scholarly journals The mRNA Export Factor Sus1 Is Involved in Spt/Ada/Gcn5 Acetyltransferase-mediated H2B Deubiquitinylation through Its Interaction with Ubp8 and Sgf11

2006 ◽  
Vol 17 (10) ◽  
pp. 4228-4236 ◽  
Author(s):  
Alwin Köhler ◽  
Pau Pascual-García ◽  
Ana Llopis ◽  
Meritxell Zapater ◽  
Francesc Posas ◽  
...  
Keyword(s):  
Mrna Export ◽  
High Salt ◽  
Nuclear Pore ◽  
Saga Complex ◽  
Histone H2b ◽  
Gal1 Promoter

Sus1 acts in nuclear mRNA export via its association with the nuclear pore-associated Sac3–Thp1–Cdc31 complex. In addition, Sus1 plays a role in transcription through its interaction with the Spt/Ada/Gcn5 acetyltransferase (SAGA) complex. Here, we have analyzed function and interaction of Sus1 within the SAGA complex. We demonstrate that Sus1 is involved in the SAGA-dependent histone H2B deubiquitinylation and maintenance of normal H3 methylation levels. By deletion analyses, we show that binding of Sus1 to SAGA depends on the deubiquitinylating enzyme Ubp8 and Sgf11. Moreover, a stable subcomplex between Sus1, Sgf11, and Ubp8 could be dissociated from SAGA under high salt conditions. In vivo recruitment of Sus1 to the activated GAL1 promoter depends on Ubp8 and vice versa. In addition, histones coenrich during SAGA purification in a Sus1–Sgf11–Ubp8-dependent way. Interestingly, sgf11 deletion enhances the mRNA export defect observed in sus1Δ cells. Thus, the Sus1–Sgf11–Ubp8 module could work at the junction between SAGA-dependent transcription and nuclear mRNA export.

scholarly journals DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Amr Nassrallah ◽  
Martin Rougée ◽  
Clara Bourbousse ◽  
Stephanie Drevensek ◽  
Sandra Fonseca ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Proteolytic Degradation ◽  
Saga Complex ◽  
Histone H2b ◽  
Evolutionarily Conserved ◽  
The Stability ◽  
Differentiation And Proliferation ◽  
Multicellular Organisms ◽  
Ubiquitination Machinery

DE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11 in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

scholarly journals SAGA-CORE subunit Spt7 is required for correct Ubp8 localization, chromatin association and deubiquitinase activity

2020 ◽  
Author(s):  
Carme Nuno-Cabanes ◽  
Varinia Garcia-Molinero ◽  
Manuel Martín-Expósito ◽  
Maria-Eugenia Gas ◽  
Paula Oliete-Calvo ◽  
...  
Keyword(s):  
Catalytic Subunit ◽  
Eukaryotic Cells ◽  
Saga Complex ◽  
Wild Type ◽  
Core Complex ◽  
Independent Manner ◽  
Histone H2b ◽  
Tetrameric Structure ◽  
Core Components

Abstract Background: Histone H2B deubiquitination is performed by numerous deubiquitinases in eukaryotic cells including Ubp8, the catalytic subunit of the tetrameric deubiquitination module (DUBm: Ubp8; Sus1; Sgf11; Sgf73) of the Spt-Ada-Gcn5 acetyltransferase (SAGA). Ubp8 is linked to the rest of SAGA through Sgf73 and is activated by the adaptors Sus1 and Sgf11. It is unknown if DUBm/Ubp8 might also work in a SAGA-independent manner. Results: Here we report that a tetrameric DUBm is assembled independently of the SAGA-CORE components SPT7, ADA1 and SPT20. In the absence of SPT7, i.e. independent of the SAGA complex, Ubp8 and Sus1 are poorly recruited to SAGA-dependent genes and to chromatin. Notably, cells lacking Spt7 or Ada1, but not Spt20, show lower levels of nuclear Ubp8 than wild type cells, suggesting a possible role for SAGA-CORE subunits in Ubp8 localization. Last, deletion of SPT7 leads to defects in Ubp8 deubiquitinase activity in in vivo and in vitroassays. Conclusions: Collectively, our studies show that the DUBm tetrameric structure can form without a complete intact SAGA-CORE complex and that it includes full length Sgf73. However, subunits of this SAGA-CORE influence DUBm association with chromatin, its localization and its activity.

