Abstract MP145: Potential Functions of the Nuclear Basket Associated With Dilated Cardiomyopathy

Objectives: NUP153 overexpression is clinically correlated with cardiomyopathies, specifically dilated cardiomyopathy (DCM). In combination with the nuclear pore complex (NPC) proteins TPR and NUP50, all three form the basket structure of the NPC that sits within the nuclear envelope. The nuclear basket regulates chromatin dynamics and nuclear export, yet its potential role in cardiopathological development and progression remains unknown. This study was thus carried out to elucidate possible functional roles for nuclear basket nucleoporins in cardiac biology. Methods: Human induced pluripotent stem cells (hiPSCs) with endogenous mEGFP tagged NUP153 were differentiated into cardiomyocytes (hiPSC-CMs) via direct differentiation. Beating started ~ Day 10 and contractile areas were analyzed for contraction velocity and magnitude. NUP153 mobility in hiPSCs and hiPSC-CMs was determined by iFRAP experiments. Bioinformatic analysis of a clinical dilated cardiomyopathy dataset (GSE29819) returned a focused ‘nuclear envelope’ gene list that was uploaded to Ingenuity Pathways Analysis for network analysis. Results: Within hiPSC-CM beating foci (A), NUP153 mobility decreased and went from a biphasic to a monophasic decay curve (B). Bioinformatic analysis of a DCM dataset (C) revealed expression changes in TPR and NUP50 , with no significant changes in NUP153 (D). Conclusions: A switch to monophasic decay kinetics of NUP153 in hiPSC-CMs suggests prioritization of stable slow pools that may reflect changes in global transcriptome regulation. In DCM, differential nucleoporin gene expression changes implicate discrete functional disruptions within the nuclear basket.

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The GLFG repetitive region of the nucleoporin Nup116p interacts with Kap95p, an essential yeast nuclear import factor.

The Journal of Cell Biology ◽

10.1083/jcb.131.6.1699 ◽

1995 ◽

Vol 131 (6) ◽

pp. 1699-1713 ◽

Cited By ~ 131

Author(s):

M K Iovine ◽

J L Watkins ◽

S R Wente

Keyword(s):

Nuclear Envelope ◽

Nuclear Import ◽

Nuclear Export ◽

Structural Basis ◽

Nuclear Pore ◽

Temperature Sensitive ◽

Repetitive Region ◽

Amino Terminal ◽

Section Electron ◽

Rna Export

Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nup116p's role in NPC function. First, deletion mutagenesis analysis of NUP116 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nup100p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nup116p's GLFG region with either Nup100p's GLFG region or Nsp1p's FXFG repetitive region abolishes the function of Nup116p. At a semipermissive growth temperature, the cells lacking Nup116p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nup116p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin beta. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

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Nuclear Export Through Nuclear Envelope Remodeling inSaccharomyces cerevisiae

10.1101/224055 ◽

2017 ◽

Author(s):

Baojin Ding ◽

Anne M. Mirza ◽

James Ashley ◽

Vivian Budnik ◽

Mary Munson

Keyword(s):

Nuclear Envelope ◽

Nuclear Export ◽

Mammalian Cells ◽

Budding Yeast ◽

Mrna Export ◽

Low Frequency ◽

Nuclear Pore ◽

Wild Type ◽

Export Pathway ◽

Wild Type Yeast

ABSTRACTIn eukaryotes, subsets of exported mRNAs are organized into large ribonucleoprotein (megaRNP) granules. How megaRNPs exit the nucleus is unclear, as their diameters are much larger than the nuclear pore complex (NPC) central channel. We previously identified a non-canonical nuclear export mechanism inDrosophila(Speese et al.,Cell2012) and mammals (Ding et al., in preparation), in which megaRNPs exit the nucleus by budding across nuclear envelope (NE) membranes. Here, we present evidence for a similar pathway in the nucleus of the budding yeast S.cerevisiae, which contain morphologically similar granules bearing mRNAs. Wild-type yeast displayed these granules at very low frequency, but this frequency was dramatically increased when the non-essential NPC protein Nup116 was deleted. These granules were not artifacts of defective NPCs; a mutation in the exportinXPO1(CRM1), in which NPCs are normal, induced similar megaRNP upregulation. We hypothesize that a non-canonical nuclear export pathway, analogous to those observed inDrosophilaand in mammalian cells, exists in yeast, and that this pathway is upregulated for use when NPCs or nuclear export are impaired.SUMMARYDing et al., describe a non-canonical mRNA export pathway in budding yeast similar to that observed inDrosophila. This pathway appears upregulated when the NPC is impaired, nuclear envelope integrity is disrupted, or the export factor Xpo1 (CRM1) is defective.

