A nuclear matrix-associated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D

One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization. All isoforms primarily localized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region. Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity.     

Cloning and characterization of Kin5, a novel Tetrahymena ciliary kinesin II

Two Tetrahymena kinesin-like proteins (klps) of the kinesin II subfamily, Kin1 and Kin2, were first identified by Brown et al. [1999: Mol Biol Cell 10: 3081-3096] and shown to be involved in ciliary morphogenesis probably as molecular motors in intraciliary transport (ICT). Using Tetrahymena genomic DNA as a template, we cloned Kin5, another kinesin II subfamily member. Kin5 is upregulated upon deciliation, suggesting that Kin5 is a ciliary protein. Kin5 is most closely related to Osm3, a Caenorhabditis elegans kinesin II; Osm3 and Kin5 have a 56% identity, which rises to 60.4% in the motor domain and a 45% identity in a 60 amino acid region of the C-terminal FERM (4.1, Ezrin, Radixin, Moesin) domain, not present in Kin1 or Kin2, which we hypothesize to be a critical domain either for dimerization or for cargo recognition in ICT. An antibody to a peptide sequence from the tail region of Kin5 localizes in a punctate pattern along the ciliary axoneme, colocalizing with an antibody to the raft protein IFT139. These findings suggest that Kin5 is an ICT motor like Osm3. Osm3 orthologs apparently transport membrane proteins and Kin5 may be the homodimeric kinesin II that performs this function in Tetrahymena cilia.     

Kin17, a mouse nuclear zinc finger protein that binds preferentially to curved DNA.

Kin17 is a 45 kDa protein encoded by the KIN17 gene located on mouse chromosome 2, band A. The kin17 amino acid sequence predicts two domains, which were shown to be functional: (i) a bipartite nuclear localization signal (NLS) that can drive the protein to the cell nucleus, (ii) a bona fide zinc finger of the C2H2 type. The zinc finger is involved in kin17 binding to double-stranded DNA since a mutant deleted of the zinc finger, kin17 delta 1, showed reduced binding. Single-stranded DNA was bound poorly by kin17. Interestingly, we found that kin17 protein showed preferential binding to curved DNA from either pBR322 or synthetic oligonucleotides. Binding of kin17 to a non-curved DNA segment increased after we had inserted into it a short curved synthetic oligonucleotide. Kin17 delta 2, a mutant deleted of 110 amino acids at the C-terminal end, still exhibited preferential binding to curved DNA and so did kin17 delta 1, suggesting that a domain recognizing curved DNA is located in the protein core.     

Kin2, the Budding Yeast Ortholog of Animal MARK/PAR-1 Kinases,Localizes to the Sites of Polarized Growth and May Regulate Septin Organization and the Cell Wall

MARK/PAR-1 protein kinases play important roles in cell polarization in animals. Kin1 and Kin2 are a pair of MARK/PAR-1 orthologs in the budding yeast Saccharomyces cerevisiae. They participate in the regulation of secretion and ER stress response. However, neither the subcellular localization of these two kinases nor whether they may have other cellular functions is clear. Here, we show that Kin2 localizes to the sites of polarized growth in addition to localization on the plasma membrane. The localization to polarity sites is mediated by two targeting domains—TD1 and TD2. TD1 locates in the N-terminal region that spans the protein kinase domain whereas TD2 locates in the C-terminal end that covers the KA1 domain. We also show that an excess of Kin2 activity impaired growth, septin organization, and chitin deposition in the cell wall. Both TD1 and TD2 contribute to this function. Moreover, we find that the C-terminal region of Kin2 interacts with Cdc11, a septin subunit, and Pea2, a component of the polarisome that is known to play a role in septin organization. These findings suggest that Kin2 may play a role in the regulation of the septin cytoskeleton and the cell wall. Finally, we show that the C-terminal region of Kin2 interacts with Rho3, a Rho GTPase, whereas the N-terminal region of Kin2 interacts with Bmh1, a 14-3-3 protein. We speculate that Kin2 may be regulated by Bmh1, Rho3, or Pea2 in vivo. Our study provides new insight in the localization, function, and regulation of Kin2.     

Mutagenic analysis of human immunodeficiency virus type 1 Vpr: role of a predicted N-terminal alpha-helical structure in Vpr nuclear localization and virion incorporation.