scholarly journals SAGA CORE subunit Spt7 is required for correct Ubp8 localization, chromatin association and deubiquitinase activity

2020 ◽  
Author(s):  
Carme Nuno-Cabanes ◽  
Varinia Garcia-Molinero ◽  
Manuel Martín-Expósito ◽  
Maria-Eugenia Gas ◽  
Paula Oliete-Calvo ◽  
...  
Keyword(s):  
Catalytic Subunit ◽  
Eukaryotic Cells ◽  
Saga Complex ◽  
Wild Type ◽  
Independent Manner ◽  
Histone H2b ◽  
Core Components

Abstract BackgroundHistone H2B deubiquitination is performed by numerous deubiquitinases in eukaryotic cells including Ubp8, the catalytic subunit of the tetrameric deubiquitination module (DUBm: Ubp8; Sus11; Sgf11; Sgf73) of the Spt-Ada- Gcn5 acetyltransferase (SAGA). Ubp8 is linked to the rest of SAGA through Sgf73 and is activated by the adaptors Sus1 and Sgf11. It is unknown if DUBm/Ubp8 might also work in a SAGA-independent manner.ResultsHere we report that a tetrameric DUBm is assembled independently of the SAGA-core components SPT7, ADA1 and SPT20. In the absence of SPT7, i.e. independent of the SAGA complex, Ubp8 and Sus1 are poorly recruited to SAGA-dependent genes and to chromatin. Notably, cells lacking Spt7 or Ada1, but not Spt20, show lower levels of nuclear Ubp8 than wild type cells, suggesting a possible role for SAGA CORE subunits in Ubp8 localization. Last, deletion of SPT7 leads to defects in Ubp8 deubiquitinase activity in in vivo and in vitro assays.ConclusionsCollectively, our studies show that a stable DUBm is assembled regardless of SAGA integrity; however its function and localization is affected by the absence of Spt7 or Ada1.

scholarly journals Crp79p, Like Mex67p, Is an Auxiliary mRNA Export Factor inSchizosaccharomyces pombe

2002 ◽  
Vol 13 (8) ◽  
pp. 2571-2584 ◽  
Author(s):  
Anjan G. Thakurta ◽  
William A. Whalen ◽  
Jin Ho Yoon ◽  
Anekella Bharathi ◽  
Libor Kozak ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Fluorescent Protein ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Rna Recognition Motif ◽  
Synthetic Lethal ◽  
Export Activity ◽  
C Terminus ◽  
Green Fluorescent

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p inSchizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)+ RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.

scholarly journals The Deubiquitylation Activity of Ubp8 Is Dependent upon Sgf11 and Its Association with the SAGA Complex

2005 ◽  
Vol 25 (3) ◽  
pp. 1173-1182 ◽  
Author(s):  
Kenneth K. Lee ◽  
Laurence Florens ◽  
Selene K. Swanson ◽  
Michael P. Washburn ◽  
Jerry L. Workman
Keyword(s):  
Gene Regulation ◽  
Ubiquitin Ligase ◽  
Histone Acetyltransferase ◽  
Saga Complex ◽  
Histone Tails ◽  
Histone H2b ◽  
Covalent Modifications ◽  
Posttranslation Modification

