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The biological effects of ionizing radiation are attributable, in large part, to induction of DNA double-strand breaks. We report here the identification of a new protein factor that reconstitutes efficient double-strand break rejoining when it is added to a reaction containing the five other polypeptides known to participate in the human nonhomologous end-joining pathway. The factor is a stable heteromeric complex of polypyrimidine tract-binding protein-associated splicing factor (PSF) and a 54-kDa nuclear RNA-binding protein (p54(nrb)). These polypeptides, to which a variety of functions have previously been attributed, share extensive homology, including tandem RNA recognition motif domains. The PSF.p54(nrb) complex cooperates with Ku protein to form a functional preligation complex with substrate DNA. Based on structural comparison with related proteins, we propose a model where the four RNA recognition motif domains in the heteromeric PSF.p54(nrb) complex cooperate to align separate DNA molecules.  相似文献   

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PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prpl8. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3' side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.  相似文献   

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PSF and p54(nrb)/NonO--multi-functional nuclear proteins   总被引:13,自引:0,他引:13  
Shav-Tal Y  Zipori D 《FEBS letters》2002,531(2):109-114
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Straub T  Knudsen BR  Boege F 《Biochemistry》2000,39(25):7552-7558
We have previously shown [Straub et al. (1998) J. Biol. Chem. 273, 26261] that the pyrimidine tract binding protein associated splicing factor PSF/p54(nrb) binds and stimulates DNA topoisomerase I. Here we show that cleavage and religation half-reactions of topoisomerase I are unaffected by PSF/p54(nrb), whereas the propensity of the enzyme to jump between separate DNA helices is stimulated. To demonstrate such an effect, topoisomerase I was first captured in suicidal cleavage of an oligonucleotide substrate. Subsequently, a cleavage/ligation equilibrium was established by adding a ligation donor under conditions allowing recleavage of the ligated substrate. Finally, a second oligonucleotide was added to the mixture, which also allowed suicidal cleavage by topoisomerase I, but did not accommodate the ligation donor of the first oligonucleotide. Thus, topoisomerase I was given the choice to engage in repeated cleavage/ligation cycles of the first oligonucleotide or to jump to the second suicide substrate and get trapped. PSF/p54(nrb) enhanced the cleavage rate of the second oligonucleotide (11-fold), suggesting that it stimulates the dissociation of topoisomerase I after ligation. Thus, stimulation of topoisomerase I catalysis by PSF/p54(nrb) seems to be affected by mobilization of the enzyme.  相似文献   

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Mammalian cells repair DNA double-strand breaks (DSBs) via efficient pathways of direct, nonhomologous DNA end joining (NHEJ) and homologous recombination (HR). Prior work has identified a complex of two polypeptides, PSF and p54(nrb), as a stimulatory factor in a reconstituted in vitro NHEJ system. PSF also stimulates early steps of HR in vitro. PSF and p54(nrb) are RNA recognition motif-containing proteins with well-established functions in RNA processing and transport, and their apparent involvement in DSB repair was unexpected. Here we investigate the requirement for p54(nrb) in DSB repair in vivo. Cells treated with siRNA to attenuate p54(nrb) expression exhibited a delay in DSB repair in a γ-H2AX focus assay. Stable knockdown cell lines derived by p54(nrb) miRNA transfection showed a significant increase in ionizing radiation-induced chromosomal aberrations. They also showed increased radiosensitivity in a clonogenic survival assay. Together, results indicate that p54(nrb) contributes to rapid and accurate repair of DSBs in vivo in human cells and that the PSF·p54(nrb) complex may thus be a potential target for radiosensitizer development.  相似文献   

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TopBP1 is a BRCT domain-rich protein that is structurally and functionally conserved throughout eukaryotic organisms. It is required for the initiation of DNA replication and for DNA repair and damage signalling. To further dissect its biological functions, we explored TopBP1-interacting proteins by co-immunoprecipitation assays and LC-ESI-MS-analyses. As TopBP1 binding partners we identified p54(nrb) and PSF, and confirmed the physical interactions by GST pull-down assays, co-immunoprecipitations and by yeast two-hybrid experiments. Recent evidence shows an involvement of p54(nrb) and PSF in DNA double-strand break repair (DSB) and radioresistance. To get a first picture of the physiological significance of the interaction of TopBP1 with p54(nrb) and PSF we investigated in real time the spatiotemporal behaviour of the three proteins after laser microirradiation of living cells. Localisation of TopBP1 at damage sites was noticed as early as 5 s following damage induction, whereas p54(nrb) and PSF localised there after 20 s. Both p54(nrb) and PSF disappeared after 20 s while TopBP1 was retained at damage sites significantly longer suggesting different functions of the proteins during DSB recognition and repair.  相似文献   

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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).  相似文献   

