
Nuclear Export ◽  
Transforming Growth Factor ◽  
Fluorescent Protein ◽  
Transforming Growth Factor Β ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Shuttling ◽  
Green Fluorescent

ABSTRACT Upon transforming growth factor β (TGF-β) stimulation, Smads accumulate in the nucleus, where they regulate gene expression. Using fluorescence perturbation experiments on Smad2 and Smad4 fused to either enhanced green fluorescent protein or photoactivatable green fluorescent protein, we have studied the kinetics of Smad nucleocytoplasmic shuttling in a quantitative manner in vivo. We have obtained rate constants for import and export of Smad2 and show that the cytoplasmic localization of Smad2 in uninduced cells reflects its nuclear export being more rapid than import. We find that TGF-β-induced nuclear accumulation of Smad2 is caused by a pronounced drop in the export rate of Smad2 from the nucleus, which is associated with a strong decrease in nuclear mobility of Smad2 and Smad4. TGF-β-induced nuclear accumulation involves neither a release from cytoplasmic retention nor an increase in Smad2 import rate. Hence, TGF-β-dependent nuclear accumulation of Smad2 is caused exclusively by selective nuclear trapping of phosphorylated, complexed Smad2. The proposed mechanism reconciles signal-dependent nuclear accumulation of Smad2 with its continuous nucleocytoplasmic cycling properties.

scholarly journals Nmd3p Is a Crm1p-Dependent Adapter Protein for Nuclear Export of the Large Ribosomal Subunit

2000 ◽  
Vol 151 (5) ◽  
pp. 1057-1066 ◽  
Author(s):  
Jennifer Hei-Ngam Ho ◽  
George Kallstrom ◽  
Arlen W. Johnson
Keyword(s):  
Nuclear Export ◽  
Ribosomal Subunit ◽  
Specific Inhibitor ◽  
Small Gtpase ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Adapter Protein ◽  
Leptomycin B ◽  
Ribosomal Subunits ◽  
Late Step

In eukaryotic cells, nuclear export of nascent ribosomal subunits through the nuclear pore complex depends on the small GTPase Ran. However, neither the nuclear export signals (NESs) for the ribosomal subunits nor the receptor proteins, which recognize the NESs and mediate export of the subunits, have been identified. We showed previously that Nmd3p is an essential protein from yeast that is required for a late step in biogenesis of the large (60S) ribosomal subunit. Here, we show that Nmd3p shuttles and that deletion of the NES from Nmd3p leads to nuclear accumulation of the mutant protein, inhibition of the 60S subunit biogenesis, and inhibition of the nuclear export of 60S subunits. Moreover, the 60S subunits that accumulate in the nucleus can be coimmunoprecipitated with the NES-deficient Nmd3p. 60S subunit biogenesis and export of truncated Nmd3p were restored by the addition of an exogenous NES. To identify the export receptor for Nmd3p we show that Nmd3p shuttling and 60S export is blocked by the Crm1p-specific inhibitor leptomycin B. These results identify Crm1p as the receptor for Nmd3p export. Thus, export of the 60S subunit is mediated by the adapter protein Nmd3p in a Crm1p-dependent pathway.

scholarly journals The Drosophila nucleoporin DNup88 localizes DNup214 and CRM1 on the nuclear envelope and attenuates NES-mediated nuclear export

2003 ◽  
Vol 163 (4) ◽  
pp. 701-706 ◽  
Author(s):  
Peggy Roth ◽  
Nikos Xylourgidis ◽  
Nafiseh Sabri ◽  
Anne Uv ◽  
Maarten Fornerod ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Export Signal ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Accumulation ◽  
Major Function ◽  
Green Fluorescent ◽  
Mutant Phenotypes

