p54(nrb) acts as a transcriptional coactivator for activation function 1 of the human androgen, receptor
The androgen receptor (AR) has two transactivation functions that have been mapped to the N- and C-terminal domains and designated as activation function-1 (AF-1) and AF-2, respectively. While the molecular basis for AF-2 function has been well studied, little is known about AF-1 coregulators. Therefore, we attempted to identify AF-1-interacting proteins from HEK293 cells by biochemical purification followed by mass fingerprinting by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Purified AF-1 region-interacting proteins were found to contain nuclear RNA-binding protein p54(nrb), polypyrimidine tract-binding protein-associated splicing factor (PSF), paraspeckle protein 1 (PSP1), and PSP2, which are assumed to be involved in pre-mRNA processing. p54(nrb) interacted with AR via the A/B domain in a ligand-dependent manner. Reflecting the physical interaction between p54(nrb) and the AR A/B domain, AR AF-1 function was potentiated by p54(nrb). Our results suggest that p54(nrb) functions as a coactivator of AR that potentiates transcription, and presumably splicing as well.
Available from: Gi Hoon Son
- "(Dong et al., 2009). In contrast to the many reported repressive effects, NonO has a transcriptional coactivator function in androgen receptor-mediated transcription (Ishitani et al., 2003; Kuwahara et al., 2006). NonO also activates transcription by enhancing the association of many transcription factors, such as E47, OTF-1 and OTF-2, with their target DNAs (Yang et al., 1997). "
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ABSTRACT: We investigated the relationship between oct4 gene expression patterns and CpG sites methylation profiles during ES cell differentiation into neurons, and identified relevant binding factor. The oct4 gene expression level gradually declined as ES cell differentiation progressed, and the CpG sites in the oct4 proximal enhancer (PE) and promoter regions were methylated in concert with ES cell differentiation. An electro-mobility shift assay (EMSA) showed that putative proteins bind to CpG sites in the oct4 PE/promoter. We purified CpG binding proteins with DNAbinding purification method, and NonO was identified by liquid chromatography-mass spectrometry. EMSA with specific competitors revealed that NonO specifically binds to the conserved CCGGTGAC sequence in the oct4 promoter. Methylation at a specific cytosine residue (CC* GGTGAC) reduced the binding affinity of NonO for the recognition sequence. Chromatin immunoprecipitation analysis confirmed that NonO binds to the unmethylated oct4 promoter. There were no changes in the NonO mRNA and protein levels between ES cells and differentiated cells. The transcriptional role of NonO in oct4 gene expression was evaluated by luciferase assays and knockdown experiments. The luciferase activity significantly increased threefold when the NonO expression vector was cotransfected with the NonO recognition sequence, indicating that NonO has a transcription activator effect on oct4 gene expression. In accordance with this effect, when NonO expression was inhibited by siRNA treatment, oct4 expression was also significantly reduced. In summary, we purified NonO, a novel protein that binds to the CpG island of oct4 promoter, and positively regulates oct4 gene expression in ES cells.
Available from: Michael Hendzel
- "About twenty years ago the group of Harris Busch purified and characterized a heterodimer consisting of a 52 and a 100 kDa subunit, most certainly corresponding to what is nowadays known as the 54 kDa nuclear RNA-binding protein (p54nrb/NONO) and the polypyrimidine tract-binding protein-associated splicing factor (PSF/SFPQ). NONO and SFPQ show 71% sequence identity and, together with paraspeckle component 1 (PSPC1), belong to a subfamily of RNA recognition motif (RRM) proteins defined by tandem RRM motifs, flanked by an additional region of sequence similarity predicted to promote formation of heteromeric complexes between each of the proteins (24). NONO and SFPQ have been implicated in nuclear retention of A- to I-edited RNA as paraspeckle components (25), pre-mRNA 3′-end formation (26), cAMP cycling (27) and transcriptional activation (28–30). Interestingly, apart from their functions in RNA biogenesis, NONO and SFPQ were reported to interact with DNA in vitro, which lead to an investigation of their function in the context of DNA repair. "
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ABSTRACT: After the generation of DNA double-strand breaks (DSBs), poly(ADP-ribose) polymerase-1 (PARP-1) is one of the first proteins
to be recruited and activated through its binding to the free DNA ends. Upon activation, PARP-1 uses NAD+ to generate large amounts of poly(ADP-ribose) (PAR), which facilitates the recruitment of DNA repair factors. Here, we identify
the RNA-binding protein NONO, a partner protein of SFPQ, as a novel PAR-binding protein. The protein motif being primarily
responsible for PAR-binding is the RNA recognition motif 1 (RRM1), which is also crucial for RNA-binding, highlighting a competition
between RNA and PAR as they share the same binding site. Strikingly, the in vivo recruitment of NONO to DNA damage sites completely depends on PAR, generated by activated PARP-1. Furthermore, we show that
upon PAR-dependent recruitment, NONO stimulates nonhomologous end joining (NHEJ) and represses homologous recombination (HR)
in vivo. Our results therefore place NONO after PARP activation in the context of DNA DSB repair pathway decision. Understanding
the mechanism of action of proteins that act in the same pathway as PARP-1 is crucial to shed more light onto the effect of
interference on PAR-mediated pathways with PARP inhibitors, which have already reached phase III clinical trials but are until
date poorly understood.
Available from: Masao Sasamata
- "Recently, it has been reported that p54 nrb regulates the activity of transcription factors such as AR, SOCS3, SOX9, and CREB    . "
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ABSTRACT: YM155, a small-molecule survivin suppressant, specifically binds to the transcription factor ILF3, which regulates the expression of survivin. In this experiment we have demonstrated that p54(nrb) binds to the survivin promoter and regulates survivin expression. p54(nrb) forms a complex with ILF3, which directly binds to YM155. YM155 induces disruption of the ILF3/p54(nrb) complex, which results in a different subcellular localization between ILF3 and p54(nrb). Thus, identification of molecular targets of YM155 in suppression of the survivin pathway, might lead to development of its use as a novel potential target in cancers.
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