A member of the Ran-binding protein family, Yrb2p, is involved in nuclear protein export

Yeast cells mutated in YRB2, which encodes a nuclear protein with similarity to other Ran-binding proteins, fail to export nuclear export signal (NES)-containing proteins including HIV Rev out of the nucleus. Unlike Xpo1p/Crm1p/exportin, an NES receptor, Yrb2p does not shuttle between the nucleus and the cytoplasm but instead remains inside the nucleus. However, by both biochemical and genetic criteria, Yrb2p interacts with Xpo1p and not with other members of the importin/karyopherin β superfamily. Moreover, the Yrb2p region containing nucleoporin-like FG repeats is important for NES-mediated protein export. Taken together, these data suggest that Yrb2p acts inside the nucleus to mediate the action of Xpo1p in at least one of several nuclear export pathways.

Expansion of a CUG trinucleotide repeat in the 3′ untranslated region of myotonic dystrophy protein kinase transcripts results in nuclear retention of transcripts

Expansion of a CTG trinucleotide repeat in the 3′ untranslated region (UTR) of DMPK, the gene encoding myotonic dystrophy protein kinase, induces the dominantly inherited neuromuscular disorder myotonic dystrophy (DM). Transcripts containing the expanded trinucleotide are abundant in differentiated cultured myoblasts, and they are spliced and polyadenylylated normally. However, mutant transcripts never reach the cytoplasm in these nonmitotic cells; instead, they form stable clusters that are tightly linked to the nuclear matrix, which can prevent effective biochemical purification of these transcripts. In DM patients, reduced DMPK protein levels, consequent to nuclear retention of mutant transcripts, are probably a cause of disease development. Formation of nuclear foci is a novel mechanism for preventing transcript export and effecting a loss of gene function.

Nuclear tRNA aminoacylation and its role in nuclear export of endogenous tRNAs in Saccharomyces cerevisiae

Nuclear tRNA aminoacylation was proposed to provide a proofreading step in Xenopus oocytes, ensuring nuclear export of functional tRNAs [Lund, E. & Dahlberg, J. E. (1998) Science 282, 2082–2085]. Herein, it is documented that tRNA aminoacylation also occurs in yeast nuclei and is important for tRNA export. We propose that tRNA aminoacylation functions in one of at least two parallel paths of tRNA export in yeast. Alteration of one aminoacyl-tRNA synthetase affects export of only cognate tRNA, whereas alterations of two other aminoacyl-tRNA synthetases affect export of both cognate and noncognate tRNAs. Saturation of tRNA export pathway is a possible explanation of this phenomenon.

The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.

The Interaction and Colocalization of Sam68 with the Splicing-associated Factor YT521-B in Nuclear Dots Is Regulated by the Src Family Kinase p59fyn

Alternative pre-mRNA splicing patterns can change an extracellular stimulus, but the signaling pathways leading to these changes are still poorly characterized. Here, we describe a tyrosine-phosphorylated nuclear protein, YT521-B, and show that it interacts with the nuclear transcriptosomal component scaffold attachment factor B, and the 68-kDa Src substrate associated during mitosis, Sam68. Northern blot analysis demonstrated ubiquitous expression, but detailed RNA in situ analysis revealed cell type specificity in the brain. YT521-B protein is localized in the nucleoplasm and concentrated in 5–20 large nuclear dots. Deletion analysis demonstrated that the formation of these dots depends on the presence of the amino-terminal glutamic acid-rich domain and the carboxyl-terminal glutamic acid/arginine-rich region. We show that the latter comprises an important protein–protein interaction domain. The Src family kinase p59fyn-mediated tyrosine phosphorylation of Sam68 negatively regulates its association with YT521-B, and overexpression of p59fyn dissolves nuclear dots containing YT521-B. In vivo splicing assays demonstrated that YT521-B modulates alternative splice site selection in a concentration-dependent manner. Together, our data indicate that YT521-B and Sam68 may be part of a signal transduction pathway that influences splice site selection.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

A role for heterodimerization in nuclear localization of a homeodomain protein

The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (β-glucuronidase) gene and showing that β-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.

