The Deubiquitylase Ubp15 Couples Transcription to mRNA Export

ABSTRACTThe nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed and exported. The role of ubiquitylation in this process is increasingly recognized as the ubiquitylation of many key players have been shown to affect mRNA nuclear export. While a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here, we identified the deubiquitylase Ubp15 as a regulator of nuclear export in Saccharomyces cerevisiae. Ubp15 interacts both with RNA polymerase II and with the nuclear pore complex, and its deletion reverts the nuclear export defect of mutants of the E3 ligase Rsp5. The deletion of UBP15 leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export.

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The deubiquitylase Ubp15 couples transcription to mRNA export

eLife ◽

10.7554/elife.61264 ◽

2020 ◽

Vol 9 ◽

Author(s):

Fanny Eyboulet ◽

Célia Jeronimo ◽

Jacques Côté ◽

François Robert

Keyword(s):

Rna Polymerase Ii ◽

Nuclear Pore Complex ◽

Nuclear Export ◽

Mrna Export ◽

Nuclear Pore ◽

Messenger Rnas ◽

E3 Ligases ◽

Nuclear Export Receptor ◽

Nascent Transcripts

Nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed, and exported. The role of ubiquitylation in this process is increasingly recognized but, while a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here we identified deubiquitylase Ubp15 as a regulator of nuclear export in Saccharomyces cerevisiae. Ubp15 interacts with both RNA polymerase II and the nuclear pore complex, and its deletion reverts the nuclear export defect of E3 ligase Rsp5 mutants. The deletion of UBP15 leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export.

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Sac3 Is an mRNA Export Factor That Localizes to Cytoplasmic Fibrils of Nuclear Pore Complex

Molecular Biology of the Cell ◽

10.1091/mbc.e02-08-0520 ◽

2003 ◽

Vol 14 (3) ◽

pp. 836-847 ◽

Cited By ~ 48

Author(s):

Elissa P. Lei ◽

Charlene A. Stern ◽

Birthe Fahrenkrog ◽

Heike Krebber ◽

Terence I. Moy ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Export ◽

Synthetic Lethality ◽

Mrna Export ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Messenger Rnas ◽

Loss Of Function ◽

Synthetic Lethal ◽

Starting Point

In eukaryotes, mRNAs are transcribed in the nucleus and exported to the cytoplasm for translation to occur. Messenger RNAs complexed with proteins referred to as ribonucleoparticles are recognized for nuclear export in part by association with Mex67, a keySaccharomyces cerevisiae mRNA export factor and homolog of human TAP/NXF1. Mex67, along with its cofactor Mtr2, is thought to promote ribonucleoparticle translocation by interacting directly with components of the nuclear pore complex (NPC). Herein, we show that the nuclear pore-associated protein Sac3 functions in mRNA export. Using a mutant allele of MTR2 as a starting point, we have identified a mutation in SAC3 in a screen for synthetic lethal interactors. Loss of function of SAC3 causes a strong nuclear accumulation of mRNA and synthetic lethality with a number of mRNA export mutants. Furthermore, Sac3 can be coimmunoprecipitated with Mex67, Mtr2, and other factors involved in mRNA export. Immunoelectron microscopy analysis shows that Sac3 localizes exclusively to cytoplasmic fibrils of the NPC. Finally, Mex67 accumulates at the nuclear rim when SAC3 is mutated, suggesting that Sac3 functions in Mex67 translocation through the NPC.

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A Role for RanBP1 in the Release of CRM1 from the Nuclear Pore Complex in a Terminal Step of Nuclear Export

The Journal of Cell Biology ◽

10.1083/jcb.145.4.645 ◽

1999 ◽

Vol 145 (4) ◽

pp. 645-657 ◽

Cited By ~ 158

Author(s):

Ralph H. Kehlenbach ◽

Achim Dickmanns ◽

Angelika Kehlenbach ◽

Tinglu Guan ◽

Larry Gerace

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Export ◽

Nuclear Pore ◽

Activated T Cells ◽

Permeabilized Cells ◽

Binding Domains ◽

Biochemical Fractionation ◽

Nuclear Export Receptor ◽

Gtpase Ran

We recently developed an assay in which nuclear export of the shuttling transcription factor NFAT (nuclear factor of activated T cells) can be reconstituted in permeabilized cells with the GTPase Ran and the nuclear export receptor CRM1. We have now used this assay to identify another export factor. After preincubation of permeabilized cells with a Ran mutant that cannot hydrolyze GTP (RanQ69L), cytosol supports NFAT export, but CRM1 and Ran alone do not. The RanQ69L preincubation leads to accumulation of CRM1 at the cytoplasmic periphery of the nuclear pore complex (NPC) in association with the p62 complex and Can/Nup214. RanGTP-dependent association of CRM1 with these nucleoporins was reconstituted in vitro. By biochemical fractionation and reconstitution, we showed that RanBP1 restores nuclear export after the RanQ69L preincubation. It also stimulates nuclear export in cells that have not been preincubated with RanQ69L. RanBP1 as well as Ran-binding domains of the cytoplasmic nucleoporin RanBP2 promote the release of CRM1 from the NPC. Taken together, our results indicate that RanGTP is important for the targeting of export complexes to the cytoplasmic side of the NPC and that RanBP1 and probably RanBP2 are involved in the dissociation of nuclear export complexes from the NPC in a terminal step of transport.

