scholarly journals The product of the Saccharomyces cerevisiae RSS1 gene, identified as a high-copy suppressor of the rat7-1 temperature-sensitive allele of the RAT7/NUP159 nucleoporin, is required for efficient mRNA export.

1996 ◽  
Vol 7 (10) ◽  
pp. 1601-1621 ◽  
Author(s):  
V Del Priore ◽  
C A Snay ◽  
A Bahr ◽  
C N Cole
Keyword(s):  
Protein Import ◽  
Mrna Export ◽  
Nuclear Accumulation ◽  
Rrna Synthesis ◽  
Growth Defect ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Extragenic Suppressor ◽  
Sensitive Allele ◽  
Nucleocytoplasmic Export

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rss1p encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rss1p partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37 degrees C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37 degrees C, the mutant Rat7-1p/Nup159-1p is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rss1p did not result in retention of the mutant Rat7-1p/Nup159-1p in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rss1p by placing the RSS1 open reading frame (ORF) under control of the GAL1 promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rss1p are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rss1p overexpression suppresses the growth defect of rat7-1 cells at 37 degrees C by acting to maintain mRNA export.

scholarly journals Monoclonal Antibodies to NTF2 Inhibit Nuclear Protein Import by Preventing Nuclear Translocation of the GTPase Ran

2000 ◽  
Vol 11 (2) ◽  
pp. 703-719 ◽  
Author(s):  
Susanne M. Steggerda ◽  
Ben E. Black ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Pore Complex ◽  
Nuclear Import ◽  
Protein Import ◽  
Nuclear Translocation ◽  
Living Cells ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Permeabilized Cells ◽  
Dependent Protein

Nuclear transport factor 2 (NTF2) is a soluble transport protein originally identified by its ability to stimulate nuclear localization signal (NLS)-dependent protein import in digitonin-permeabilized cells. NTF2 has been shown to bind nuclear pore complex proteins and the GDP form of Ran in vitro. Recently, it has been reported that NTF2 can stimulate the accumulation of Ran in digitonin-permeabilized cells. Evidence that NTF2 directly mediates Ran import or that NTF2 is required to maintain the nuclear concentration of Ran in living cells has not been obtained. Here we show that cytoplasmic injection of anti-NTF2 mAbs resulted in a dramatic relocalization of Ran to the cytoplasm. This provides the first evidence that NTF2 regulates the distribution of Ran in vivo. Moreover, anti-NTF2 mAbs inhibited nuclear import of both Ran and NLS-containing protein in vitro, suggesting that NTF2 stimulates NLS-dependent protein import by driving the nuclear accumulation of Ran. We also show that biotinylated NTF2-streptavidin microinjected into the cytoplasm accumulated at the nuclear envelope, indicating that NTF2 can target a binding partner to the nuclear pore complex. Taken together, our data show that NTF2 is an essential regulator of the Ran distribution in living cells and that NTF2-mediated Ran nuclear import is required for NLS-dependent protein import.

scholarly journals The Nsp1p Carboxy-Terminal Domain Is Organized into Functionally Distinct Coiled-Coil Regions Required for Assembly of Nucleoporin Subcomplexes and Nucleocytoplasmic Transport

2001 ◽  
Vol 21 (23) ◽  
pp. 7944-7955 ◽  
Author(s):  
Susanne M. Bailer ◽  
Carolin Balduf ◽  
Ed Hurt
Keyword(s):  
Protein Import ◽  
Coiled Coil ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pores ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal

ABSTRACT Nucleoporin Nsp1p, which has four predicted coiled-coil regions (coils 1 to 4) in the essential carboxy-terminal domain, is unique in that it is part of two distinct nuclear pore complex (NPC) subcomplexes, Nsp1p-Nup57p-Nup49p-Nic96p and Nsp1p-Nup82p-Nup159p. As shown by in vitro reconstitution, coiled-coil region 2 (residues 673 to 738) is sufficient to form heterotrimeric core complexes and can bind either Nup57p or Nup82p. Accordingly, interaction of Nup82p with Nsp1p coil 2 is competed by excess Nup57p. Strikingly, coil 3 and 4 mutants are still assembled into the core Nsp1p-Nup57p-Nup49p complex but no longer associate with Nic96p. Consistently, the Nsp1p-Nup57p-Nup49p core complex dissociates from the nuclear pores in nsp1coil 3 and 4 mutant cells, and as a consequence, defects in nuclear protein import are observed. Finally, the nsp1-L640Stemperature-sensitive mutation, which maps in coil 1, leads to a strong nuclear mRNA export defect. Thus, distinct coiled-coil regions within Nsp1p-C have separate functions that are related to the assembly of different NPC subcomplexes, nucleocytoplasmic transport, and incorporation into the nuclear pores.