Homologous pairing in stretched supercoiled DNA

By using elastic measurements on single DNA molecules, we show that stretching a negatively supercoiled DNA activates homologous pairing in physiological conditions. These experiments indicate that a stretched unwound DNA locally denatures to alleviate the force-driven increase in torsional stress. This is detected by hybridization with 1 kb of homologous single-stranded DNA probes. The stretching force involved (≈2 pN) is small compared with those typically developed by molecular motors, suggesting that this process may be relevant to DNA processing in vivo. We used this technique to monitor the progressive denaturation of DNA as it is unwound and found that distinct, stable denaturation bubbles formed, beginning in A+T-rich regions.

Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

Electron microscopy of human skin fibroblasts syringe-loaded with human immunodeficiency virus type 1 protease (HIV-1 PR) revealed several effects on nuclear architecture. The most dramatic is a change from a spherical nuclear morphology to one with multiple lobes or deep invaginations. The nuclear matrix collapses or remains only as a peripheral rudiment, with individual elements thicker than in control cells. Chromatin organization and distribution is also perturbed. Attempts to identify a major nuclear protein whose cleavage by the protease might be responsible for these alterations were unsuccessful. Similar changes were observed in SW 13 T3 M [vimentin+] cells, whereas no changes were observed in SW 13 [vimentin−] cells after microinjection of protease. Treatment of SW 13 [vimentin−] cells, preinjected with vimentin to establish an intermediate filament network, with HIV-1 PR resulted in alterations in chromatin staining and distribution, but not in nuclear shape. These same changes were produced in SW 13 [vimentin−] cells after the injection of a mixture of vimentin peptides, produced by the cleavage of vimentin to completion by HIV-1 PR in vitro. Similar experiments with 16 purified peptides derived from wild-type or mutant vimentin proteins and five synthetic peptides demonstrated that exclusively N-terminal peptides were capable of altering chromatin distribution. Furthermore, two separate regions of the N-terminal head domain are primarily responsible for perturbing nuclear architecture. The ability of HIV-1 to affect nuclear organization via the liberation of vimentin peptides may play an important role in HIV-1-associated cytopathogenesis and carcinogenesis.

Disruption of the FG nucleoporin NUP98 causes selective changes in nuclear pore complex stoichiometry and function

The NUP98 gene encodes precursor proteins that generate two nucleoplasmically oriented nucleoporins, NUP98 and NUP96. By using gene targeting, we have selectively disrupted the murine NUP98 protein, leaving intact the expression and localization of NUP96. We show that NUP98 is essential for mouse gastrulation, a developmental stage that is associated with rapid cell proliferation, but dispensable for basal cell growth. NUP98−/− cells had an intact nuclear envelope with a normal number of embedded nuclear pore complexes. Typically, NUP98-deficient cells contained on average approximately 5-fold more cytoplasmic annulate lamellae than control cells. We found that a set of cytoplasmically oriented nucleoporins, including NUP358, NUP214, NUP88, and p62, assembled inefficiently into nuclear pores of NUP98−/− cells. Instead, these nucleoporins were prominently associated with the annulate lamellae. By contrast, a group of nucleoplasmically oriented nucleoporins, including NUP153, NUP50, NUP96, and NUP93, had no affinity for annulate lamellae and assembled normally into nuclear pores. Mutant pores were significantly impaired in transport receptor-mediated docking of proteins with a nuclear localization signal or M9 import signal and showed weak nuclear import of such substrates. In contrast, the ability of mutant pores to import ribosomal protein L23a and spliceosome protein U1A appeared intact. These observations show that NUP98 disruption selectively impairs discrete protein import pathways and support the idea that transport of distinct import complexes through the nuclear pore complex is mediated by specific subsets of nucleoporins.

