Bipartite nuclear localization signal of matrin 3 is essential for vertebrate cells

Matrin 3, a nuclear matrix protein has potential (1) to withhold promiscuously edited RNAs within the nucleus in cooperation with p54(nrb) and PSF, (2) to mediate NMDA-induced neuronal death, and (3) to modulate promoter activity of genes proximal to matrix/scaffold attachment region (MAR/SAR). We identified a bipartite nuclear localization signal (NLS) of chicken matrin 3 (cmatr3) at residues 583-602. By expressing green fluorescent protein (GFP) fused to the NLS mutant in chicken DT40 cells, we showed an essential role of the NLS for cell proliferation. Furthermore, we showed that both clusters of basic amino acids and a linker of the bipartite NLS were essential and sufficient for the nuclear import of GFP. Exogenous cmatr3 rescued the HeLa cells where human matrin 3 was suppressed by RNA interference, but cmatr3 containing deletions at either of the basic amino acid clusters or the linker could not.     

The RING domain and first zinc finger of TRAF6 coordinate signaling by interleukin-1, lipopolysaccharide, and RANKL

TRAF6, a crucial adaptor molecule in innate and adaptive immunity, contains three distinct functional domains. The C-terminal TRAF domain facilitates oligomerization and sequence-specific interaction with receptors or other adaptor proteins. In conjunction with the dimeric E2 enzyme Ubc13-Uev1A, the N-terminal RING domain of TRAF6 functions as an E3 ubiquitin (Ub) ligase that facilitates its own site-specific ubiquitination through the generation of a Lys-63-linked poly-Ub chain. This modification does not cause its proteasomal degradation but rather serves as a scaffold to activate both the IKK and stress kinase pathways. Connecting the N-and C-terminal regions, the four internal zinc finger (ZF) motifs have yet to be functionally defined. In this study, we examined the role of the ZF domains in interleukin-1, lipopolysaccharide, and RANKL signaling by reconstitution of TRAF6-deficient cells with point mutations or deletions of these ZF motifs. Although ZF domains 2-4 are dispensable for activating IKK, p38, and JNK by interleukin-1 and lipopolysaccharide, the first ZF domain together with an intact RING domain of TRAF6 is essential for activating these pathways. Furthermore, TRAF6 autoubiquitination and its interaction with Ubc13 are dependent on ZF1 and an intact RING domain. Additionally, expression of TRAF6 lacking ZF2-4 in TRAF6-deficient monocytes rescues RANKL-mediated osteoclast differentiation and LPS-stimulated interleukin-6 production. These data provide evidence for the critical role of the Ub ligase activity of TRAF6, which is coordinated via the RING domain and ZF1 to supply the necessary elements in signaling by cytokines dependent upon TRAF6.     

Ciz1, Cip1 interacting zinc finger protein 1 binds the consensus DNA sequence ARYSR(0–2)YYAC

The matrin 3 family of nuclear proteins consists of members with potentially diverse activities. Matrin 3 and NP220 share RNA-binding domains, and NP220 has been shown to recognize and bind to the DNA sequence, CCCCC (G/C). We have isolated and characterized another member of the matrin 3 family, designated NP94, from a medulloblastoma. This protein, also named Ciz1, has previously been characterized for its ability to interact with p21(Cip1/Waf1) and contains 3 zinc finger domains and a matrin 3-homologous domain 3. Our immunofluorescence and Northern blot analysis data indicate that Ciz1 is localized in the nucleus and is expressed in a wide range of tissues, especially the pancreas and the brain; within the brain, the highest message levels are found in the cerebellum. A modified selected and amplified binding (SAAB) sequence method was used to identify DNA sequences recognized by Ciz1. From the analysis of the retrieved SAAB sequences and verification using electrophoretic mobility shift assays, we formulated a consensus DNA sequence, ARYSR(0-2)YYAC, recognized by Ciz1. The potential activities of Ciz1, including those involved in brain tumorigenesis, are discussed.     

Active nuclear import and export pathways regulate E2F-5 subcellular localization

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.     

Matrin 3 is a Ca2+/calmodulin-binding protein cleaved by caspases

Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca(2+)-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca(2+)/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca(2+)-dependent interaction with CaM and caspase-mediated cleavage.     

Neural cell surface differentiation antigen gp130(RB13-6) induces fibroblasts and glioma cells to express astroglial proteins and invasive properties.