ABSTRACT Covalent modifications of the histone tails and the cross talk between these modifications are hallmark features of gene regulation. The SAGA histone acetyltransferase complex is one of the most well-characterized complexes involved in these covalent modifications. The recent finding that the removal of the ubiquitin group from H2B is performed by a component of SAGA, Ubp8, is intriguing as it assigns two posttranslation modification processes to one complex. In this work, we characterize the association of Ubp8 with SAGA and the effect that acetylation and deubiquitylation have on one another in vitro and in vivo. We found not only that Ubp8 is a part of the SAGA complex, but also that its deubiquitylation activity requires Ubp8's association with SAGA. Furthermore, we found that the Ubp8 association with SAGA requires Sgf11 and that this requirement is reciprocal. We also found that the acetylation and deubiquitylation activities of SAGA are independent of one another. However, we found that preacetylating histone H2B inhibited subsequent deubiquitylation. Additionally, we found that increasing the ubiquitylation state of H2B inhibited the expression of the ARG1 gene, whose repression was previously shown to require the RAD6 ubiquitin ligase. Taken together, these data indicate that the expression of some genes, including ARG1, is regulated by a balance of histone H2B ubiquitylation in the cell.

scholarly journals The Fission Yeast Nup107-120 Complex Functionally Interacts with the Small GTPase Ran/Spi1 and Is Required for mRNA Export, Nuclear Pore Distribution, and Proper Cell Division

2004 ◽  
Vol 24 (14) ◽  
pp. 6379-6392 ◽  
Author(s):  
Siau Wei Baï ◽  
Jacques Rouquette ◽  
Makoto Umeda ◽  
Wolfgang Faigle ◽  
Damarys Loew ◽  
...  
Keyword(s):  
Cell Division ◽  
Mrna Export ◽  
Small Gtpase ◽  
Pore Distribution ◽  
Open Reading Frames ◽  
Nuclear Pore ◽  
Restrictive Temperature ◽  
Functional Studies ◽  
Gtpase Ran

ABSTRACT We have characterized Schizosaccharomyces pombe open reading frames encoding potential orthologues of constituents of the evolutionarily conserved Saccharomyces cerevisiae Nup84 vertebrate Nup107-160 nuclear pore subcomplex, namely Nup133a, Nup133b, Nup120, Nup107, Nup85, and Seh1. In spite of rather weak sequence conservation, in vivo analyses demonstrated that these S. pombe proteins are localized at the nuclear envelope. Biochemical data confirmed the organization of these nucleoporins within conserved complexes. Although examination of the S. cerevisiae and S. pombe deletion mutants revealed different viability phenotypes, functional studies indicated that the involvement of this complex in nuclear pore distribution and mRNA export has been conserved between these highly divergent yeasts. Unexpectedly, microscopic analyses of some of the S. pombe mutants revealed cell division defects at the restrictive temperature (abnormal septa and mitotic spindles and chromosome missegregation) that were reminiscent of defects occurring in several S. pombe GTPase Ran (RanSp)/Spi1 cycle mutants. Furthermore, deletion of nup120 moderately altered the nuclear location of RanSp/Spi1, whereas overexpression of a nonfunctional RanSp/Spi1-GFP allele was specifically toxic in the Δnup120 and Δnup133b mutant strains, indicating a functional and genetic link between constituents of the S. pombe Nup107-120 complex and of the RanSp/Spi1 pathway.

scholarly journals DET1-mediated degradation of a SAGA-like deubiquitination module controls H2Bub homeostasis

2018 ◽  
Author(s):  
Amr Nassrallah ◽  
Martin Rougée ◽  
Clara Bourbousse ◽  
Stéphanie Drevensek ◽  
Sandra Fonseca ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Proteolytic Degradation ◽  
Saga Complex ◽  
Histone H2b ◽  
Evolutionarily Conserved ◽  
The Stability ◽  
Differentiation And Proliferation ◽  
Multicellular Organisms ◽  
Ubiquitination Machinery