Many cellular responses rely on the control of nucleocytoplasmic transport of transcriptional regulators. The Drosophila nucleoporin Nup88 is selectively required for nuclear accumulation of Rel proteins and full activation of the innate immune response. Here, we investigate the mechanisms underlying its role in nucleocytoplasmic transport. Nuclear import of an nuclear localization signal-enhanced green fluorescent protein (NLS-EGFP) reporter is not affected in DNup88 (members only; mbo) mutants, whereas the level of CRM1-dependent EGFP-nuclear export signal (EGFP-NES) export is increased. We show that the nuclear accumulation of the Drosophila Rel protein Dorsal requires CRM1. DNup88 binds to DNup214 and DCRM1 in vitro, and both proteins become mislocalized from the nuclear rim into the nucleus of mbo mutants. Overexpression of DNup88 is sufficient to relocalize DNup214 and CRM1 on the nuclear envelope and revert the mutant phenotypes. We propose that a major function of DNup88 is to anchor DNup214 and CRM1 on the nuclear envelope and thereby attenuate NES-mediated nuclear export.

A global survey of CRM1-dependent nuclear export sequences in the human deubiquitinase family

2011 ◽  
Vol 441 (1) ◽  
pp. 209-217 ◽  
Author(s):  
Iraia García-Santisteban ◽  
Sonia Bañuelos ◽  
Jose A. Rodríguez
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
The Novel ◽  
Sequence Motifs ◽  
Leptomycin B ◽  
Specific Amino Acid ◽  
Green Fluorescent ◽  
Nuclear Export Receptor

The mechanisms that regulate the nucleocytoplasmic localization of human deubiquitinases remain largely unknown. The nuclear export receptor CRM1 binds to specific amino acid motifs termed NESs (nuclear export sequences). By using in silico prediction and experimental validation of candidate sequences, we identified 32 active NESs and 78 inactive NES-like motifs in human deubiquitinases. These results allowed us to evaluate the performance of three programs widely used for NES prediction, and to add novel information to the recently redefined NES consensus. The novel NESs identified in the present study reveal a subset of 22 deubiquitinases bearing motifs that might mediate their binding to CRM1. We tested the effect of the CRM1 inhibitor LMB (leptomycin B) on the localization of YFP (yellow fluorescent protein)- or GFP (green fluorescent protein)-tagged versions of six NES-bearing deubiquitinases [USP (ubiquitin-specific peptidase) 1, USP3, USP7, USP21, CYLD (cylindromatosis) and OTUD7B (OTU-domain-containing 7B)]. YFP–USP21 and, to a lesser extent, GFP–OTUD7B relocated from the cytoplasm to the nucleus in the presence of LMB, revealing their nucleocytoplasmic shuttling capability. Two sequence motifs in USP21 had been identified during our survey as active NESs in the export assay. Using site-directed mutagenesis, we show that one of these motifs mediates USP21 nuclear export, whereas the second motif is not functional in the context of full-length USP21.

scholarly journals Live-Cell Fluorescence Imaging Reveals the Dynamics of Protein Kinase CK2 Individual Subunits

2003 ◽  
Vol 23 (3) ◽  
pp. 975-987 ◽  
Author(s):  
Odile Filhol ◽  
Arsenio Nueda ◽  
Véronique Martel ◽  
Delphine Gerber-Scokaert ◽  
Maria José Benitez ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nuclear Localization ◽  
Protein Kinase Ck2 ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Nuclear Accumulation ◽  
Nucleocytoplasmic Trafficking ◽  
Green Fluorescent ◽  
Nucleocytoplasmic Distribution ◽  
Kinase Ck2

ABSTRACT Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

scholarly journals Cse1p Is Involved in Export of Yeast Importin α from the Nucleus

1998 ◽  
Vol 18 (11) ◽  
pp. 6805-6815 ◽  
Author(s):  
Jens Solsbacher ◽  
Patrick Maurer ◽  
F. Ralf Bischoff ◽  
Gabriel Schlenstedt
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Wild Type ◽  
Importin Α ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin. Importin α binds to the NLS and to importin β, which carries it through the nuclear pore complex (NPC). Importin disassembles in the nucleus, evidently by binding of RanGTP to importin β. The importin subunits are exported separately. We investigated the role of Cse1p, theSaccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin α). Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC. We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells. Srp1p but not importin β accumulated in nuclei ofcse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways. Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP. The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p. Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p. The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

Sign in / Sign up

Export Citation Format

Share Document