scholarly journals The nuclear basket mediates perinuclear mRNA scanning in budding yeast

2015 ◽  
Vol 211 (6) ◽  
pp. 1131-1140 ◽  
Author(s):  
Mark-Albert Saroufim ◽  
Pierre Bensidoun ◽  
Pascal Raymond ◽  
Samir Rahman ◽  
Matthew R. Krause ◽  
...  
Keyword(s):  
Nuclear Periphery ◽  
Nuclear Pore ◽  
Messenger Rnas ◽  
Nuclear Pores ◽  
Transport Channel ◽  
Mrna Binding Protein ◽  
Live Cell Microscopy ◽  
Scanning Behavior ◽  
Mrna Binding ◽  
Cell Microscopy

After synthesis and transit through the nucleus, messenger RNAs (mRNAs) are exported to the cytoplasm through the nuclear pore complex (NPC). At the NPC, messenger ribonucleoproteins (mRNPs) first encounter the nuclear basket where mRNP rearrangements are thought to allow access to the transport channel. Here, we use single mRNA resolution live cell microscopy and subdiffraction particle tracking to follow individual mRNAs on their path toward the cytoplasm. We show that when reaching the nuclear periphery, RNAs are not immediately exported but scan along the nuclear periphery, likely to find a nuclear pore allowing export. Deletion or mutation of the nuclear basket proteins MLP1/2 or the mRNA binding protein Nab2 changes the scanning behavior of mRNPs at the nuclear periphery, shortens residency time at nuclear pores, and results in frequent release of mRNAs back into the nucleoplasm. These observations suggest a role for the nuclear basket in providing an interaction platform that keeps RNAs at the periphery, possibly to allow mRNP rearrangements before export.

Sequential degradation of proteins from the nuclear envelope during apoptosis

2001 ◽  
Vol 114 (20) ◽  
pp. 3643-3653 ◽  
Author(s):  
Madeleine Kihlmark ◽  
Gabriela Imreh ◽  
Einar Hallberg
Keyword(s):  
Nuclear Envelope ◽  
Apoptotic Cell ◽  
Nuclear Periphery ◽  
Condensed Chromatin ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Nuclear Pores ◽  
Double Labeling ◽  
Lamin B ◽  
Nuclear Apoptosis

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.

scholarly journals A Distinct and Parallel Pathway for the Nuclear Import of an mRNA-binding Protein

1997 ◽  
Vol 139 (7) ◽  
pp. 1645-1653 ◽  
Author(s):  
Lucy F. Pemberton ◽  
Jonathan S. Rosenblum ◽  
Günter Blobel
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Binding Protein ◽  
Nuclear Pore ◽  
Polyadenylated Rna ◽  
Mrna Binding Protein ◽  
Parallel Pathway ◽  
Rna Export ◽  
Mrna Binding ◽  
Transport Factors

Three independent pathways of nuclear import have so far been identified in yeast, each mediated by cognate nuclear transport factors, or karyopherins. Here we have characterized a new pathway to the nucleus, mediated by Mtr10p, a protein first identified in a screen for strains defective in polyadenylated RNA export. Mtr10p is shown to be responsible for the nuclear import of the shuttling mRNA-binding protein Npl3p. A complex of Mtr10p and Npl3p was detected in cytosol, and deletion of Mtr10p was shown to lead to the mislocalization of nuclear Npl3p to the cytoplasm, correlating with a block in import. Mtr10p bound peptide repeat-containing nucleoporins and Ran, suggesting that this import pathway involves a docking step at the nuclear pore complex and is Ran dependent. This pathway of Npl3p import is distinct and does not appear to overlap with another known import pathway for an mRNA-binding protein. Thus, at least two parallel pathways function in the import of mRNA-binding proteins, suggesting the need for the coordination of these pathways.

scholarly journals Evolutionary conserved protein motifs drive attachment of the plant nucleoskeleton at nuclear pores

2021 ◽  
Author(s):  
Sarah Mermet ◽  
Maxime Voisin ◽  
Joris Mordier ◽  
Tristan Dubos ◽  
Sylvie Tutois ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Evolutionary History ◽  
Nuclear Periphery ◽  
Land Plants ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Protein Motifs ◽  
Peptide Motifs ◽  
Functional Regions ◽  
Computational Predictions

ABSTRACTThe nucleoskeleton forms a filamentous meshwork under the nuclear envelope and contributes to the regulation of nuclear morphology and gene expression. To understand how the Arabidopsis nucleoskeleton physically connects to the nuclear periphery, we investigated the nucleoskeleton protein KAKU4 and sought for functional regions responsible for its localization at the nuclear periphery. Computational predictions identified three evolutionary conserved peptide motifs within the N-terminal region of KAKU4. Functional analysis revealed that these motifs are required for homomerization of KAKU4, interaction with the nucleoskeleton proteins CROWDED NUCLEI (CRWN) and localization at the nuclear periphery. We find that similar protein motifs are present in NUP82 and NUP136, two plant specific nucleoporins from the Nuclear Pore Complex (NPC) basket. These conserved motifs allow the two nucleoporins to bind CRWN proteins, thus revealing a physical link between the nucleoskeleton and nuclear pores in plants. Finally, whilst NUP82, NUP136 and KAKU4 have a common evolutionary history predating non-vascular land plants, KAKU4 mainly localizes outside the NPC suggesting neofunctionalization of an ancient nucleoporin into a new nucleoskeleton component.