SummaryDE-ETIOLATED 1 (DET1) is an evolutionarily conserved component of the ubiquitination machinery that mediates the destabilization of key regulators of cell differentiation and proliferation in multicellular organisms. In this study, we provide evidence from Arabidopsis that DET1 is essential for the regulation of histone H2B monoubiquitination (H2Bub) over most genes by controlling the stability of a plant deubiquitination module (DUBm). In contrast with yeast and metazoan DUB modules that are associated with the large SAGA complex, the Arabidopsis DUBm only comprises three proteins (hereafter named SGF11, ENY2 and UBP22) and appears to act independently as a major H2Bub deubiquitinase activity. Our study further unveils that DET1-DDB1-Associated-1 (DDA1) protein interacts with SGF11in vivo, linking the DET1 complex to light-dependent ubiquitin-mediated proteolytic degradation of the DUBm. Collectively, these findings uncover a signaling path controlling DUBm availability, potentially adjusting H2Bub turnover capacity to the cell transcriptional status.

scholarly journals Mutational Uncoupling of the Role of Sus1 in Nuclear Pore Complex Targeting of an mRNA Export Complex and Histone H2B Deubiquitination

2009 ◽  
Vol 284 (18) ◽  
pp. 12049-12056 ◽  
Author(s):  
Christoph Klöckner ◽  
Maren Schneider ◽  
Sheila Lutz ◽  
Divyang Jani ◽  
Dieter Kressler ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Histone H2b

scholarly journals Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export

2001 ◽  
Vol 155 (3) ◽  
pp. 339-354 ◽  
Author(s):  
Sanjay Vasu ◽  
Sundeep Shah ◽  
Arturo Orjalo ◽  
Minkyu Park ◽  
Wolfgang H. Fischer ◽  
...  
Keyword(s):  
Binding Site ◽  
Nuclear Export ◽  
Protein Import ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Egg Extracts ◽  
Rna Export ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.

scholarly journals Differential Targeting of Nuclear Pore Complex Proteins in Poliovirus-Infected Cells

2007 ◽  
Vol 82 (4) ◽  
pp. 1647-1655 ◽  
Author(s):  
Nogi Park ◽  
Pavan Katikaneni ◽  
Tim Skern ◽  
Kurt E. Gustin
Keyword(s):  
Nuclear Pore Complex ◽  
Defense Mechanisms ◽  
Guanidine Hydrochloride ◽  
Mrna Export ◽  
Differential Sensitivity ◽  
Nuclear Pore ◽  
Nucleocytoplasmic Trafficking ◽  
Synthesis Inhibitor ◽  
Infected Cells

ABSTRACT Poliovirus disrupts nucleocytoplasmic trafficking and results in the cleavage of two nuclear pore complex (NPC) proteins, Nup153 and Nup62. The NPC is a 125-MDa complex composed of multiple copies of 30 different proteins. Here we have extended the analysis of the NPC in infected cells by examining the status of Nup98, an interferon-induced NPC protein with a major role in mRNA export. Our results indicate that Nup98 is targeted for cleavage after infection but that this occurs much more rapidly than it does for Nup153 and Nup62. In addition, we find that cleavage of these NPC proteins displays differential sensitivity to the viral RNA synthesis inhibitor guanidine hydrochloride. Inhibition of nuclear import and relocalization of host nuclear proteins to the cytoplasm were only apparent at later times after infection when all three nucleoporins (Nups) were cleaved. Surprisingly, analysis of the distribution of mRNA in infected cells revealed that proteolysis of Nup98 did not result in an inhibition of mRNA export. Cleavage of Nup98 could be reconstituted by the addition of purified rhinovirus type 2 2Apro to whole-cell lysates prepared from uninfected cells, suggesting that the 2A protease has a role in this process in vivo. These results indicate that poliovirus differentially targets subsets of NPC proteins at early and late times postinfection. In addition, targeting of interferon-inducible NPC proteins, such as Nup98, may be an additional weapon in the arsenal of poliovirus and perhaps other picornaviruses to overcome host defense mechanisms.

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