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

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.

Download Full-text

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.

Download Full-text

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.

Download Full-text

The Deubiquitylase Ubp15 Couples Transcription to mRNA Export

10.1101/2020.08.20.258822 ◽

2020 ◽

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

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.

Download Full-text

Mex67p of Schizosaccharomyces pombeInteracts with Rae1p in Mediating mRNA Export

Molecular and Cellular Biology ◽

10.1128/mcb.20.23.8767-8782.2000 ◽

2000 ◽

Vol 20 (23) ◽

pp. 8767-8782 ◽

Cited By ~ 52

Author(s):

Jin Ho Yoon ◽

Dona C. Love ◽

Anjan Guhathakurta ◽

John A. Hanover ◽

Ravi Dhar

Keyword(s):

Nuclear Export ◽

Rna Binding ◽

Fluorescent Protein ◽

Synthetic Lethality ◽

Sequence Similarity ◽

Mrna Export ◽

Nuclear Periphery ◽

Nuclear Pore ◽

Multicopy Suppressor ◽

Accessory Factor

ABSTRACT We identified the Schizosaccharomyces pombe mex67 gene (spmex67) as a multicopy suppressor of rae1-167 nup184-1 synthetic lethality and the rae1-167 tsmutation. spMex67p, a 596-amino-acid-long protein, has considerable sequence similarity to the Saccharomyces cerevisiae Mex67p (scMex67p) and human Tap. In contrast toscMEX67, spmex67 is essential for neither growth nor nuclear export of mRNA. However, an spmex67 null mutation (Δmex67) is synthetically lethal with therae1-167 mutation and accumulates poly(A)+ RNA in the nucleus. We identified a central region (149 to 505 amino acids) within spMex67p that associates with a complex containing Rae1p that complements growth and mRNA export defects of therae1-167 Δmex67 synthetic lethality. This region is devoid of RNA-binding, N-terminal nuclear localization, and the C-terminal nuclear pore complex-targeting regions. The (149–505)-green fluorescent protein (GFP) fusion is found diffused throughout the cell. Overexpression of spMex67p inhibits growth and mRNA export and results in the redistribution of the diffused localization of the (149–505)-GFP fusion to the nucleus and the nuclear periphery. These results suggest that spMex67p competes for essential mRNA export factor(s). Finally, we propose that the 149–505 region of spMex67p could act as an accessory factor in Rae1p-dependent transport and that spMex67p participates at various common steps with Rae1p export complexes in promoting the export of mRNA.

Download Full-text

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.

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.e01-11-0133 ◽

2002 ◽

Vol 13 (8) ◽

pp. 2571-2584 ◽

Cited By ~ 8

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.

Download Full-text

Nuclear accumulation of CHMP7 initiates nuclear pore complex injury and subsequent TDP-43 dysfunction in sporadic and familial ALS

Science Translational Medicine ◽

10.1126/scitranslmed.abe1923 ◽

2021 ◽

Vol 13 (604) ◽

pp. eabe1923

Author(s):

Alyssa N. Coyne ◽

Victoria Baskerville ◽

Benjamin L. Zaepfel ◽

Dennis W. Dickson ◽

Frank Rigo ◽

...

Keyword(s):

Nuclear Pore Complex ◽

Nuclear Export ◽

Induced Pluripotent Stem Cell ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Human Motor Cortex ◽

Sporadic Als ◽

Human Neurons ◽

Induced Pluripotent ◽

And Function

Alterations in the components [nucleoporins (Nups)] and function of the nuclear pore complex (NPC) have been implicated as contributors to the pathogenesis of genetic forms of neurodegeneration including C9orf72 amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). We hypothesized that Nup alterations and the consequential loss of NPC function may lie upstream of TDP-43 dysfunction and mislocalization widely observed in ALS, FTD, and related neurodegenerative diseases. Here, we provide evidence that CHMP7, a critical mediator of NPC quality control, is increased in nuclei of C9orf72 and sporadic ALS induced pluripotent stem cell (iPSC)–derived spinal neurons (iPSNs) and postmortem human motor cortex before the emergence of Nup alterations. Inhibiting the nuclear export of CHMP7 triggered Nup reduction and TDP-43 dysfunction and pathology in human neurons. Knockdown of CHMP7 alleviated disease-associated Nup alterations, deficits in Ran GTPase localization, defects in TDP-43–associated mRNA expression, and downstream glutamate-induced neuronal death. Thus, our data support a role for altered CHMP7-mediated Nup homeostasis as a prominent initiating pathological mechanism for familial and sporadic ALS and highlight the potential for CHMP7 as therapeutic target.

Download Full-text

Formation of Tap/NXT1 Heterodimers Activates Tap-Dependent Nuclear mRNA Export by Enhancing Recruitment to Nuclear Pore Complexes

Molecular and Cellular Biology ◽

10.1128/mcb.22.1.245-256.2002 ◽

2002 ◽

Vol 22 (1) ◽

pp. 245-256 ◽

Cited By ~ 79

Author(s):

Heather L. Wiegand ◽

Glen A. Coburn ◽

Yan Zeng ◽

Yibin Kang ◽

Hal P. Bogerd ◽

...