scholarly journals Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I

2015 ◽  
Vol 35 (13) ◽  
pp. 2321-2331 ◽  
Author(s):  
Krysta L. Engel ◽  
Sarah L. French ◽  
Olga V. Viktorovskaya ◽  
Ann L. Beyer ◽  
David A. Schneider
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Initiation Factor ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
Protein Levels ◽  
Pol I ◽  
Low Levels ◽  
Sensitive Allele ◽  
Polymerase I

Spt6 (suppressor ofTy6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele,spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression ofRRN3rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA.

scholarly journals A Novel Fluorescence-based Genetic Strategy Identifies Mutants ofSaccharomyces cerevisiaeDefective for Nuclear Pore Complex Assembly

1998 ◽  
Vol 9 (9) ◽  
pp. 2439-2461 ◽  
Author(s):  
Mirella Bucci ◽  
Susan R. Wente
Keyword(s):  
Fluorescent Protein ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pore Complexes ◽  
Specific Subset ◽  
Powerful Approach ◽  
Sensitive Allele ◽  
Green Fluorescent ◽  
Pore Complexes ◽  
Carboxy Terminal

Nuclear pore complexes (NPCs) are large proteinaceous portals for exchanging macromolecules between the nucleus and the cytoplasm. Revealing how this transport apparatus is assembled will be critical for understanding the nuclear transport mechanism. To address this issue and to identify factors that regulate NPC formation and dynamics, a novel fluorescence-based strategy was used. This approach is based on the functional tagging of NPC proteins with the green fluorescent protein (GFP), and the hypothesis that NPC assembly mutants will have distinct GFP-NPC signals as compared with wild-type (wt) cells. By fluorescence-activated cell sorting for cells with low GFP signal from a population of mutagenized cells expressing GFP-Nup49p, three complementation groups were identified: two correspond to mutantnup120 and gle2 alleles that result in clusters of NPCs. Interestingly, a third group was a novel temperature-sensitive allele of nup57. The lowered GFP-Nup49p incorporation in the nup57-E17 cells resulted in a decreased fluorescence level, which was due in part to a sharply diminished interaction between the carboxy-terminal truncated nup57pE17and wt Nup49p. Interestingly, thenup57-E17 mutant also affected the incorporation of a specific subset of other nucleoporins into the NPC. Decreased levels of NPC-associated Nsp1p and Nup116p were observed. In contrast, the localizations of Nic96p, Nup82p, Nup159p, Nup145p, and Pom152p were not markedly diminished. Coincidentally, nuclear import capacity was inhibited. Taken together, the identification of such mutants with specific perturbations of NPC structure validates this fluorescence-based strategy as a powerful approach for providing insight into the mechanism of NPC biogenesis.

scholarly journals A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes.

1995 ◽  
Vol 129 (4) ◽  
pp. 939-955 ◽  
Author(s):  
L C Gorsch ◽  
T C Dockendorff ◽  
C N Cole
Keyword(s):  
Messenger Rna ◽  
Mrna Export ◽  
Temperature Shift ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nuclear Pore Complexes ◽  
Reporter Protein ◽  
Direct Role ◽  
Pore Complexes

