Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease

ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting. Other proteins identified were the spliceosomal components hnRNP A2/B1, hnRNP H3, and TLS/FUS. PSF is also known to interact with PTB, another member of the hnRNP family of proteins, and we further demonstrated that PTB interacts with ZNF198. The interaction between TLS/FUS and ZNF198 was confirmed using Western blot analysis. In 293 cells expressing the ZNF198/FGFR1 fusion protein, neither PSF nor PTB binds to the fusion protein, possibly because of their differential localization in the cell.     

Nuclear relocalization of the pre-mRNA splicing factor PSF during apoptosis involves hyperphosphorylation, masking of antigenic epitopes, and changes in protein interactions

The spatial nuclear organization of regulatory proteins often reflects their functional state. PSF, a factor essential for pre-mRNA splicing, is visualized by the B92 mAb as discrete nuclear foci, which disappeared during apoptosis. Because this mode of cell death entails protein degradation, it was considered that PSF, which like other splicing factors is sensitive to proteolysis, might be degraded. Nonetheless, during the apoptotic process, PSF remained intact and was N-terminally hyperphosphorylated on serine and threonine residues. Retarded gel migration profiles suggested differential phosphorylation of the molecule in mitosis vs. apoptosis and under-phosphorylation during blockage of cells at G1/S. Experiments with the use of recombinant GFP-tagged PSF provided evidence that in the course of apoptosis the antigenic epitopes of PSF are masked and that PSF reorganizes into globular nuclear structures. In apoptotic cells, PSF dissociated from PTB and bound new partners, including the U1--70K and SR proteins and therefore may acquire new functions.     

BRK phosphorylates PSF promoting its cytoplasmic localization and cell cycle arrest

BReast tumor Kinase (BRK) also known as protein kinase 6 (PTK6) is a non-receptor tyrosine kinase overexpressed in the majority of human breast carcinoma. The expression of BRK is a known prognostic marker of breast carcinoma. BRK has been shown to lie downstream of epidermal growth factor (EGF) signaling and mediate effects on cell proliferation and migration. To identify BRK substrates and interacting proteins, we undertook a proteomic approach. BRK immune complexes were purified from the BT-20 breast cancer cell line and analyzed by mass spectrometry. Herein, we report the identification of PSF, the polypyrimidine tract-binding (PTB) protein-associated splicing factor, as a BRK-interacting protein and substrate. BRK and PSF co-eluted in a large protein complex that was regulated by EGF stimulation. Furthermore, BRK and PSF co-immunoprecipitated in BT-20 cells and we defined the interaction as being an SH3 domain–polyproline interaction. The C-terminal tyrosines of PSF were the site of phosphorylation by BRK. Moreover, tyrosine phosphorylation of PSF was also observed upon EGF stimulation, consistent with a role of PSF and BRK downstream of the EGF receptor. Interestingly, the tyrosine phosphorylation promoted the cytoplasmic relocalization of PSF, impaired its binding to polypyrimidine RNA, and led to cell cycle arrest. Our findings show that BRK targets the PSF RNA-binding protein during EGF stimulation.     

Evidence that a nuclear matrix protein participates in premessenger RNA splicing

The role of nuclear matrix proteins in premessenger RNA splicing has been investigated using antibodies raised against isolated rat liver nuclear matrix and cross-reactive with a 65-kDa HeLa cell nuclear matrix protein (IGA-65). IGA-65 is an internal nuclear matrix component which can be solubilized as a component of nuclear splicing extracts, by the action of endogenous ribonucleases, EDTA, and DTT during extract preparation. Preincubation of splicing extract with antibodies against IGA-65 (anti-IGA-65) inhibited in vitro splicing of exogenous adenovirus precursor RNA. Furthermore, assembly of precursor RNA into active spliceosome complexes was inhibited by pretreatment of extracts with anti-IGA-65, suggesting a role for IGA-65 during early spliceosome assembly. The IGA-65 present in splicing extracts was distinguishable from known U-snRNP and hnRNP proteins on protein gels. Furthermore, electrophoresis of splicing extract on native gels indicated that IGA-65 was present in protein complexes different from those containing U-snRNPs or hnRNP C protein. The data support identification of complexes containing IGA-65 as nuclear factors involved in pre-mRNA splicing and, by extension, suggest a role for the nuclear matrix during processing in vivo.     

