Identification of the polypyrimidine tract binding protein-associated splicing factor.p54(nrb) complex as a candidate DNA double-strand break rejoining factor

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.     

Purification and cDNA cloning of HeLa cell p54nrb, a nuclear protein with two RNA recognition motifs and extensive homology to human splicing factor PSF and Drosophila NONA/BJ6.

While searching for a human homolog of the S.cerevisiae splicing factor PRP18, we found a polypeptide that reacted strongly with antibodies against PRP18. We purified this polypeptide from HeLa cells using a Western blot assay, and named it p54nrb (for nuclear RNA-binding protein, 54 kDa). cDNAs encoding p54nrb were cloned with probes derived from partial sequence of the purified protein. These cDNAs have identical coding sequences but differ as a result of alternative splicing in the 5' untranslated region. The cDNAs encode a 471 aa polypeptide that contains two RNA recognition motifs (RRMs). Human p54nrb has no homology to yeast PRP18, except for a common epitope, but is instead 71% identical to human splicing factor PSF within a 320 aa region that includes both RRMs. In addition, both p54nrb and PSF are rich in Pro and Gln residues outside the main homology region. The Drosophila puff-specific protein BJ6, one of three products encoded by the alternatively spliced no-on-transient A gene (nonA), which is required for normal vision and courtship song, is 42% identical to p54nrb in the same 320 aa region. The striking homology between p54nrb, PSF, and NONA/BJ6 defines a novel phylogenetically conserved protein segment, termed DBHS domain (for Drosophila behavior, human splicing), which may be involved in regulating diverse pathways at the level of pre-mRNA splicing.     

PSF and p54nrb bind a conserved stem in U5 snRNA

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.     

PSF/p54(nrb) stimulates "jumping" of DNA topoisomerase I between separate DNA helices

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.     

p54nrb/NonO and PSF promote U snRNA nuclear export by accelerating its export complex assembly

The assembly of spliceosomal U snRNPs in metazoans requires nuclear export of U snRNA precursors. Four factors, nuclear cap-binding complex (CBC), phosphorylated adaptor for RNA export (PHAX), the export receptor CRM1 and RanGTP, gather at the m⁷G-cap-proximal region and form the U snRNA export complex. Here we show that the multifunctional RNA-binding proteins p54nrb/NonO and PSF are U snRNA export stimulatory factors. These proteins, likely as a heterodimer, accelerate the recruitment of PHAX, and subsequently CRM1 and Ran onto the RNA substrates in vitro, which mediates efficient U snRNA export in vivo. Our results reveal a new layer of regulation for U snRNA export and, hence, spliceosomal U snRNP biogenesis.     

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).     

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.     

Involvement of p54(nrb), a PSF partner protein,in DNA double-strand break repair and radioresistance

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.     

Proteomic identification of PSF and p54(nrb) as TopBP1-interacting proteins

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.