In a screen for Saccharomyces cerevisiae genes required for nucleocytoplasmic transport of messenger RNA, we identified the RAT7 gene (ribonucleic acid trafficking), which encodes an essential protein of 1,460 amino acids. Rat7p is located at the nuclear rim in a punctate pattern characteristic of nucleoporins. Furthermore, the central third of Rat7p contains 22 XXFG and three XFXFG degenerate repeats that are similar to signature GLFG and XFXFG repeats present in a majority of yeast and some mammalian nucleoporins sequenced to date. Shift of a strain bearing the temperature-sensitive rat7-1 allele from 23 degrees C to 37 degrees C resulted in rapid (within 15 minutes) cessation of mRNA export, but did not cause concomitant cytoplasmic accumulation of a reporter protein bearing a nuclear localization signal. This suggests that Rat7p may play a direct role in nucleocytoplasmic export of RNA. Immunofluorescence and thin section electron microscopy revealed that in rat7-1 cells grown at 23 degrees C, the majority of nuclear pore complexes (NPCs) were clustered on one side of the nucleus. No ultrastructural abnormalities of the nuclear envelope were seen. Interestingly, shifting rat7-1 cells to 37 degrees C for 1 h caused the NPCs to disperse, restoring near wild-type NPC distribution. After this temperature shift, the mutant Rat7p was no longer detectable by immunofluorescence.

scholarly journals Dominant suppressors of yeast actin mutations that are reciprocally suppressed.

Genetics ◽  
1989 ◽  
Vol 121 (4) ◽  
pp. 675-683
Author(s):  
A E Adams ◽  
D Botstein
Keyword(s):  
High Temperature ◽  
Linkage Group ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Mutant Library ◽  
Suppressor Mutations ◽  
Extragenic Suppressor ◽  
New Gene ◽  
Allele Specificity

Abstract A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.

scholarly journals Nuclear transport defects and nuclear envelope alterations are associated with mutation of the Saccharomyces cerevisiae NPL4 gene.

1996 ◽  
Vol 7 (11) ◽  
pp. 1835-1855 ◽  
Author(s):  
C DeHoratius ◽  
P A Silver
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Protein Import ◽  
Nuclear Protein ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Temperature Sensitive Mutants ◽  
Nuclear Protein Import

To identify components involved in nuclear protein import, we used a genetic selection to isolate mutants that mislocalized a nuclear-targeted protein. We identified temperature-sensitive mutants that accumulated several different nuclear proteins in the cytoplasm when shifted to the semipermissive temperature of 30 degrees C; these were termed npl (nuclear protein localization) mutants. We now present the properties of yeast strains bearing mutations in the NPL4 gene and report the cloning of the NPL4 gene and the characterization of the Np14 protein. The npl4-1 mutant was isolated by the previously described selection scheme. The second allele, npl4-2, was identified from an independently derived collection of temperature-sensitive mutants. The npl4-1 and npl4-2 strains accumulate nuclear-targeted proteins in the cytoplasm at the nonpermissive temperature consistent with a defect in nuclear protein import. Using an in vitro nuclear import assay, we show that nuclei prepared from temperature-shifted npl4 mutant cells are unable to import nuclear-targeted proteins, even in the presence of cytosol prepared from wild-type cells. In addition, npl4-2 cells accumulate poly(A)+ RNA in the nucleus at the nonpermissive temperature, consistent with a failure to export mRNA from the nucleus. The npl4-1 and npl4-2 cells also exhibit distinct, temperature-sensitive structural defects: npl4-1 cells project extra nuclear envelope into the cytoplasm, whereas npl4-2 cells from nuclear envelope herniations that appear to be filled with poly(A)+ RNA. The NPL4 gene encodes an essential M(r) 64,000 protein that is located at the nuclear periphery and localizes in a pattern similar to nuclear pore complex proteins. Taken together, these results indicate that this gene encodes a novel nuclear pore complex or nuclear pore complex-associated component required for nuclear membrane integrity and nuclear transport.

scholarly journals Characterization of an essential Saccharomyces cerevisiae gene related to RNA processing: cloning of RNA1 and generation of a new allele with a novel phenotype.

1985 ◽  
Vol 5 (5) ◽  
pp. 907-915 ◽  
Author(s):  
N S Atkinson ◽  
R W Dunst ◽  
A K Hopper
Keyword(s):  
Rna Processing ◽  
Elevated Temperatures ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Multicopy Plasmid ◽  
Recessive Lethal ◽  
Recessive Lethal Mutation ◽  
Sensitive Allele