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The Portal Vertex of KSHV Promotes Docking of Capsids at the Nuclear Pores

Viruses ◽

10.3390/v13040597 ◽

2021 ◽

Vol 13 (4) ◽

pp. 597

Author(s):

Daniela Dünn-Kittenplon ◽

Asaf Ashkenazy-Titelman ◽

Inna Kalt ◽

Jean-Paul Lellouche ◽

Yaron Shav-Tal ◽

...

Keyword(s):

Nuclear Envelope ◽

Nuclear Export ◽

Nuclear Pore ◽

Salt Treatment ◽

Reading Frame ◽

Protein Subunits ◽

Tegument Proteins ◽

Dead End ◽

Lower Extent ◽

Icosahedral Capsid

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a cancer-related herpesvirus. Like other herpesviruses, the KSHV icosahedral capsid includes a portal vertex, composed of 12 protein subunits encoded by open reading frame (ORF) 43, which enables packaging and release of the viral genome into the nucleus through the nuclear pore complex (NPC). Capsid vertex-specific component (CVSC) tegument proteins, which directly mediate docking at the NPCs, are organized on the capsid vertices and are enriched on the portal vertex. Whether and how the portal vertex is selected for docking at the NPC is unknown. Here, we investigated the docking of incoming ORF43-null KSHV capsids at the NPCs, and describe a significantly lower fraction of capsids attached to the nuclear envelope compared to wild-type (WT) capsids. Like WT capsids, nuclear envelope-associated ORF43-null capsids co-localized with different nucleoporins (Nups) and did not detach upon salt treatment. Inhibition of nuclear export did not alter WT capsid docking. As ORF43-null capsids exhibit lower extent of association with the NPCs, we conclude that although not essential, the portal has a role in mediating the interaction of the CVSC proteins with Nups, and suggest a model whereby WT capsids can dock at the nuclear envelope through a non-portal penton vertex, resulting in an infection ‘dead end’.

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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.

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Nuclear Envelope Dynamics During Mitosis

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100103188 ◽

1988 ◽

Vol 46 ◽

pp. 224-225

Author(s):

Brian Burke

Keyword(s):

Nuclear Envelope ◽

Membrane Vesicles ◽

Nuclear Pore ◽

Nuclear Pore Complexes ◽

Animal Cells ◽

Interphase Chromosome ◽

Lamin B ◽

Chromosome Architecture ◽

Complex Membrane ◽

Pore Complexes

The nuclear envelope is a complex membrane structure that forms the boundary of the nuclear compartment in eukaryotes. It regulates the passage of macromolecules between the two compartments and may be important for organizing interphase chromosome architecture. In interphase animal cells it forms a remarkably stable structure consisting of a double membrane ouerlying a protein meshwork or lamina and penetrated by nuclear pore complexes. The latter form the channels for nucleocytoplasmic exchange of macromolecules, At the onset of mitosis, however, it rapidly disassembles, the membranes fragment to yield small vesicles and the lamina, which is composed of predominantly three polypeptides, lamins R, B and C (MW approx. 74, 68 and 65 kDa respectiuely), breaks down. Lamins B and C are dispersed as monomers throughout the mitotic cytoplasm, while lamin B remains associated with the nuclear membrane vesicles.

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Faculty Opinions recommendation of The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1023280.285732 ◽

2005 ◽

Author(s):

Mary Dasso

Keyword(s):

Nuclear Envelope ◽

Nuclear Pore Complex ◽

Nuclear Pore ◽

Complex Assembly ◽

Nuclear Envelope Formation

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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.