Characterization of the nuclear export signal of polypyrimidine tract-binding protein.

The polypyrimidine tract-binding protein (PTB) is a nuclear protein that regulates alternative splicing. In addition, it plays a role in the cytoplasm during infection by some viruses and functions as a positive effector of hepatitis B virus RNA export. Thus, it presumably contains a nuclear export signal (NES). Using a heterokaryon export assay in transfected cultured cells, we have shown that the N-terminal 25 amino acid residues of PTB function as an autonomous NES, with residues 11-16 being important for its activity. Unlike the heteronuclear ribonucleoprotein A1 NES, this NES is separable from the nuclear localization signal, which spans the entire N-terminal 60 residues of PTB. The PTB NES cannot be shown to bind to CAS or Crm1, cellular receptors known to export proteins from the nucleus, and it functions in the presence of leptomycin B, a specific inhibitor of Crm1-dependent export. PTB deleted of its NES, unlike wild type PTB, does not stimulate the export of hepatitis B virus RNA. Therefore, the PTB NES is a functionally important domain of this multifunctional protein that utilizes an unknown export receptor.     

Formation of nuclear splicing factor compartments is independent of lamins A/C

Nuclear lamins are major architectural elements of the mammalian cell nucleus, and they have been implicated in the functional organization of the nuclear interior, possibly by providing structural support for nuclear compartments. Colocalization studies have suggested a structural role for lamins in the formation and maintenance of pre-mRNA splicing factor compartments. Here, we have directly tested this hypothesis by analysis of embryonic fibroblasts from knock-out mice lacking A- and C-type lamins. We show that the morphology and cellular properties of splicing factor compartments are independent of A- and C-type lamins. Genetic loss of lamins A/C has no effect on the cellular distribution of several pre-mRNA splicing factors and does not affect the compartment morphology as examined by light and electron microscopy. The association of splicing factors with the nuclear matrix fraction persists in the absence of lamins A/C. Live cell microscopy demonstrates that the intranuclear positional stability of splicing factor compartments is maintained and that the exchange dynamics of SF2/ASF between the compartments and the nucleoplasm is not affected by loss of lamin A/C. Our results demonstrate that formation and maintenance of intranuclear splicing factor compartments is independent of lamins A/C, and they argue against an essential structural role of lamins A/C in splicing factor compartment morphology.     

Chromium-induced cross-linking of nuclear proteins and DNA

The in vivo cross-linking of proteins to DNA in intact Novikoff ascites hepatoma cells exposed to the chromium salt K2CrO4 was studied. DNA-protein complexes were assayed by high speed centrifugation of cells solubilized in buffered 4% sodium dodecyl sulfate and by electrophoretic identification of proteins associated with DNA-containing pellets. Further evidence of DNA-protein complexes, not dissociable in this buffer, was obtained by CsCl gradient centrifugation. Time dependence experiments showed that detectable cross-linking occurred after cells were exposed to chromium salt for at least 4 h, and the amount of DNA-protein complexes increased with longer incubation times. Complex formation occurred only with chromium salt concentrations of 200 microM or greater, and maximal cross-linking was effected at 5 mM. Immunotransfer methodology employing antibodies to nuclear matrix fraction and lamins was used to identify some of the polypeptides comprising the cross-linked complexes. These studies indicated specificity of chromium-induced complex formation within the nuclear protein fractions assayed. Our results document the ability of chromate to produce specific DNA-protein cross-links in living cells.     

