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A crystallographic study of human NONO (p54(nrb)): overcoming pathological problems with purification, data collection and noncrystallographic symmetry.

ABSTRACT: Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54(nrb)) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that L-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis (b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed.

SUBMITTER: Knott GJ

PROVIDER: S-EPMC5038197 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Nuclear scaffold protein p54<sup>nrb</sup>/NONO facilitates the hypoxia-enhanced progression of hepatocellular carcinoma.

Project description:Hypoxia and related oxidative stress are closely related to the development and treatment of hepatocellular carcinoma (HCC). However, the mechanism mediated by hypoxia in HCC has not yet been elucidated. Here, we found multifunction scaffold protein p54nrb/NONO exerted pleiotropic effects to regulate hypoxia transcription signals, thereby enhancing the progression of liver cancer. Extensive analysis of clinical data demonstrated that NONO was significantly upregulated and represented as a poor prognostic indicator of HCC. The crucial role of NONO in driving angiogenesis and glycolysis, two well-known cancer phenotypes mediated by hypoxia, was examined in vitro an in vivo. Mechanistically, NONO interacted with and stabilized both HIF-1 and HIF-2 complexes thus activating the transcription of hypoxia-induced genes. Besides, NONO bound pre-mRNA and subsequent mRNA of these genes to facilitate them splicing and mRNA stability, respectively. Thus, NONO knockout seriously disrupted the expression of a cluster of HIF-1/2 targets and impeded hypoxia-enhanced progression in HCC. In conclusion, NONO functioned as a multipurpose scaffold that interacted with HIF-1/2 complex and their downstream transcripts to facilitate the expression of hypoxia-induced genes, allowing malignant proliferation, indicating that NONO might be a potential therapeutic target for HCC.

| S-EPMC8211563 | biostudies-literature

RNA-binding protein p54<sup>nrb</sup>/NONO potentiates nuclear EGFR-mediated tumorigenesis of triple-negative breast cancer.

Project description:Nuclear-localized epidermal growth factor receptor (EGFR) highly correlates with the malignant progression and may be a promising therapeutic target for breast cancer. However, molecular mechanisms of nuclear EGFR in triple-negative breast cancer (TNBC) have not been fully elucidated. Here, we performed gene-annotation enrichment analysis for the interactors of nuclear EGFR and found that RNA-binding proteins (RBPs) were closely associated with nuclear EGFR. We further demonstrated p54nrb/NONO, one of the RBPs, significantly interacted with nuclear EGFR. NONO was upregulated in 80 paired TNBC tissues and indicated a poor prognosis. Furthermore, NONO knockout significantly inhibited TNBC proliferation in vitro and in vivo. Mechanistically, NONO increased the stability of nuclear EGFR and recruited CREB binding protein (CBP) and its accompanying E1A binding protein p300, thereby enhancing the transcriptional activity of EGFR. In turn, EGFR positively regulated the affinity of NONO to mRNAs of nuclear EGFR downstream genes. Furthermore, the results indicated that the nuclear EGFR/NONO complex played a critical role in tumorigenesis and chemotherapy resistance. Taken together, our findings indicate that NONO enhances nuclear EGFR-mediated tumorigenesis and may be a potential therapeutic target for TNBC patients with nuclear EGFR expression.

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Regulation of PCGEM1 by p54/nrb in prostate cancer.

Project description:PCGEM1 is a long non-coding RNA (lncRNA) that is often upregulated in prostate cancer. However, little is known how PCGEM1 is regulated. In the present study, we show transcriptional regulation of PCGEM1 in response to androgen deprivation by p54/nrb. While ectopic expression of p54/nrb increases, suppression of p54/nrb by RNAi or knockout (KO) reduces PCGEM1. Moreover, rescue experiments indicate that re-expression of p54/nrb in KO cells restores the ability to induce PCGEM1, leading to upregulation of the androgen receptor splice variant AR3 which has been shown to play a role in castration resistance. Finally, 3,3'-Diindolylmethane (DIM), a known chemoprevention agent, is capable of suppressing PCGEM1 expression by preventing the interaction of p54/nrb with the PCGEM1 promoter. In particular, DIM reduces tumor growth by suppression of PCGEM1 and promoting apoptosis in the castrated xenograft mouse model. Together, these results demonstrate a novel mechanism of p54/nrb-mediated expression of PCGEM1 and AR3, contributing to castration resistance in prostate cancer.

| S-EPMC5041109 | biostudies-literature

Detection of translational noncrystallographic symmetry in Patterson functions.

Project description:Detection of translational noncrystallographic symmetry (TNCS) can be critical for success in crystallographic phasing, particularly when molecular-replacement models are poor or anomalous phasing information is weak. If the correct TNCS is detected then expected intensity factors for each reflection can be refined, so that the maximum-likelihood functions underlying molecular replacement and single-wavelength anomalous dispersion use appropriate structure-factor normalization and variance terms. Here, an analysis of a curated database of protein structures from the Protein Data Bank to investigate how TNCS manifests in the Patterson function is described. These studies informed an algorithm for the detection of TNCS, which includes a method for detecting the number of vectors involved in any commensurate modulation (the TNCS order). The algorithm generates a ranked list of possible TNCS associations in the asymmetric unit for exploration during structure solution.