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Distinct Basket Nucleoporins roles in Nuclear Pore Function and Gene Expression: Tpr is an integral component of the TREX-2 mRNA export pathway

10.1101/685263 ◽

2019 ◽

Cited By ~ 1

Author(s):

Vasilisa Aksenova ◽

Hang Noh Lee ◽

Alexandra Smith ◽

Shane Chen ◽

Prasanna Bhat ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Export ◽

Protein Modification ◽

Mrna Export ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Nucleocytoplasmic Trafficking ◽

Pore Complexes ◽

Slow Turnover

AbstractNuclear pore complexes (NPCs) are important for many processes beyond nucleocytoplasmic trafficking, including protein modification, chromatin remodeling, transcription, mRNA processing and mRNA export. The multi-faceted nature of NPCs and the slow turnover of their components has made it difficult to understand the role of basket nucleoporins (Nup153, Nup50 and Tpr) in these diverse processes. To address this question, we used anAuxin-InducedDegron (AID) system to distinguish roles of basket nucleoporins: Loss of individual nucleoporins caused distinct alteration in patterns of nucleocytoplasmic trafficking and gene expression. Importantly, Tpr elimination caused rapid and pronounced changes in transcriptomic profiles within two hours of auxin addition. These changes were dissimilar to shifts observed after loss of Nup153 or Nup50, but closely related to changes after depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, GANP association to NPCs was specifically disrupted upon TPR depletion. Together, our findings demonstrate a unique and pivotal role of Tpr in regulating gene expression through GANP- and/or NXF1-dependent mRNA nuclear export.

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The Great Escape: mRNA Export through the Nuclear Pore Complex

International Journal of Molecular Sciences ◽

10.3390/ijms222111767 ◽

2021 ◽

Vol 22 (21) ◽

pp. 11767

Author(s):

Paola De Magistris

Keyword(s):

Conformational Changes ◽

Nuclear Pore Complex ◽

Nuclear Export ◽

Messenger Rna ◽

Mrna Export ◽

Protein Translation ◽

Nuclear Pore ◽

Body Of Knowledge ◽

Transport Receptors ◽

Basic Characteristics

Nuclear export of messenger RNA (mRNA) through the nuclear pore complex (NPC) is an indispensable step to ensure protein translation in the cytoplasm of eukaryotic cells. mRNA is not translocated on its own, but it forms ribonuclear particles (mRNPs) in association with proteins that are crucial for its metabolism, some of which; like Mex67/MTR2-NXF1/NXT1; are key players for its translocation to the cytoplasm. In this review, I will summarize our current body of knowledge on the basic characteristics of mRNA export through the NPC. To be granted passage, the mRNP cargo needs to bind transport receptors, which facilitate the nuclear export. During NPC transport, mRNPs undergo compositional and conformational changes. The interactions between mRNP and the central channel of NPC are described; together with the multiple quality control steps that mRNPs undergo at the different rings of the NPC to ensure only proper export of mature transcripts to the cytoplasm. I conclude by mentioning new opportunities that arise from bottom up approaches for a mechanistic understanding of nuclear export.

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RAE1 Is a Shuttling mRNA Export Factor That Binds to a GLEBS-like NUP98 Motif at the Nuclear Pore Complex through Multiple Domains

The Journal of Cell Biology ◽

10.1083/jcb.145.2.237 ◽

1999 ◽

Vol 145 (2) ◽

pp. 237-254 ◽

Cited By ~ 159

Author(s):

Colin E.J. Pritchard ◽

Maarten Fornerod ◽

Lawryn H. Kasper ◽

Jan M.A. van Deursen

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Export ◽

Mrna Export ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Xenopus Laevis Oocytes ◽

Binding Motif ◽

Binding Studies ◽

Direct Role ◽

Multiple Domains

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p–Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non–WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98–RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.