The PSF.p54nrb complex is a novel Mnk substrate that binds the mRNA for tumor necrosis factor alpha

To identify new potential substrates for the MAP kinase signal-integrating kinases (Mnks), we employed a proteomic approach. The Mnks are targeted to the translational machinery through their interaction with the cap-binding initiation factor complex. We tested whether proteins retained on cap resin were substrates for the Mnks in vitro, and identified one such protein as PSF (the PTB (polypyrimidine tract-binding protein)-associated splicing factor). Mnks phosphorylate PSF at two sites in vitro, and our data show that PSF is an Mnk substrate in vivo. We also demonstrate that PSF, together with its partner, p54(nrb), binds RNAs that contain AU-rich elements (AREs), such as those for proinflammatory cytokines (e.g. tumor necrosis factor alpha (TNFalpha)). Indeed, PSF associates specifically with the TNFalpha mRNA in living cells. PSF is phosphorylated at two sites by the Mnks. Our data show that Mnk-mediated phosphorylation increases the binding of PSF to the TNFalpha mRNA in living cells. These findings identify a novel Mnk substrate. They also suggest that the Mnk-catalyzed phosphorylation of PSF may regulate the fate of specific mRNAs by modulating their binding to PSF.p54(nrb).     

Over-expression of SR-cyclophilin, an interaction partner of nuclear pinin, releases SR family splicing factors from nuclear speckles

Pre-mRNA splicing takes place within a dynamic ribonucleoprotein particle called the spliceosome and occurs in an ordered pathway. Although it is known that spliceosome consists of five small nuclear RNAs and at least 50 proteins, little is known about how the interaction among the proteins changes during splicing. Here we identify that SR-cyp, a Moca family of nuclear cyclophilin, interacts and colocalizes with nuclear pinin (pnn), a SR-related protein involving in pre-mRNA splicing. Nuclear pnn interacts with SR-cyp via its C-terminal RS domain. Upon SR-cyp over-expression, however, the subnuclear distribution of nuclear pnn is altered, resulting in its redistribution from nuclear speckles to a diffuse nucleoplasmic form. The diffuse subnuclear distribution of nuclear pnn is not due to epitope masking, accelerated protein turnover or post-translational modification. Furthermore, we find that SR-cyp regulates the subnuclear distribution of other SR family proteins, including SC35 and SRm300, in a similar manner as it does on nuclear pnn. This result is significant because it suggests that SR-cyp plays a general role in modulating the distribution pattern of SR-like and SR proteins, similar to that of Clk (cdc2-like kinase)/STY on SR family splicing factors. SR-cyp might direct its effect via either alteration of protein folding/conformation or of protein-protein interaction and thus may add another control level of regulation of SR family proteins and modification of their functions.     

The fate of dsRNA in the nucleus: a p54(nrb)-containing complex mediates the nuclear retention of promiscuously A-to-I edited RNAs

How do cells discriminate between selectively edited mRNAs that encode new protein isoforms, and dsRNA-induced, promiscuously edited RNAs that encode nonfunctional, mutant proteins? We have developed a Xenopus oocyte model system which shows that a variety of hyperedited, inosine-containing RNAs are specifically retained in the nucleus. To uncover the mechanism of inosine-induced retention, HeLa cell nuclear extracts were used to isolate a multiprotein complex that binds specifically and cooperatively to inosine-containing RNAs. This complex contains the inosine-specific RNA binding protein p54(nrb), the splicing factor PSF, and the inner nuclear matrix structural protein matrin 3. We provide evidence that one function of the complex identified here is to anchor hyperedited RNAs to the nuclear matrix, while allowing selectively edited mRNAs to be exported.     

核内小体的复杂结构与多样化功能

核内小体是定位于细胞核内的无膜结构,为多蛋白-RNA复合体,通过招募相关蛋白参与基因转录、RNA剪切、表观遗传调控、肿瘤发生与抑制及抗病毒防御等多种细胞活动。明确核内小体蛋白的形成过程、功能和调控机制对研究相关疾病与病毒-宿主作用机制均具有重要意义。以下以几种核内小体蛋白为例,对核内小体的形成方式、结构与功能进行综述,并重点阐述其在抗病毒感染中的重要作用,期望为宿主抗病毒免疫机制研究提供一个新的靶标。