Keyword(s):

Nuclear Export ◽

Mrna Export ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Protein Functions ◽

Cellular Mrnas ◽

Pore Complexes

ABSTRACT The Tap protein has been shown to activate the nuclear export of mRNA species bearing retroviral constitutive transport elements and is also believed to play an essential role in the sequence nonspecific export of cellular mRNAs. However, it has remained unclear how Tap activity is regulated in vivo. Here, we report that the small NXT1/p15-1 protein functions as a critical cofactor for Tap-mediated mRNA export in both human and invertebrate cells. In the absence of NXT1 binding, the Tap protein is unable to effectively interact with components of the nuclear pore complex and both Tap nucleocytoplasmic shuttling and the nuclear export of mRNA molecules tethered to Tap are therefore severely attenuated. Formation of a Tap/NXT1 heterodimer enhances nucleoporin binding both in vitro and in vivo and induces the formation of a Tap/NXT1/nucleoporin ternary complex that is likely to be a key intermediate in the process of nuclear mRNA export. The critical importance of NXT1 for the nuclear export of poly(A)+ RNA is emphasized by the finding that specific inhibition of the expression of the Drosophila homolog of human NXT1, by using RNA interference, results in the nuclear accumulation of poly(A)+ RNA in cultured insect cells. These data suggest that NXT1 may act as a molecular switch that regulates the ability of Tap to mediate nuclear mRNA export by controlling the interaction of Tap with components of the nuclear pore.

Download Full-text

The ESCRT-III protein VPS4, but not CHMP4B or CHMP2B, is pathologically increased in familial and sporadic ALS neuronal nuclei

Acta Neuropathologica Communications ◽

10.1186/s40478-021-01228-0 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Alyssa N. Coyne ◽

Jeffrey D. Rothstein

Keyword(s):

Nuclear Pore Complex ◽

Mammalian Cells ◽

Nuclear Accumulation ◽

Nuclear Pore ◽

Dependent Manner ◽

Structured Illumination Microscopy ◽

Neuronal Nuclei ◽

Complex Injury ◽

Sporadic Als ◽

Human Neurons

AbstractNuclear pore complex injury has recently emerged as an early and significant contributor to familial and sporadic ALS disease pathogenesis. However, the molecular events leading to this pathological phenomenon characterized by the reduction of specific nucleoporins from neuronal nuclear pore complexes remain largely unknown. This is due in part to a lack of knowledge regarding the biological pathways and proteins underlying nuclear pore complex homeostasis specifically in human neurons. We have recently uncovered that aberrant nuclear accumulation of the ESCRT-III protein CHMP7 initiates nuclear pore complex in familial and sporadic ALS neurons. In yeast and non-neuronal mammalian cells, nuclear relocalization of CHMP7 has been shown to recruit the ESCRT-III proteins CHMP4B, CHMP2B, and VPS4 to facilitate nuclear pore complex and nuclear envelope repair and homeostasis. Here, using super resolution structured illumination microscopy, we find that neither CHMP4B nor CHMP2B are increased in ALS neuronal nuclei. In contrast, VPS4 expression is significantly increased in ALS neuronal nuclei prior to the emergence of nuclear pore injury in a CHMP7 dependent manner. However, unlike our prior CHMP7 knockdown studies, impaired VPS4 function does not mitigate alterations to the NPC and the integral transmembrane nucleoporin POM121. Collectively our data suggest that while alterations in VPS4 subcellular localization appear to be coincident with nuclear pore complex injury, therapeutic efforts to mitigate this pathogenic cascade should be targeted towards upstream events such as the nuclear accumulation of CHMP7 as we have previously described.

Download Full-text

WRN modulates translation by influencing mRNA export from the nucleus through its interaction with the export receptor NXF1 in HeLa cancer cell

10.21203/rs.3.rs-33612/v2 ◽

2020 ◽

Author(s):

Juan Manuel Iglesias-Pedraz ◽

Diego Matia Fossatti Jara ◽

Valeria Del Carmen Valle-Riestra Felice ◽

Sergio Rafael Cruz Visalaya ◽

Jose Antonio Ayala Felix ◽

...

Keyword(s):

Oxidative Stress ◽

Protein Synthesis ◽

Cancer Cells ◽

Nuclear Export ◽

Werner Syndrome ◽

Mrna Export ◽

Premature Aging ◽

Metabolic Shift ◽

Loss Of Function ◽

Global Protein Synthesis

Abstract Background The Werner syndrome protein (WRN) belongs to the RecQ family of helicases and its loss of function results in the premature aging disease Werner syndrome (WS). We previously demonstrated that an early cellular change induced by WRN depletion is a posttranscriptional decrease in the levels of enzymes involved in metabolic pathways that control macromolecular synthesis and protect from oxidative stress. This metabolic shift is tolerated by normal cells but causes mitochondria dysfunction and acute oxidative stress in rapidly growing cancer cells, thereby suppressing their proliferation.Results To identify the mechanism underlying this metabolic shift, we examined global protein synthesis and mRNA nucleocytoplasmic distribution after WRN knockdown. We determined that WRN depletion in HeLa cells attenuates global protein synthesis without affecting the level of key components of the mRNA export machinery. We further observed that WRN depletion affects the nuclear export of mRNAs and demonstrated that WRN directly interacts with mRNA and the mRNA export receptor Nuclear Export Factor 1 (NXF1).Conclusions Our findings suggest that WRN influences the export of mRNAs from the nucleus through its interaction with the NXF1 export receptor thereby affecting cellular proteostasis. In summary, we identified a new partner and a novel function of WRN, which is especially important for the proliferation of cancer cells.

Download Full-text