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A nuclear export sequence promotes CRM1-dependent targeting of the nucleoporin Nup214 to the nuclear pore complex

Journal of Cell Science ◽

10.1242/jcs.258095 ◽

2021 ◽

Vol 134 (6) ◽

Author(s):

Mohamed Hamed ◽

Birgit Caspar ◽

Sarah A. Port ◽

Ralph H. Kehlenbach

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Export ◽

Nuclear Pore ◽

Wild Type ◽

Leptomycin B ◽

Nuclear Export Sequence ◽

Binding Partners ◽

Dependent Protein ◽

Nuclear Export Receptor ◽

Cytoplasmic Side

ABSTRACT Nup214 is a major nucleoporin on the cytoplasmic side of the nuclear pore complex with roles in late steps of nuclear protein and mRNA export. It interacts with the nuclear export receptor CRM1 (also known as XPO1) via characteristic phenylalanine-glycine (FG) repeats in its C-terminal region. Here, we identify a classic nuclear export sequence (NES) in Nup214 that mediates Ran-dependent binding to CRM1. Nup214 versions with mutations in the NES, as well as wild-type Nup214 in the presence of the selective CRM1 inhibitor leptomycin B, accumulate in the nucleus of Nup214-overexpressing cells. Furthermore, physiological binding partners of Nup214, such as Nup62 and Nup88, are recruited to the nucleus together with Nup214. Nuclear export of mutant Nup214 can be rescued by artificial nuclear export sequences at the C-terminal end of Nup214, leading also to a correct localization of Nup88. Our results suggest a function of the Nup214 NES in the biogenesis of the nuclear pore complex and/or in terminal steps of CRM1-dependent protein export.

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The L1 stalk is required for efficient export of nascent large ribosomal subunits in yeast

10.1101/629261 ◽

2019 ◽

Author(s):

Sharmishtha Musalgaonkar ◽

Joshua J. Black ◽

Arlen W. Johnson

Keyword(s):

Ribosomal Protein ◽

Nuclear Pore Complex ◽

Nuclear Export ◽

Nuclear Pore ◽

Structural Studies ◽

Ribosomal Subunits

AbstractThe ribosomal protein Rpl1 (uL1 in universal nomenclature) is essential in yeast and constitutes part of the L1 stalk which interacts with E site ligands on the ribosome. Structural studies of nascent pre-60S complexes in yeast have shown that a domain of the Crm1-dependent nuclear export adapter Nmd3, binds in the E site and interacts with Rpl1, inducing closure of the L1 stalk. Based on this observation, we decided to reinvestigate the role of the L1 stalk in nuclear export of pre-60S subunits despite previous work showing that Rpl1-deficient ribosomes are exported from the nucleus and engage in translation. Large cargoes, such as ribosomal subunits, require multiple export factors to facilitate their transport through the nuclear pore complex. Here, we show that pre-60S subunits lacking Rpl1 or truncated for the RNA of the L1 stalk are exported inefficiently. Surprisingly, this is not due to a measurable defect in recruitment of Nmd3 but appears to result from inefficient recruitment of the Mex67-Mtr2 heterodimer.

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CRM1 promotes capsid disassembly and nuclear envelope translocation of adenovirus independently of its export function

Journal of Virology ◽

10.1128/jvi.01273-21 ◽

2021 ◽

Author(s):

Floriane Lagadec ◽

Irene Carlon-Andres ◽

Jessica Ragues ◽

Sarah Port ◽

Harald Wodrich ◽

...

Keyword(s):

Nuclear Export ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Microtubule Organizing Center ◽

Leptomycin B ◽

Pore Complexes ◽

Nuclear Export Receptor ◽

Mitotic Cells ◽

Capsid Disassembly

After receptor-mediated endocytosis and endosomal escape, adenoviral capsids can travel via microtubule organizing centers to the nuclear envelope. Upon capsid disassembly, viral genome import into nuclei of interphase cells then occurs through nuclear pore complexes, involving the nucleoporins Nup214 and Nup358. Import also requires the activity of the classic nuclear export receptor CRM1, as it is blocked by the selective inhibitor leptomycin B. We have now used artificially enucleated as well as mitotic cells to analyze the role of an intact nucleus in different steps of the viral life cycle. In enucleated U2OS cells, viral capsids traveled to the microtubule organizing center, whereas their removal from this complex was blocked, suggesting that this step required nuclear factors. In mitotic cells, on the other hand, CRM1 promoted capsid disassembly and genome release, suggesting a role of this protein that does not require intact nuclear envelopes or nuclear pore complexes and is distinct from its function as a nuclear export receptor. Similar to enucleation, inhibition of CRM1 by leptomycin B also leads to an arrest of adenoviral capsids at the microtubule organizing center. In a small-scale screen using leptomycin B-resistant versions of CRM1, we identified a mutant, CRM1 W142A P143A, that is compromised with respect to adenoviral capsid disassembly, both in interphase and in mitotic cells. Strikingly, this mutant is capable of exporting cargo proteins out of the nucleus of living cells or digitonin-permeabilized cells, pointing to a role of the mutated region that is not directly linked to nuclear export. IMPORTANCE A role of nucleoporins and of soluble transport factors in adenoviral genome import into the nucleus of infected cells in interphase has previously been established. The nuclear export receptor CRM1 promotes genome import, but its precise function is not known. Using enucleated and mitotic cells, we showed that CRM1 does not simply function by exporting a crucial factor out of the nucleus that would then trigger capsid disassembly and genome import. Instead, CRM1 has an export-independent role, a notion that is also supported by a mutant, CRM1 W142A P143A, which is export-competent but deficient in viral capsid disassembly, both in interphase and in mitotic cells.

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