scholarly journals Live imaging of single nuclear pores reveals unique assembly kinetics and mechanism in interphase

2010 ◽  
Vol 191 (1) ◽  
pp. 15-22 ◽  
Author(s):  
Elisa Dultz ◽  
Jan Ellenberg
Keyword(s):  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Constant Rate ◽  
The Reformation ◽  
Assembly Kinetics ◽  
Live Cell Microscopy ◽  
Pore Complexes ◽  
Cell Microscopy

In metazoa, new nuclear pore complexes (NPCs) form at two different cell cycle stages: at the end of mitosis concomitant with the reformation of the nuclear envelope and during interphase. However, the mechanisms of these assembly processes may differ. In this study, we apply high resolution live cell microscopy to analyze the dynamics of single NPCs in living mammalian cells during interphase. We show that nuclear growth and NPC assembly are correlated and occur at a constant rate throughout interphase. By analyzing the kinetics of individual NPC assembly events, we demonstrate that they are initiated by slow accumulation of the membrane nucleoporin Pom121 followed by the more rapid association of the soluble NPC subcomplex Nup107–160. This inverse order of recruitment and the overall much slower kinetics compared with postmitotic NPC assembly support the conclusion that the two processes occur by distinct molecular mechanisms.

scholarly journals A Pre-mRNA-Binding Protein Accompanies the RNA from the Gene through the Nuclear Pores and into Polysomes

Cell ◽  
1996 ◽  
Vol 84 (2) ◽  
pp. 253-264 ◽  
Author(s):  
Neus Visa ◽  
Alla T Alzhanova-Ericsson ◽  
Xin Sun ◽  
Elena Kiseleva ◽  
Birgitta Björkroth ◽  
...  
Keyword(s):  
Binding Protein ◽  
Nuclear Pores ◽  
Mrna Binding Protein ◽  
Mrna Binding

scholarly journals High-Throughput Fluorescence Anisotropy Screen for Inhibitors of the Oncogenic mRNA Binding Protein, IMP-1

2013 ◽  
Vol 19 (3) ◽  
pp. 427-436 ◽  
Author(s):  
Lily Mahapatra ◽  
Chengjian Mao ◽  
Neal Andruska ◽  
Chen Zhang ◽  
David J. Shapiro
Keyword(s):  
Small Molecule ◽  
High Throughput ◽  
Fluorescence Anisotropy ◽  
Messenger Rnas ◽  
High Throughput Screen ◽  
Alternative Approach ◽  
Mrna Binding Protein ◽  
Elevated Expression ◽  
Mrna Binding

Cancer cell proliferation is regulated by oncogenes, such as c-Myc. An alternative approach to directly targeting individual oncogenes is to target IMP-1, an oncofetal protein that binds to and stabilizes messenger RNAs (mRNAs), leading to elevated expression of c-Myc and other oncogenes. Expression of IMP-1 is tightly correlated with a poor prognosis and reduced survival in ovarian, lung, and colon cancer. Small-molecule inhibitors of IMP-1 have not been reported. We established a fluorescence anisotropy/polarization microplate assay (FAMA) for analyzing binding of IMP-1 to a fluorescein-labeled 93 nucleotide c-Myc mRNA target (flMyc), developed the assay as a highly robust (Z′ factor = 0.60) FAMA-based high-throughput screen for inhibitors of binding of IMP-1 to flMyc, and carried out a successful pilot screen of 17,600 small molecules. Our studies support rapidly filtering out toxic nonspecific inhibitors using an early cell-based assay in control cells lacking the target protein. The physiologic importance of verified hits from the in vitro high-throughput screen was demonstrated by identification of the first small-molecule IMP-1 inhibitor, a lead compound that selectively inhibits proliferation of IMP-1–positive cancer cells with very little or no effect on proliferation of IMP-1–negative cells.

scholarly journals Structural and biochemical analyses of the DEAD-box ATPase Sub2 in association with THO or Yra1

