Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway

Thomas, Franziska; Kutay, Ulrike

doi:10.1242/jcs.00464

Cited by 167 publications

(201 citation statements)

References 48 publications

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“…It has been shown previously that in vertebrates, the large 60 S preribosome subunit is exported via CRM1 and the transport adaptor NMD3 (40,42). We therefore investigated the role of the Nup214-Nup88 subcomplex in 60 S preribosomal nuclear export by studying the localization of GFP-tagged NMD3 (40).…”

Section: Resultsmentioning

confidence: 98%

“…The NLS-eGFP insert was amplified by PCR from pBSSK using the forward primer 5Ј-CCCCTCGAGGTCGACGGTATC-3Ј and a reverse primer containing a NotI site, 5Ј-ATATATATAGCGGCCGC-TTAGTTTCTAGACTTGTACAGCTC-3Ј, and subcloned into pCDNA3 by digestion with HindIII and NotI. GFP-NMD3 and rpL29-GFP were a kind gift from U. Kutay (40). To create a RNAi-insensitive Nup214 expressing plasmid, DpnI-mediated site-directed mutagenesis was performed on pBluescriptKS(Ϫ)CAN (47) creating four silent mutations in the target sequence TCACATCCGCTAGCAACAC.…”

Section: Methodsmentioning

confidence: 99%

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Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Bernad¹,

Engelsma

Sanderson³

et al. 2006

Journal of Biological Chemistry

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The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a kinetic/hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. The transport receptor CRM1 facilitates export of a large variety of cargoes. Export of the preribosomal 60 S subunit follows this pathway through the adaptor protein NMD3. Using RNA interference, we depleted two FG-containing cytoplasmically oriented NPC complexes, Nup214-Nup88 and Nup358, and investigated CRM1-mediated export. A dramatic defect in NMD3-mediated export of preribosomes was found in Nup214-Nup88-depleted cells, whereas only minor export defects were evident in other CRM1 cargoes or upon depletion of Nup358. We show that the large C-terminal FG domain of Nup214 is not accessible to freely diffusing molecules from the nucleus, indicating that it does not conduct 60 S preribosomes through the NPC. Consistently, derivatives of Nup214 lacking the FG-repeat domain rescued the 60 S export defect. We show that the coiled-coil region of Nup214 is sufficient for 60 S nuclear export, coinciding with recruitment of Nup88 to the NPC. Our data indicate that Nup214 plays independent roles in NPC function by participating in the kinetic/ hydrophobic barrier through its FG-rich domain and by enabling NPC gating through association with Nup88.

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Section: Resultsmentioning

confidence: 98%

Section: Methodsmentioning

confidence: 99%

Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Bernad¹,

Engelsma

Sanderson³

et al. 2006

Journal of Biological Chemistry

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“…Hence, the abnormal GTP/ GDP ratio in the gua1-G388D mutant could contribute to the observed defects in tRNA maturation by interfering with Ran function. Defects in Ran-dependent transport of ribosomal processing factors or ribosomal subunit precursors (Thomas and Kutay 2003;Todaka et al 2005) could likewise be involved in the reduced levels of ribosomal subunits seen in gua1-G388D cells.…”

Section: Resultsmentioning

confidence: 99%

Guanine Nucleotide Pool Imbalance Impairs Multiple Steps of Protein Synthesis and Disrupts GCN4 Translational Control in Saccharomyces cerevisiae

et al. 2011

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Purine nucleotides are structural components of the genetic material, function as phosphate donors, participate in cellular signaling, are cofactors in enzymatic reactions, and constitute the main carriers of cellular energy. Thus, imbalances in A/G nucleotide biosynthesis affect nearly the whole cellular metabolism and must be tightly regulated. We have identified a substitution mutation (G388D) that reduces the activity of the GMP synthase Gua1 in budding yeast and the total G-nucleotide pool, leading to precipitous reductions in the GDP/GTP ratio and ATP level in vivo. gua1-G388D strongly reduces the rate of growth, impairs general protein synthesis, and derepresses translation of GCN4 mRNA, encoding a transcriptional activator of diverse amino acid biosynthetic enzymes. Although processing of pre-tRNA i Met and other tRNA precursors, and the aminoacylation of tRNA iMet are also strongly impaired in gua1-G388D cells, tRNA i GTP complex (TC) and the multifactor complex (MFC) required for translation initiation accumulate $10-fold in gua1-G388D cells and, to a lesser extent, in wild-type (WT) cells treated with 6-azauracil (6AU). Consistently, addition of an external supply of guanine reverts all the phenotypes of gua1-G388D cells, but not those of gua1-G388D Dhpt1 mutants unable to refill the internal GMP pool through the salvage pathway. These and other findings suggest that a defect in guanine nucleotide biosynthesis evokes a reduction in the rate of general protein synthesis by impairing multiple steps of the process, disrupts the gene-specific reinitiation mechanism for translation of GCN4 mRNA and has far-reaching effects in cell biology and metabolism.

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“…Nmd3 is an essential nucleocytoplasmic shuttling protein that contains a leucine-rich NES and is required for Crm1-dependent export of the 60S subunit (Ho et al, 2000b;Gadal et al, 2001;Thomas and Kutay, 2003;Trotta et al, 2003). However, several questions arise regarding the export of ribosomal subunits.…”

Section: Introductionmentioning

confidence: 99%

Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

Hung

Patel

et al. 2008

MBoC

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We previously showed that nuclear export of the large (60S) ribosomal subunit relies on Nmd3 in a Crm1-dependent manner. Recently the general mRNA export factor, the Mtr2/Mex67 heterodimer, was shown to act as an export receptor in parallel with Crm1. These observations raise the possibility that nuclear export of the 60S subunit in Saccharomyces cerevisiae requires multiple export receptors. Here, we show that the previously characterized 60S subunit biogenesis factor, Arx1, also acts as an export receptor for the 60S subunit. We found that deletion of ARX1 was synthetic lethal with nmd3 and mtr2 mutants and was synthetic sick with several nucleoporin mutants. Deletion of ARX1 led to accumulation of pre-60S particles in the nucleus that were enriched for Nmd3, Crm1, Mex67, and Mtr2, suggesting that in the absence of Arx1, 60S export is impaired even though the subunit is loaded with export receptors. Finally, Arx1 interacted with several nucleoporins in yeast two-hybrid as well as in vitro assays. These results show that Arx1 can directly bridge the interaction between the pre-60S particle and the NPC and thus is a third export receptor for the 60S subunit in yeast.

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Biogenesis and nuclear export of ribosomal subunits in higher eukaryotes depend on the CRM1 export pathway

Cited by 167 publications

References 48 publications

Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Nup214-Nup88 Nucleoporin Subcomplex Is Required for CRM1-mediated 60 S Preribosomal Nuclear Export

Guanine Nucleotide Pool Imbalance Impairs Multiple Steps of Protein Synthesis and Disrupts GCN4 Translational Control in Saccharomyces cerevisiae

Arx1 Is a Nuclear Export Receptor for the 60S Ribosomal Subunit in Yeast

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