Transient expression of the differentiation and tumor cell surface antigen gp130(RB13-6) characterizes a subset of rat glial progenitor cells susceptible to ethylnitrosourea-induced neurooncogenesis. gp130(RB13-6) is as a member of an emerging protein family of ecto-phosphodiesterases/nucleotide pyrophosphatases that includes PC-1 and the tumor cell motility factor autotaxin. We have investigated the potential role of gp130(RB13-6) in glial differentiation by transfection of three cell lines of different origin that do not express endogenous gp130(RB13-6) (NIH-3T3 mouse fibroblasts; C6 and BT7Ca rat glioma cells) with the cDNA encoding gp130(RB13-6). The effect of gp130(RB13-6) expression was analyzed in terms of overall cell morphology, the expression of glial cell-specific marker proteins, and invasiveness. Transfectant sublines, consisting of 100% gp130(RB13-6)-positive cells, exhibited an altered, bipolar morphology. Fascicular aggregates of fibroblastoid cells subsequently developed into mesh-like patterns. Contrary to the parental NIH-3T3 and BT7Ca cells, the transfectant cells invaded into collagen type I. As shown by immunofluorescence staining of the transfectant sublines as well as of primary cultures composed of gp130(RB13-6)-positive and -negative cells, expression of gp130(RB13-6) induced coexpression of proteins typical for glial cells and their precursors, i.e., glial fibrillary acidic protein, the low affinity nerve growth factor receptor, and the neural proteins Thy-1, Ran-2, and S-100. In accordance with its expression in the immature rat nervous system, gp130(RB13-6) may thus have a significant role in the glial differentiation program and its subversion in neurooncogenesis.     

RB and RB2/p130 genes demonstrate both specific and overlapping functions during the early steps of in vitro neural differentiation of marrow stromal stem cells

Marrow stromal stem cells (MSCs) are stem-like cells that are currently being tested for their potential use in cell therapy for a number of human diseases. MSCs can differentiate into both mesenchymal and nonmesenchymal lineages. In fact, in addition to bone, cartilage and fat, it has been demonstrated that MSCs are capable of differentiating into neurons and astrocytes. RB and RB2/p130 genes are involved in the differentiation of several systems. For this reason, we evaluated the role of RB and RB2/p130 in the differentiation and apoptosis of MSCs under experimental conditions that allow for MSC differentiation toward the neuron-like phenotype. To this end, we ectopically expressed either RB or RB2/p130 and monitored proliferation, differentiation and apoptosis in rat primary MSC cultures induced to differentiate toward the neuron-like phenotype. Both RB and RB2/P130 decreased cell proliferation rate. In pRb-overexpressing cells, the arrest of cell growth was also observed in the presence of the HDAC-inhibitor TSA, suggesting that its antiproliferative activity does not rely upon the HDAC pathway, while the addition of TSA to pRb2/p130-overexpressing cells relieved growth inhibition. TUNEL reactions and studies on the expression of genes belonging to the Bcl-2 family showed that while RB protected differentiating MSCs from apoptosis, RB2/p130 induced an increase of apoptosis compared to controls. The effects of both RB and RB2/p130 on programmed cell death appeared to be HDAC- independent. Molecular analysis of neural differentiation markers and immunocytochemistry revealed that RB2/p130 contributes mainly to the induction of generic neural properties and RB triggers cholinergic differentiation. Moreover, the differentiation potentials of RB2/p130 and RB appear to rely, at least in part, on the activity of HDACs.     

Role of RB and RB2/P130 genes in marrow stromal stem cells plasticity

Marrow stromal cells (MSCs) are stem-like cells having a striking somatic plasticity. In fact, besides differentiating into mesenchymal lineages (bone, cartilage, and fat), they are capable of differentiating into neurons and astrocytes in vitro and in vivo. The RB and RB2/P130 genes, belonging to the retinoblastoma gene family, play a key role in neurogenesis, and for this reason, we investigated their role in neural commitment and differentiation of MSCs. In MSCs that were either uncommitted or committed toward neural differentiation, we ectopically expressed RB and RB2/P130 genes and analyzed their role in regulating the cell cycle, apoptosis and differentiation. In uncommitted MSCs, the activity of RB and RB2/P130 appeared limited to negatively regulating cell cycle progression, having no role in apoptosis and differentiation (toward either mesenchymal or neural lineages). On the other hand, in MSCs committed toward the neural phenotype, both RB and RB2/P130 reduced cell proliferation rate and affected the apoptotic process. RB protected differentiating cells from programmed cell death. On the contrary, RB2/P130 increased the percentage of cells in apoptosis. All of these activities were accomplished mainly in an HDAC-independent way. The retinoblastoma genes also influenced differentiation in neural committed MSCs. RB2/P130 contributes mainly to the induction of generic neural properties, while RB triggers cholinergic differentiation. These differentiating activities are HDAC-dependent. Our research shows that there is a critical temporal requirement for the RB genes during neuronal differentiation of MSCs: they are not required for cell commitment but play a role in the maturation process. For the above reasons, RB and RB2/P130 may have a role in neural differentiation but not in neural determination.     

