scholarly journals Imaging within single NPCs reveals NXF1’s role in mRNA export on the cytoplasmic side of the pore

2019 ◽  
Vol 218 (9) ◽  
pp. 2962-2981 ◽  
Author(s):  
Rakefet Ben-Yishay ◽  
Amir Mor ◽  
Amit Shraga ◽  
Asaf Ashkenazy-Titelman ◽  
Noa Kinor ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Protein Interactions ◽  
Rna Binding ◽  
Mrna Export ◽  
Living Cells ◽  
Nuclear Pore ◽  
Protein Protein Interactions ◽  
Superresolution Microscopy ◽  
Directional Flow ◽  
Cytoplasmic Side

Translocation of mRNA through the nuclear pore complex (NPC) requires interactions with different NPC regions. To determine the interactions that are crucial for effective mRNA export in living cells, we examined mRNA export within individual pores by applying various types of mRNA export blocks that stalled mRNPs at different stages of transition. Focusing on the major mRNA export factor NXF1, we found that initial mRNP binding to the NPC did not require NXF1 in the NPC, whereas release into the cytoplasm did. NXF1 localization in the NPC did not require RNA or RNA binding. Superresolution microscopy showed that NXF1 consistently occupied positions on the cytoplasmic side of the NPC. Interactions with specific nucleoporins were pinpointed using FLIM-FRET for measuring protein–protein interactions inside single NPCs, showing that Dbp5 helicase activity of mRNA release is conserved in yeast and humans. Altogether, we find that specific interactions on the cytoplasmic side of the NPC are fundamental for the directional flow of mRNA export.

scholarly journals The Dbp5 cycle at the nuclear pore complex during mRNA export I: dbp5 mutants with defects in RNA binding and ATP hydrolysis define key steps for Nup159 and Gle1

2011 ◽  
Vol 25 (10) ◽  
pp. 1052-1064 ◽  
Author(s):  
C. A. Hodge ◽  
E. J. Tran ◽  
K. N. Noble ◽  
A. R. Alcazar-Roman ◽  
R. Ben-Yishay ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Rna Binding ◽  
Atp Hydrolysis ◽  
Mrna Export ◽  
Nuclear Pore ◽  
Key Steps

scholarly journals Nuclear pore complex assembly and maintenance in POM121- and gp210-deficient cells

2006 ◽  
Vol 173 (4) ◽  
pp. 477-483 ◽  
Author(s):  
Fabrizia Stavru ◽  
Gitte Nautrup-Pedersen ◽  
Volker C. Cordes ◽  
Dirk Görlich
Keyword(s):  
Lipid Bilayer ◽  
Nuclear Pore Complex ◽  
Protein Interactions ◽  
Human Fibroblasts ◽  
Nucleation Site ◽  
Free Form ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Protein Protein Interactions ◽  
Pore Complexes

So far, POM121 and gp210 are the only known anchoring sites of vertebrate nuclear pore complexes (NPCs) within the lipid bilayer of the nuclear envelope (NE) and, thus, are excellent candidates for initiating the NPC assembly process. Indeed, we demonstrate that POM121 can recruit several nucleoporins, such as Nup62 or Nup358, to ectopic assembly sites. It thus appears to act as a nucleation site for the assembly of NPC substructures. Nonetheless, we observed functional NPCs and intact NEs in severely POM121-depleted cells. Double knockdowns of gp210 and POM121 in HeLa cells, as well as depletion of POM121 from human fibroblasts, which do not express gp210, further suggest that NPCs can assemble or at least persist in a POM121- and gp210-free form. This points to extensive redundancies in protein–protein interactions within NPCs and suggests that vertebrate NPCs contain additional membrane-integral nucleoporins for anchorage within the lipid bilayer of the NE. In Stavru et al. (on p. 509 of this issue), we describe such an additional transmembrane nucleoporin as the metazoan orthologue of yeast Ndc1p.

scholarly journals Mex67p of Schizosaccharomyces pombeInteracts with Rae1p in Mediating mRNA Export

2000 ◽  
Vol 20 (23) ◽  
pp. 8767-8782 ◽  
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.

scholarly journals A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells

PLoS ONE ◽  
2010 ◽  
Vol 5 (2) ◽  
pp. e9344 ◽  
Author(s):  
Carina Banning ◽  
Jörg Votteler ◽  
Dirk Hoffmann ◽  
Herwig Koppensteiner ◽  
Martin Warmer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Protein Interactions ◽  
Living Cells ◽  
Protein Protein Interactions

A guide-tag system controlling client enrichment into Y15 peptide-based granules for an in-cell protein recruitment assay

2021 ◽  
Author(s):  
Takayuki Miki ◽  
Masahiro Hashimoto ◽  
Taichi Nakai ◽  
Hisakazu Mihara
Keyword(s):  
Protein Interactions ◽  
Living Cells ◽  
The Self ◽  
Cell Protein ◽  
Protein Protein Interactions ◽  
Self Assembling ◽  
Client Proteins ◽  
Protein Recruitment ◽  
Peptide Tag

A series of guide-tags that can control the enrichment of client proteins into artificial scaffolds constituted by the self-assembling Y15 peptide tag facilitates the analysis of protein–protein interactions in living cells.

Femtosecond UV-laser pulses to unveil protein–protein interactions in living cells

2015 ◽  
Vol 73 (3) ◽  
pp. 637-648 ◽  
Author(s):  
Francesco Itri ◽  
Daria M. Monti ◽  
Bartolomeo Della Ventura ◽  
Roberto Vinciguerra ◽  
Marco Chino ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Laser Pulses ◽  
Living Cells ◽  
Uv Laser ◽  
Protein Protein Interactions
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