The RNA1 gene product is believed to be involved in RNA metabolism due to the phenotype of a single conditionally lethal, temperature-sensitive allele, rna1-1. We cloned the RNA1 gene and determined that it produces a 1,400-nucleotide polyadenylated transcript. On a multicopy plasmid, the mutant rna1-1 allele partially complements the rna1-1 temperature-sensitive growth defect. This suggests that the temperature-sensitive nature of the rna1-1 allele results from the synthesis of a product with lowered activity or stability at elevated temperatures or from a decrease in synthesis of the rna1-1 product at the restrictive temperature. A chromosomal disruption of RNA1 behaves as a recessive lethal mutation. Haploids bearing the disruption were isolated by sporulating a diploid heterozygous for the disrupted allele and the rna1-1 allele and possessing an episomal copy of the RNA1 gene. Analysis of the rescued haploids bearing the chromosomal disruption indicated that the recessive lethal phenotype of the RNA1 disruption is not merely due to a block in spore germination. Unexpectedly, diploids heterozygous for the disruption and the rna1-1 alleles become aneuploid for chromosome XIII at a frequency of 2 to 5%. It appears that the disrupted RNA1 allele on a multicopy plasmid also promotes aneuploidy for chromosome XIII. Promotion of aneuploidy seems to be a phenotype of this particular allele of RNA1.

scholarly journals RPM2, independently of its mitochondrial RNase P function, suppresses an ISP42 mutant defective in mitochondrial import and is essential for normal growth.

1995 ◽  
Vol 15 (9) ◽  
pp. 4763-4770 ◽  
Author(s):  
C K Kassenbrock ◽  
G J Gao ◽  
K R Groom ◽  
P Sulo ◽  
M G Douglas ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Normal Growth ◽  
Rnase P ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Mitochondrial Trna ◽  
Mitochondrial Protein Import ◽  
Established Role ◽  
Fermentative Yeast

RPM2 is identified here as a high-copy suppressor of isp42-3, a temperature-sensitive mutant allele of the mitochondrial protein import channel component, Isp42p. RPM2 already has an established role as a protein component of yeast mitochondrial RNase P, a ribonucleoprotein enzyme required for the 5' processing of mitochondrial precursor tRNAs. A relationship between mitochondrial tRNA processing and protein import is not readily apparent, and, indeed, the two functions can be separated. Truncation mutants lacking detectable RNase P activity still suppress the isp42-3 growth defect. Moreover, RPM2 is required for normal fermentative yeast growth, even though mitochondrial RNase P activity is not. The portion of RPM2 required for normal growth and suppression of isp42-3 is the same. We conclude that RPM2 is a multifunctional gene. We find Rpm2p to be a soluble protein of the mitochondrial matrix and discuss models to explain its suppression of isp42-3.

scholarly journals REF1/Aly and the additional exon junction complex proteins are dispensable for nuclear mRNA export

2002 ◽  
Vol 159 (4) ◽  
pp. 579-588 ◽  
Author(s):  
David Gatfield ◽  
Elisa Izaurralde
Keyword(s):  
Essential Role ◽  
Mrna Export ◽  
Adaptor Protein ◽  
Protein S ◽  
Nuclear Accumulation ◽  
Nuclear Pore ◽  
Exon Junction Complex ◽  
Exon Junction ◽  
Cellular Mrnas ◽  
Additional Exon

The metazoan proteins UAP56, REF1, and NXF1 are thought to bind sequentially to mRNA to promote its export to the cytoplasm: UAP56 is thought to recruit REF1 to nascent mRNA; REF1 acts as an adaptor protein mediating the association of NXF1 with mRNA, whereas NXF1 translocates the mRNA across the nuclear pore complex. REF1 is a component of the exon–exon junction complex (EJC); thus, the EJC is thought to play a role in the export of spliced mRNA. NXF1 and UAP56 are essential for mRNA export. An essential role for metazoan REF1 or the additional EJC proteins in this process has not been established. Contrary to expectation, we show that REF1 and the additional components of the EJC are dispensable for export of bulk mRNA in Drosophila cells. Only when REF1 and RNPS1 are codepleted, or when all EJC proteins are simultaneously depleted is a partial nuclear accumulation of polyadenylated RNAs observed. Because a significant fraction of bulk mRNA is detected in the cytoplasm of cells depleted of all EJC proteins, we conclude that additional adaptor protein(s) mediate the interaction between NXF1 and cellular mRNAs in metazoa. Our results imply that the essential role of UAP56 in mRNA export is not restricted to the recruitment of REF1.

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