A Family of Novel DNA-Binding Nuclear Proteins Having Polypyrimidine Tract-Binding Motif and Arginine/Serine-Rich Motif

NP220 is a DNA-binding nuclear protein originally cloned from human cell lines. Human NP220 (hNP220) has an arginine/serine-rich motif found in non-small nuclear RNP splicing factors (SR proteins) and shares three domains (MH1, MH2 and MH3) with an acidic nuclear matrix protein, matrin 3. The MH2 domain repeats three times and has homology to the polypyrimidine tract-binding motif of heterogeneous nuclear RNP I/L. NP220 also has a DNA-binding domain and nine repeats of the sequence LVTVDEVIEEEDL (acidic repeat). We have now isolated mouse equivalents of NP220 (mNP220s) and found that NP220s form a family of proteins with four members produced by alternative splicing of a common pre-mRNA. Two longer forms (NP220α and NP220β) have all functional domains mentioned above while two shorter forms (NP220γ and NP220δ) lack the DNA-binding domain and the acidic repeat. The structural aspects of NP220s are distinct from that of the SR proteins but rather resemble U2AF and Tra2 which activate a specific 3′-splicing site of specific genes in response to differentiation-dependent signals.     

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.     

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).     

p130RB2 positively contributes to ATR activation in response to replication stress via the RPA32-ETAA1 axis

Ataxia-telangiectasia mutated and Rad3-related (ATR) kinase is a crucial regulator of the cell cycle checkpoint and activated in response to DNA replication stress by two independent pathways via RPA32-ETAA1 and TopBP1. However, the precise activation mechanism of ATR by the RPA32-ETAA1 pathway remains unclear. Here, we show that p130RB2, a member of the retinoblastoma protein family, participates in the pathway under hydroxyurea-induced DNA replication stress. p130RB2 binds to ETAA1, but not TopBP1, and depletion of p130RB2 inhibits the RPA32-ETAA1 interaction under replication stress. Moreover, p130RB2 depletion reduces ATR activation accompanied by phosphorylation of its targets RPA32, Chk1, and ATR itself. It also causes improper re-progression of S phase with retaining single-stranded DNA after cancelation of the stress, which leads to an increase in the anaphase bridge phenotype and a decrease in cell survival. Importantly, restoration of p130RB2 rescued the disrupted phenotypes of p130RB2 knockdown cells. These results suggest positive involvement of p130RB2 in the RPA32-ETAA1-ATR axis and proper re-progression of the cell cycle to maintain genome integrity.     

Zif268基因突变体的克隆与真核表达载体的构建

以小鼠转录因子Zif268的三锌指DNA结合区为模板,利用重叠(Overlap)PCR技术,获得Zif268关键氨基酸位点同时突变的三锌指突变体ZF123、2ZF123。以ZFl23、2ZF123为模板,PCR扩增获得TAT—ZF123,TAT—2ZF123序列。构建表达质拉PET—28—a^ —TAT—ZF123,pET—28—a^ —TAT—2ZF123。为利用HIVTAT蛋白的跨膜功能,实现ZF123、2ZF123在哺乳动物细胞中的表达打下基础。     

Zinc Finger-like Motif Conserved in a Family of RNA Binding Proteins

NP220s compose a family of RNA binding proteins together with matrin 3, one of major proteins of the nuclear matrix. They have repeats of RNA recognition motif (RRM; MH2) homologous to RRM in heterogeneous nuclear RNPs I/L in addition to MH1 and MH3 with unknown function. In search of additional homologous sequences, we found the reported sequence of rat matrin 3 is partially incorrect. Correction of this sequence showed that the NP220 family has a fourth homologous motif with the characteristics of a Cys₂-His₂ zinc finger-like motif. The sequence of this motif is perfectly conserved in human and mouse NP220s despite their 75% overall sequence homology.