The Vpr gene product of human immunodeficiency virus type 1 is a virion-associated protein that is important for efficient viral replication in nondividing cells such as macrophages. At the cellular level, Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Incorporation of Vpr into viral particles requires a determinant within the p6 domain of the Gag precursor polyprotein Pr55gag. In the present study, we have used site-directed mutagenesis to identify a domain(s) of Vpr involved in virion incorporation and nuclear localization. Truncations of the carboxyl (C)-terminal domain, rich in basic residues, resulted in a less stable Vpr protein and in the impairment of both virion incorporation and nuclear localization. However, introduction of individual substitution mutations in this region did not impair Vpr nuclear localization and virion incorporation, suggesting that this region is necessary for the stability and/or optimal protein conformation relevant to these Vpr functions. In contrast, the substitution mutations within the amino (N)-terminal region of Vpr that is predicted to adopt an alpha-helical structure (extending from amino acids 16 to 34) impaired both virion incorporation and nuclear localization, suggesting that this structure may play a pivotal role in modulating both of these biological properties. These results are in agreement with a recent study showing that the introduction of proline residues in this predicted alpha-helical region abolished Vpr virion incorporation, presumably by disrupting this secondary structure (S. Mahalingam, S. A. Khan, R. Murali, M. A. Jabbar, C. E. Monken, R. G. Collman, and A. Srinivasan, Proc. Natl. Acad. Sci. USA 92:3794-3798, 1995). Interestingly, our results show that two Vpr mutants harboring single amino acid substitutions (L to F at position 23 [L23F] and A30F) on the hydrophobic face of the predicted helix coded for relatively stable proteins that retained their ability to translocate to the nucleus but exhibited dramatic reduction in Vpr incorporation, suggesting that this hydrophobic face might mediate protein-protein interactions required for Vpr virion incorporation but not nuclear localization. Furthermore, a single mutation (E25K) located on the hydrophilic face of this predicted alpha-helical structure affected not only virion incorporation but also nuclear localization of Vpr. The differential impairment of Vpr nuclear localization and virion incorporation by mutations in the predicted N-terminal alpha-helical region suggests that this region of Vpr plays a role in both of these biological functions of Vpr.     

Functional interaction of Nic96p with a core nucleoporin complex consisting of Nsp1p, Nup49p and a novel protein Nup57p.

Nic96p has been isolated previously in a complex together with the nuclear pore proteins Nsp1p, Nup49p and a p54 polypeptide. In a genetic screen for Nsp1p-interacting components, we now find NIC96, as well as a novel gene NUP57 which encodes the p54 protein (called Nup57p). Nup57p which is essential for cell growth contains GLFG repeats in the N-terminal half and heptad repeats in the C-terminal half. The domain organization of Nic96p is more complex: N-terminally located heptad repeats mediate binding to a trimeric Nsp1p-Nup49p-Nup57p complex, but are not required for the formation of this core complex; single amino acid substitutions in the central domain yield thermosensitive mutants, which do not impair interaction with the Nsp1 complex; the C-terminal domain is neither essential nor required for binding to the nucleoporin complex, but strikingly mutations in this part cause synthetic lethality with nsp1 and nup57 mutant alleles. Since a strain in which the Nic96p heptad repeats were deleted shows, similar to nsp1 and nup49 mutants, cytoplasmic mislocalization of a nuclear reporter protein, we propose that the interaction of the heterotrimeric Nsp1p-Nup49p-Nup57p core complex with Nic96p is required for protein transport into the nucleus.     

A novel conserved nuclear localization signal is recognized by a group of yeast importins

Nucleo-cytoplasmic transport of proteins is mostly mediated by specific interaction between transport receptors of the importin beta family and signal sequences present in their cargo. While several signal sequences, in particular the classical nuclear localization signal (NLS) recognized by the heterodimeric importin alpha/beta complex are well known, the signals recognized by other importin beta-like transport receptors remain to be characterized in detail. Here we present the systematic analysis of the nuclear import of Saccharomyces cerevisiae Asr1p, a nonessential alcohol-responsive Ring/PHD finger protein that shuttles between nucleus and cytoplasm but accumulates in the nucleus upon alcohol stress. Nuclear import of Asr1p is constitutive and mediated by its C-terminal domain. A short sequence comprising residues 243-280 is sufficient and necessary for active targeting to the nucleus. Moreover, the nuclear import signal is conserved from yeast to mammals. In vitro, the nuclear localization signal of Asr1p directly interacts with the importins Kap114p, Kap95p, Pse1p, Kap123p, or Kap104p, interactions that are sensitive to the presence of RanGTP. In vivo, these importins cooperate in nuclear import. Interestingly, the same importins mediate nuclear transport of histone H2A. Based on mutational analysis and sequence comparison with a region mediating nuclear import of histone H2A, we identified a novel type of NLS with the consensus sequence R/KxxL(x)(n)V/YxxV/IxK/RxxxK/R that is recognized by five yeast importins and connects them into a highly efficient network for nuclear import of proteins.     