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Bioinformatic Analysis of Structure and Function of LIM Domains of Human Zyxin Family Proteins

International Journal of Molecular Sciences ◽

10.3390/ijms22052647 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2647

Author(s):

M. Quadir Siddiqui ◽

Maulik D. Badmalia ◽

Trushar R. Patel

Keyword(s):

Nucleic Acid ◽

Nuclear Export ◽

Consensus Sequence ◽

Nuclear Export Signal ◽

Bioinformatic Analysis ◽

Nucleic Acid Binding ◽

Protein Protein Interaction ◽

Lim Domains ◽

Protein Nucleic Acid ◽

And Function

Members of the human Zyxin family are LIM domain-containing proteins that perform critical cellular functions and are indispensable for cellular integrity. Despite their importance, not much is known about their structure, functions, interactions and dynamics. To provide insights into these, we used a set of in-silico tools and databases and analyzed their amino acid sequence, phylogeny, post-translational modifications, structure-dynamics, molecular interactions, and functions. Our analysis revealed that zyxin members are ohnologs. Presence of a conserved nuclear export signal composed of LxxLxL/LxxxLxL consensus sequence, as well as a possible nuclear localization signal, suggesting that Zyxin family members may have nuclear and cytoplasmic roles. The molecular modeling and structural analysis indicated that Zyxin family LIM domains share similarities with transcriptional regulators and have positively charged electrostatic patches, which may indicate that they have previously unanticipated nucleic acid binding properties. Intrinsic dynamics analysis of Lim domains suggest that only Lim1 has similar internal dynamics properties, unlike Lim2/3. Furthermore, we analyzed protein expression and mutational frequency in various malignancies, as well as mapped protein-protein interaction networks they are involved in. Overall, our comprehensive bioinformatic analysis suggests that these proteins may play important roles in mediating protein-protein and protein-nucleic acid interactions.

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Visualizing Association of the Retroviral Gag Protein with Unspliced Viral RNA in the Nucleus

mBio ◽

10.1128/mbio.00524-20 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 3

Author(s):

Rebecca J. Kaddis Maldonado ◽

Breanna Rice ◽

Eunice C. Chen ◽

Kevin M. Tuffy ◽

Estelle F. Chiari ◽

...

Keyword(s):

Plasma Membrane ◽

Nuclear Envelope ◽

Rous Sarcoma Virus ◽

Nuclear Export ◽

Live Cell ◽

Viral Rna ◽

Wild Type ◽

Gag Protein ◽

Rous Sarcoma ◽

Sarcoma Virus

ABSTRACT Packaging of genomic RNA (gRNA) by retroviruses is essential for infectivity, yet the subcellular site of the initial interaction between the Gag polyprotein and gRNA remains poorly defined. Because retroviral particles are released from the plasma membrane, it was previously thought that Gag proteins initially bound to gRNA in the cytoplasm or at the plasma membrane. However, the Gag protein of the avian retrovirus Rous sarcoma virus (RSV) undergoes active nuclear trafficking, which is required for efficient gRNA encapsidation (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc Natl Acad Sci U S A 99:3944–3949, 2002, https://doi.org/10.1073/pnas.062652199; R. Garbitt-Hirst, S. P. Kenney, and L. J. Parent, J Virol 83:6790–6797, 2009, https://doi.org/10.1128/JVI.00101-09). These results raise the intriguing possibility that the primary contact between Gag and gRNA might occur in the nucleus. To examine this possibility, we created a RSV proviral construct that includes 24 tandem repeats of MS2 RNA stem-loops, making it possible to track RSV viral RNA (vRNA) in live cells in which a fluorophore-conjugated MS2 coat protein is coexpressed. Using confocal microscopy, we observed that both wild-type Gag and a nuclear export mutant (Gag.L219A) colocalized with vRNA in the nucleus. In live-cell time-lapse images, the wild-type Gag protein trafficked together with vRNA as a single ribonucleoprotein (RNP) complex in the nucleoplasm near the nuclear periphery, appearing to traverse the nuclear envelope into the cytoplasm. Furthermore, biophysical imaging methods suggest that Gag and the unspliced vRNA physically interact in the nucleus. Taken together, these data suggest that RSV Gag binds unspliced vRNA to export it from the nucleus, possibly for packaging into virions as the viral genome. IMPORTANCE Retroviruses cause severe diseases in animals and humans, including cancer and acquired immunodeficiency syndromes. To propagate infection, retroviruses assemble new virus particles that contain viral proteins and unspliced vRNA to use as gRNA. Despite the critical requirement for gRNA packaging, the molecular mechanisms governing the identification and selection of gRNA by the Gag protein remain poorly understood. In this report, we demonstrate that the Rous sarcoma virus (RSV) Gag protein colocalizes with unspliced vRNA in the nucleus in the interchromatin space. Using live-cell confocal imaging, RSV Gag and unspliced vRNA were observed to move together from inside the nucleus across the nuclear envelope, suggesting that the Gag-gRNA complex initially forms in the nucleus and undergoes nuclear export into the cytoplasm as a viral ribonucleoprotein (vRNP) complex.

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