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Intensity statistics in the presence of translational noncrystallographic symmetry.

Project description:In the case of translational noncrystallographic symmetry (tNCS), two or more copies of a component in the asymmetric unit of the crystal are present in a similar orientation. This causes systematic modulations of the reflection intensities in the diffraction pattern, leading to problems with structure determination and refinement methods that assume, either implicitly or explicitly, that the distribution of intensities is a function only of resolution. To characterize the statistical effects of tNCS accurately, it is necessary to determine the translation relating the copies, any small rotational differences in their orientations, and the size of random coordinate differences caused by conformational differences. An algorithm to estimate these parameters and refine their values against a likelihood function is presented, and it is shown that by accounting for the statistical effects of tNCS it is possible to unmask the competing statistical effects of twinning and tNCS and to more robustly assess the crystal for the presence of twinning.

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Noncrystallographic symmetry-constrained map obtained by direct density optimization.

Project description:Noncrystallographic symmetry (NCS) averaging following molecular-replacement phasing is generally the major technique used to solve a structure with several molecules in one asymmetric unit, such as a spherical icosahedral viral particle. As an alternative method to NCS averaging, a new approach to optimize or to refine the electron density directly under NCS constraints is proposed. This method has the same effect as the conventional NCS-averaging method but does not include the process of Fourier synthesis to generate the electron density from amplitudes and the corresponding phases. It has great merit for the solution of structures with limited data that are either twinned or incomplete at low resolution. This method was applied to the case of the T = 1 shell-domain subviral particle of Penaeus vannamei nodavirus with data affected by twinning using the REFMAC5 refinement software.

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

Project description: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 gamma-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.

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p54(nrb) associates with the 5' splice site within large transcription/splicing complexes.

Project description:The functional coupling of transcription and splicing has been reported both in vivo and in vitro, but the molecular mechanisms governing these interactions remain largely unknown. Here we show that p54(nrb), a transcription/splicing factor, associates with the 5' splice site (SS) within large complexes present in HeLa cell nuclear extracts, in which the hyperphosphorylated form of RNA polymerase II (RNAPIIO) is associated with U1 or U1 and U2 snRNPs. These RNAPIIO-snRNP complexes also contain other transcription/splicing factors, such as PSF and TLS, as well as transcription factors that interact with RNAPIIO during elongation, including P-TEFb, TAT-SF1 and TFIIF. The presence of these factors in functional elongation complexes, demonstrated using an immobilized DNA template assay, strongly suggests that the RNAPIIO-snRNP complexes reflect physiologically relevant interactions between the transcription and splicing machineries. Our finding that both p54(nrb) and PSF, which bind the C-terminal domain of the largest subunit of RNAPII, can interact directly with the 5' SS indicates that these factors may mediate contacts between RNAPII and snRNPs during the coupled transcription/splicing process.

| S-EPMC394241 | biostudies-other

Flexible torsion-angle noncrystallographic symmetry restraints for improved macromolecular structure refinement.

Project description:One of the great challenges in refining macromolecular crystal structures is a low data-to-parameter ratio. Historically, knowledge from chemistry has been used to help to improve this ratio. When a macromolecule crystallizes with more than one copy in the asymmetric unit, the noncrystallographic symmetry relationships can be exploited to provide additional restraints when refining the working model. However, although globally similar, NCS-related chains often have local differences. To allow for local differences between NCS-related molecules, flexible torsion-based NCS restraints have been introduced, coupled with intelligent rotamer handling for protein chains, and are available in phenix.refine for refinement of models at all resolutions.

| S-EPMC4014122 | biostudies-literature

Transcriptional activity of the murine retinol-binding protein gene is regulated by a multiprotein complex containing HMGA1, p54 nrb/NonO, protein-associated splicing factor (PSF) and steroidogenic factor 1 (SF1)/liver receptor homologue 1 (LRH-1).

Project description:Retinol-binding protein (RBP4) transports retinol in the circulation from hepatic stores to peripheral tissues. Since little is known regarding the regulation of this gene, we analysed the cis-regulatory sequences of the mouse RBP4 gene. Our data show that transcription of the gene is regulated through a bipartite promoter: a proximal region necessary for basal expression and a distal segment responsible for cAMP-induction. This latter region contains several binding sites for the structural HMGA1 proteins, which are important to promoter regulation. We further demonstrate that HMGA1s play a key role in basal and cAMP-induction of Rbp4 transcription and the RBP4 and HMGA1 genes are coordinately regulated in vitro and in vivo. HMGA1 acts to recruit transcription factors to the RBP4 promoter and we specifically identified p54(nrb)/NonO and protein-associated splicing factor (PSF) as components that interact with this complex. Steroidogenic factor 1 (SF1) or the related liver receptor homologue 1 (LRH-1) are also associated with this complex upon cAMP-induction. Depletion of SF1/LRH-1 by RNA interference resulted in a dramatic loss of cAMP-induction. Collectively, our results demonstrate that basal and cAMP-induced Rbp4 transcription is regulated by a multiprotein complex that is similar to ones that modulate expression of genes of steroid hormone biosynthesis. Since genes related to glucose metabolism are regulated in a similar fashion, this suggests that Rbp4 expression may be regulated as part of a network of pathways relevant to the onset of type 2 diabetes.

| S-EPMC2753725 | biostudies-literature

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