The double-stranded RNA binding domain of human Dicer functions as a nuclear localization signal

Dicer is a key player in microRNA (miRNA) and RNA interference (RNAi) pathways, processing miRNA precursors and double-stranded RNA into ∼21-nt-long products ultimately triggering sequence-dependent gene silencing. Although processing of substrates in vertebrate cells occurs in the cytoplasm, there is growing evidence suggesting Dicer is also present and functional in the nucleus. To address this possibility, we searched for a nuclear localization signal (NLS) in human Dicer and identified its C-terminal double-stranded RNA binding domain (dsRBD) as harboring NLS activity. We show that the dsRBD-NLS can mediate nuclear import of a reporter protein via interaction with importins β, 7, and 8. In the context of full-length Dicer, the dsRBD-NLS is masked. However, duplication of the dsRBD localizes the full-length protein to the nucleus. Furthermore, deletion of the N-terminal helicase domain results in partial accumulation of Dicer in the nucleus upon leptomycin B treatment, indicating that CRM1 contributes to nuclear export of Dicer. Finally, we demonstrate that human Dicer has the ability to shuttle between the nucleus and the cytoplasm. We conclude that Dicer is a shuttling protein whose steady-state localization is cytoplasmic.     

Overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates nuclear mRNA export

Human TAP and its yeast orthologue Mex67p are members of the multigene family of NXF proteins. A conserved feature of NXFs is a leucine-rich repeat domain (LRR) followed by a region related to the nuclear transport factor 2 (the NTF2-like domain). The NTF2-like domain of metazoan NXFs heterodimerizes with a protein known as p15 or NXT. A C-terminal region related to ubiquitin-associated domains (the UBA-like domain) is present in most, but not all NXF proteins. Saccharomyces cerevisiae Mex67p and Caenorhabditis elegans NXF1 are essential for the export of messenger RNA from the nucleus. Human TAP mediates the export of simian type D retroviral RNAs bearing the constitutive transport element, but the precise role of TAP and p15 in mRNA nuclear export has not yet been established. Here we show that overexpression of TAP/p15 heterodimers bypasses nuclear retention and stimulates the export of mRNAs that are otherwise exported inefficiently. This stimulation of mRNA export is strongly reduced by removing the UBA-like domain of TAP and abolished by deleting the LRR domain or the NTF2-like domain. Similar results are obtained when TAP/p15 heterodimers are directly tethered to the RNA export cargo. Our data indicate that formation of TAP/p15 heterodimers is required for TAP-mediated export of mRNA and show that the LRR domain of TAP plays an essential role in this process.     

Nuclear import and DNA-binding activity of RFX1. Evidence for an autoinhibitory mechanism.

RFX1 binds and regulates the enhancers of a number of viruses and cellular genes. RFX1 belongs to the evolutionarily conserved RFX protein family that shares a DNA-binding domain and a conserved C-terminal region. In RFX1 this conserved region mediates dimerization, and is followed by a unique C-terminal tail, containing a highly acidic stretch. In HL-60 cells nuclear translocation of RFX1 is regulated by protein kinase C with unknown mechanisms. By confocal fluorescence microscopy, we have identified a nonclassical nuclear localization signal (NLS) at the extreme C-terminus. The adjacent 'acidic region', which showed no independent NLS activity, potentiated the function of the NLS. Subcellular fractionation showed that the tight association of RFX1 with the nucleus is mediated by its DNA-binding domain and enhanced by the dimerization domain. In contrast, the acidic region inhibited nuclear association, by down-regulating the DNA-binding activity of RFX1. These data suggest an autoinhibitory interaction, which may regulate the function of RFX1 at the level of DNA binding. The C-terminal tail thus constitutes a composite localization domain, which on the one hand mediates nuclear import of RFX1, and on the other hand inhibits its association with the nucleus and binding to DNA. The participation of the acidic region in both activities suggests a mechanism by which the nuclear import and DNA-binding activity of RFX1 may be coordinately regulated by phosphorylation by kinases such as PKC.     

Cloning and characterization of a novel p21(Cip1/Waf1)-interacting zinc finger protein, ciz1.

p21(Cip1/Waf1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. Here, we report a novel p21(Cip1/Waf1)-interacting protein, Ciz1 (for Cip1 interacting zinc finger protein), which contains polyglutamine repeats and glutamine-rich region in the N-terminus as well as three zinc-finger motifs and one MH3 (matrin 3-homologous domain 3) in the C-terminal region. Ciz1 bound to the N-terminal, the CDK2-interacting part of p21(Cip1/Waf1), and the interaction was disrupted by the overexpression of CDK2. A region of about 150 amino acids containing the first zinc-finger motif in Ciz1 was the binding site for p21(Cip1/Waf1). When Ciz1 and p21(Cip1/Waf1) were individually overexpressed in U2-OS cells, they mostly localized in the nucleus. However, coexpression of Ciz1 induced cytoplasmic distribution of p21(Cip1/Waf1). These data indicate that Ciz1 is a unique nuclear protein that regulates the cellular localization of p21(Cip1/Waf1).