Prespliceosomal Assembly on Microinjected Precursor mRNA Takes Place in Nuclear Speckles

Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. They have been shown to serve neighboring active genes as a reservoir of these factors. In this study, we show that, in HeLa cells, the (pre)spliceosomal assembly on precursor mRNA (pre-mRNA) is associated with the speckles. For this purpose, we used microinjection of splicing competent and mutant adenovirus pre-mRNAs with differential splicing factor binding, which form different (pre)spliceosomal complexes and followed their sites of accumulation. Splicing competent pre-mRNAs are rapidly targeted into the speckles, but the targeting is temperature-dependent. The polypyrimidine tract sequence is required for targeting, but, in itself, is not sufficient. The downstream flanking sequences are particularly important for the targeting of the mutant pre-mRNAs into the speckles. In supportive experiments, the behavior of the speckles was followed after the microinjection of antisense deoxyoligoribonucleotides complementary to the specific domains of snRNAs. Under these latter conditions prespliceosomal complexes are formed on endogenous pre-mRNAs. We conclude that the (pre)spliceosomal complexes on microinjected pre-mRNA are formed inside the speckles. Their targeting into and accumulation in the speckles is a result of the cumulative loading of splicing factors to the pre-mRNA and the complexes formed give rise to the speckled pattern observed.

Different dynamics in nuclear entry of subunits of the repair/transcription factor TFIIH

We report here the different ways in which four subunits of the basal transcription/repair factor TFIIH (XPB, XPD, p62 and p44) and the damage recognition XPC repair protein can enter the nucleus. We examined their nuclear localization by transiently expressing the gene products tagged with the enhanced green fluorescent protein (EGFP) in transfected 3T3 cells. In agreement with the identification of more than one putative nuclear localization signal (NLS) in their protein sequences, XPB, XPC, p62 and p44 chimeras were rapidly sorted to the nucleus. In contrast, the XPD–EGFP chimeras appeared mainly localized in the cytoplasm, with a minor fraction of transfectants showing the EGFP-based fluorescence also in the nucleus. The ability of the XPD chimeras to enter the nucleus was confirmed by western blotting on fractionated cell extracts and by functional complementation of the repair defect in the UV5 rodent cells, mutated in the XPD homologous gene. By deletion mutagenesis, we were unable to identify any sequence specific for nuclear localization. In particular, deletion of the putative NLS failed to affect subcellular localization and, conversely, the C-terminal part of XPD containing the putative NLS showed no specific nuclear accumulation. These findings suggest that the nuclear entry of XPD depends on its complexation with other proteins in the cytoplasm, possibly other components of the TFIIH complex.

Participation of the nuclear cap binding complex in pre-mRNA 3′ processing

Communication between the 5′ and 3′ ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5′ end is bound by the nuclear cap binding complex (CBC). This RNA–protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3′ end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5′ and 3′ ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3′ processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of “closed-loops,” due to interactions between factors bound at their 5′ and 3′ ends.

Genetic Interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during Nuclear Fusion in Saccharomyces cerevisiae

During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Δ, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Δ and sec72Δ, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein–protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.

The Nucleoporin Nup153 Plays a Critical Role in Multiple Types of Nuclear Export

The fundamental process of nucleocytoplasmic transport takes place through the nuclear pore. Peripheral pore structures are presumably poised to interact with transport receptors and their cargo as these receptor complexes first encounter the pore. One such peripheral structure likely to play an important role in nuclear export is the basket structure located on the nuclear side of the pore. At present, Nup153 is the only nucleoporin known to localize to the surface of this basket, suggesting that Nup153 is potentially one of the first pore components an RNA or protein encounters during export. In this study, anti-Nup153 antibodies were used to probe the role of Nup153 in nuclear export in Xenopus oocytes. We found that Nup153 antibodies block three major classes of RNA export, that of snRNA, mRNA, and 5S rRNA. Nup153 antibodies also block the NES protein export pathway, specifically the export of the HIV Rev protein, as well as Rev-dependent RNA export. Not all export was blocked; Nup153 antibodies did not impede the export of tRNA or the recycling of importin β to the cytoplasm. The specific antibodies used here also did not affect nuclear import, whether mediated by importin α/β or by transportin. Overall, the results indicate that Nup153 is crucial to multiple classes of RNA and protein export, being involved at a vital juncture point in their export pathways. This juncture point appears to be one that is bypassed by tRNA during its export. We asked whether a physical interaction between RNA and Nup153 could be observed, using homoribopolymers as sequence-independent probes for interaction. Nup153, unlike four other nucleoporins including Nup98, associated strongly with poly(G) and significantly with poly(U). Thus, Nup153 is unique among the nucleoporins tested in its ability to interact with RNA and must do so either directly or indirectly through an adaptor protein. These results suggest a unique mechanistic role for Nup153 in the export of multiple cargos.