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yi Ren ◽  
Philip Schmiege ◽  
Günter Blobel
Keyword(s):  
Reaction Mechanisms ◽  
Nuclear Pore ◽  
Closed Conformation ◽  
Dead Box ◽  
Messenger Ribonucleoprotein ◽  
Mrna Binding Protein ◽  
The Dead ◽  
Biochemical Analyses ◽  
Mrna Binding ◽  
Open Configuration

mRNA is cotranscrptionally processed and packaged into messenger ribonucleoprotein particles (mRNPs) in the nucleus. Prior to export through the nuclear pore, mRNPs undergo several obligatory remodeling reactions. In yeast, one of these reactions involves loading of the mRNA-binding protein Yra1 by the DEAD-box ATPase Sub2 as assisted by the hetero-pentameric THO complex. To obtain molecular insights into reaction mechanisms, we determined crystal structures of two relevant complexes: a THO hetero-pentamer bound to Sub2 at 6.0 Å resolution; and Sub2 associated with an ATP analogue, RNA, and a C-terminal fragment of Yra1 (Yra1-C) at 2.6 Å resolution. We found that the 25 nm long THO clamps Sub2 in a half-open configuration; in contrast, when bound to the ATP analogue, RNA and Yra1-C, Sub2 assumes a closed conformation. Both THO and Yra1-C stimulated Sub2’s intrinsic ATPase activity. We propose that THO surveys common landmarks in each nuclear mRNP to localize Sub2 for targeted loading of Yra1.

scholarly journals Interaction between the Shuttling mRNA Export Factor Gle1 and the Nucleoporin hCG1: A Conserved Mechanism in the Export of Hsp70 mRNA

2005 ◽  
Vol 16 (9) ◽  
pp. 4304-4315 ◽  
Author(s):  
Frederic Kendirgi ◽  
Deborah J. Rexer ◽  
Abel R. Alcázar-Román ◽  
Halina M. Onishko ◽  
Susan R. Wente
Keyword(s):  
Mrna Export ◽  
Nuclear Pore ◽  
Messenger Rnas ◽  
Hsp70 Mrna ◽  
Amino Acid Region ◽  
Hsp70 Protein ◽  
Physical Interactions ◽  
Mrna Binding

Translocation of messenger RNAs through the nuclear pore complex (NPC) requires coordinated physical interactions between stable NPC components, shuttling transport factors, and mRNA-binding proteins. In budding yeast (y) and human (h) cells, Gle1 is an essential mRNA export factor. Nucleocytoplasmic shuttling of hGle1 is required for mRNA export; however, the mechanism by which hGle1 associates with the NPC is unknown. We have previously shown that the interaction of hGle1 with the nucleoporin hNup155 is necessary but not sufficient for targeting hGle1 to NPCs. Here, we report that the unique C-terminal 43 amino acid region of the hGle1B isoform mediates binding to the C-terminal non-FG region of the nucleoporin hCG1/NPL1. Moreover, hNup155, hGle1B, and hCG1 formed a heterotrimeric complex in vitro. This suggested that these two nucleoporins were required for the NPC localization of hGle1. Using an siRNA-based approach, decreased levels of hCG1 resulted in hGle1 accumulation in cytoplasmic foci. This was coincident with inhibition of heat shock-induced production of Hsp70 protein and export of the Hsp70 mRNA in HeLa cells. Because this closely parallels the role of the hCG1 orthologue yNup42/Rip1, we speculate that hGle1-hCG1 function in the mRNA export mechanism is highly conserved.

The Formation, Structure, Distribution and Frequency of Nuclear Pores

1971 ◽  
Vol 29 ◽  
pp. 518-519
Author(s):  
G. G. Maul
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Membrane ◽  
Dynamic Structure ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Filter Function ◽  
Etching Technique ◽  
Selective Filter ◽  
Membranous Part

The chromatin of eukaryotic cells is separated from the cytoplasm by a double membrane. One obvious structural specialization of the nuclear membrane is the presence of pores which have been implicated to facilitate the selective nucleocytoplasmic exchange of a variety of large molecules. Thus, the function of nuclear pores has mainly been regarded to be a passive one. Non-membranous diaphragms, radiating fibers, central rings, and other pore-associated structures were thought to play a role in the selective filter function of the nuclear pore complex. Evidence will be presented that suggests that the nuclear pore is a dynamic structure which is non-randomly distributed and can be formed during interphase, and that a close relationship exists between chromatin and the membranous part of the nuclear pore complex.Octagonality of the nuclear pore complex has been confirmed by a variety of techniques. Using the freeze-etching technique, it was possible to show that the membranous part of the pore complex has an eight-sided outline in human melanoma cells in vitro. Fibers which traverse the pore proper at its corners are continuous and indistinguishable from chromatin at the nucleoplasmic side, as seen in conventionally fixed and sectioned material. Chromatin can be seen in octagonal outline if serial sections are analyzed which are parallel but do not include nuclear membranes (Fig. 1). It is concluded that the shape of the pore rim is due to fibrous material traversing the pore, and may not have any functional significance. In many pores one can recognize a central ring with eight fibers radiating to the corners of the pore rim. Such a structural arrangement is also found to connect eight ribosomes at the nuclear membrane.

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