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The U1 snRNP-A (U1A) protein has been known for many years as a component of the U1 snRNP. We have previously described a form of U1A present in human cells in significant amounts that is not associated with the U1 snRNP or U1 RNA but instead is part of a novel complex of non-snRNP proteins that we have termed snRNP-free U1A, or SF-A. Antibodies that specifically recognize this complex inhibit in vitro splicing and polyadenylation of pre-mRNA, suggesting that this complex may play an important functional role in these mRNA-processing activities. This finding was underscored by the determination that one of the components of this complex is the polypyrimidine-tract-binding protein-associated splicing factor, PSF. In order to further our studies on this complex and to determine the rest of the components of the SF-A complex, we prepared several stable HeLa cell lines that overexpress a tandem-affinity-purification-tagged version of U1A (TAP-tagged U1A). Nuclear extract was prepared from one of these cell lines, line 107, and affinity purification was performed along with RNase treatment. We have used mass spectrometry analysis to identify the candidate factors that associate with U1A. We have now identified and characterized PSF, p54(nrb), and p68 as novel components of the SF-A complex. We have explored the function of this complex in RNA processing, specifically cleavage and polyadenylation, by performing immunodepletions followed by reconstitution experiments, and have found that p54(nrb) is critical.  相似文献   

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Ha K  Takeda Y  Dynan WS 《DNA Repair》2011,10(3):252-259
PSF (gene name SFPQ) is a member of a small family of proteins with dual functions in RNA biogenesis and DNA repair. PSF and PSF-containing complexes stimulate double-strand break repair in cell free systems, most likely via direct interaction with the repair substrate. Prior in vitro studies are, however, insufficient to demonstrate whether PSF contributes to DNA repair in living cells. Here, we investigate the effect of miRNA-mediated PSF knockdown in human (HeLa) cells. We find that PSF is essential for reproductive viability. To circumvent this and investigate the DNA damage sensitivity phenotype, we established a genetic rescue assay based on co-transfection of PSF miRNA and mutant PSF expression constructs. Mutational analysis suggests that sequences required for viability and radioresistance are partially separable, and that the latter requires a unique N-terminal PSF domain. As an independent means to investigate PSF sequences involved in DNA repair, we established an assay based on real-time relocalization of PSF-containing complexes to sites of dense, laser-induced DNA damage in living cells. We show that relocalization is driven by sequences in PSF, rather than its dimerization partner, p54(nrb)/NONO, and that sequences required for relocalization reside in the same N-terminal domain that contributes to radioresistance. Further evidence for the importance of PSF sequences in mediating relocalization is provided by observations that PSF promotes relocalization of a third protein, PSPC1, under conditions where p54(nrb) is limiting. Together, these observations support the model derived from prior biochemical studies that PSF influences repair via direct, local, interaction with the DNA substrate.  相似文献   

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Synthetic oligonucleotides are used to regulate gene expression through different mechanisms. Chemical modifications of the backbone of the nucleic acid and/or of the 2′ moiety of the ribose can increase nuclease stability and/or binding affinity of oligonucleotides to target molecules. Here we report that transfection of 2′-F-modified phosphorothioate oligonucleotides into cells can reduce the levels of P54nrb and PSF proteins through proteasome-mediated degradation. Such deleterious effects of 2′-F-modified oligonucleotides were observed in different cell types from different species, and were independent of oligonucleotide sequence, positions of the 2′-F-modified nucleotides in the oligonucleotides, method of delivery or mechanism of action of the oligonucleotides. Four 2′-F-modified nucleotides were sufficient to cause the protein reduction. P54nrb and PSF belong to Drosophila behavior/human splicing (DBHS) family. The third member of the family, PSPC1, was also reduced by the 2′-F-modified oligonucleotides. Preferential association of 2′-F-modified oligonucleotides with P54nrb was observed, which is partially responsible for the protein reduction. Consistent with the role of DBHS proteins in double-strand DNA break (DSB) repair, elevated DSBs were observed in cells treated with 2′-F-modified oligonucleotides, which contributed to severe impairment in cell proliferation. These results suggest that oligonucleotides with 2′-F modifications can cause non-specific loss of cellular protein(s).  相似文献   

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《Gene》1997,185(2):181-186
Bovine adenovirus type 2 (BAV2) is a medium size double-stranded DNA virus which infects both bovine and ovine species, resulting in mild respiratory and gastrointestinal disorders. To better understand the virus and its growth characterisitics in Madin-Darby bovine kidney (MDBK) cells, we have cloned and sequenced the extreme right-end segment of the BAV2 genome (90.5–100 map units). Analysis of the nucleotide sequence revealed 40 potential open reading frames (ORFs) with coding capacity for polypeptides that are 25 or more amino acid (aa) residues long. Six of these ORFs encode polypeptides that show homology to well-characterized early region 4 (E4) proteins of human adenovirus type 2 (Ad2) and Ad12. ORF1 has the potential to encode a 114 aa long polypeptide that is 54% homologous to the E4 14 kDa protein of Ad2. ORF2 encodes a 78 aa long polypeptide that exhibits 40% homology to the E4 13 kDa protein of Ad2. ORFs 3–6 encode polypeptides that have homology to the E4 34 kDa protein encoded by ORF6 of Ad2 and Ad12. ORFs 3, 4 and 5 encode 128, 96 and 31 aa long polypeptides, respectively. The 128-aa polypeptide exhibits 59% homology, while the 96 and 31 aa long polypeptides exhibit 61% and 70% homology to the E4 34 kDa protein, respectively. ORF6 has the potential to encode a 57 aa long polypeptide that has 67% homology to the E4 34 kDa protein of Ad2 and 50% homology to the E4 34 kDa protein of Ad12.  相似文献   

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