Tumor Necrosis Factor Receptor Associated Factors (TRAFs) 2 and 3 Form a Transcriptional Complex with Phosho-RNA Polymerase II and p65 in CD40 Ligand Activated Neuro2a Cells.

The tumor necrosis factor receptor-associated factors (TRAFs) have been classically described as adaptor proteins that function as solely cytosolic signaling intermediates for the TNF receptor superfamily, Toll-like receptors (TLRs), NOD, like receptors (NLRs), cytokine receptors, and others. In this study, we show for the first time that TRAFs are present within the cytoplasm and nucleus of Neuro2a cells and primary cortical neurons, and that TRAF2 and TRAF3 translocate into the nucleus within minutes of CD40L stimulation. Analysis of the transcriptional regulatory potential of TRAFs by luciferase assay revealed that each of the TRAFs differentially functions as a transcriptional activator or repressor in a cell-specific manner. Interestingly, ChIP-qPCR data demonstrate that TRAFs 2/3, p65, and pRNAPol II form part of a transcriptional complex on the Icam-1 gene promoter upon CD40L stimulation. We further determined that TRAF2 recruitment to the nucleus is critical for the ubiquitination of H2b, a transcription permissive epigenetic modification. Our findings demonstrate for the first time that TRAFs 2/3 participate in the formation of a CD40L-induced transcriptional complex in neuronal cells.

Human 4E-T represses translation of bound mRNAs and enhances microRNA-mediated silencing.

A key player in translation initiation is eIF4E, the mRNA 5' cap-binding protein. 4E-Transporter (4E-T) is a recently characterized eIF4E-binding protein, which regulates specific mRNAs in several developmental model systems. Here, we first investigated the role of its enrichment in P-bodies and eIF4E-binding in translational regulation in mammalian cells. Identification of the conserved C-terminal sequences that target 4E-T to P-bodies was enabled by comparison of vertebrate proteins with homologues in Drosophila (Cup and CG32016) and Caenorhabditis elegans by sequence and cellular distribution. In tether function assays, 4E-T represses bound mRNA translation, in a manner independent of these localization sequences, or of endogenous P-bodies. Quantitative polymerase chain reaction and northern blot analysis verified that bound mRNA remained intact and polyadenylated. Ectopic 4E-T reduces translation globally in a manner dependent on eIF4E binding its consensus Y30X4L site. In contrast, tethered 4E-T continued to repress translation when eIF4E-binding was prevented by mutagenesis of YX4L, and modestly enhanced the decay of bound mRNA, compared with wild-type 4E-T, mediated by increased binding of CNOT1/7 deadenylase subunits. As depleting 4E-T from HeLa cells increased steady-state translation, in part due to relief of microRNA-mediated silencing, this work demonstrates the conserved yet unconventional mechanism of 4E-T silencing of particular subsets of mRNAs.

Chromophore maturation and fluorescence fluctuation spectroscopy of fluorescent proteins in a cell-free expression system.

Cell-free synthesis, a method for the rapid expression of proteins, is increasingly used to study interactions of complex biological systems. GFP and its variants have become indispensable for fluorescence studies in live cells and are equally attractive as reporters for cell-free systems. This work investigates the use of fluorescence fluctuation spectroscopy (FFS) as a tool for quantitative analysis of protein interactions in cell-free expression systems. We also explore chromophore maturation of fluorescent proteins, which is of crucial importance for fluorescence studies. A droplet sample protocol was developed that ensured sufficient oxygenation for chromophore maturation and ease of manipulation for titration studies. The kinetics of chromophore maturation of EGFP, EYFP, and mCherry were analyzed as a function of temperature. A strong increase in the rate from room temperature to 37°C was observed. We further demonstrate that all EGFP proteins fully mature in the cell-free solution and that brightness is a robust parameter specifying stoichiometry. Finally, FFS is applied to study the stoichiometry of the nuclear transport factor 2 in a cell-free system over a broad concentration range. We conclude that combining cell-free expression and FFS provides a powerful technique for quick, quantitative study of chromophore maturation and protein-protein interaction.

Translocación nuclear del receptor de glucocorticoides en fibroblastos de pacientes asmáticos con poliposis nasal insensible al tratamiento glucocorticoideo / Nuclear Translocation of the Glucocorticoid Receptor in Fibroblasts of Asthmatic Patients with Nasal Polyposis Insensitive to Glucocorticoid Treatment

Introducción: La poliposis nasal (PN) se trata con glucorticoides (GC) tópicos. En algunos pacientes eltratamiento es ineficaz requiriéndose cirugía endoscópica nasosinusal. Para ejercer su función, el GCprecisa unirse al receptor de GC (RG) y este desplazarse al núcleo celular. Nuestro objetivo fue establecersi la pobre respuesta a los GC es debida a una alteración en la translocación del RG al núcleo.Métodos: Se realizaron cultivos celulares de fibroblastos nasales de 7 controles sanos y 12 pacientes conPN y asma. Los fibroblastos se incubaron con budesonida o dexametasona (10-7M) durante diferentestiempos (30 min a 4 h) y la translocación del RG se analizó mediante inmunocitoquímica.Resultados: Ambos GC indujeron translocación del RG en todos los grupos, doblando su concentración enel núcleo (30 min) respecto al basal.Noencontramos diferencias en la translocación delRGentre controlesy pacientes ni relación con la gravedad del asma o la intolerancia a los antiinflamatorios no esteroideos(AINE). En los sujetos atópicos se observó una disminución de la translocación con budesonida (1 h, 3 h y4 h, p < 0,05) y dexametasona (30 min y 2 h, p < 0,05).Conclusiones: La insensibilidad al tratamiento con GC no parece responder a alteraciones en la translocacióndel RG al núcleo. Tampoco la gravedad del asma ni la intolerancia a los AINE parecen influiren la translocación del RG. La asociación entre atopía y la alteración en la translocación del RG mereceestudiarse más profundamente(AU)

Background: Nasal polyposis (NP) is treated with topical glucocorticoids (GC). Some patients requireendoscopic nasal surgery because GC treatment is ineffective. To exert its function, the GC needs to bindwith the GC receptor (GR) and the GC-GR complex moves to the cell nucleus. Our aim was to establishwhether the poor response to GC is due to an alteration in the translocation of the GR to the nucleus.Methods: We performed nasal fibroblast cell cultures from seven healthy controls and 12 patients withNP and asthma. Fibroblasts were incubated with budesonide or dexamethasone (10-7M) for differenttimes (30 min to 4 h) and GR translocation was analyzed by immunocytochemistry.Results: Both GC induced GR translocation in every group, doubling its concentration in the cellnucleus (30 min) compared to baseline. There were no differences in GR translocation between controlsand patients, nor differences related to the severity of asthma or intolerance to non-steroidal antiinflammatory drugs (NSAIDs). Atopic subjects showed a decrease in GR translocation with budesonide (1 h, 3 h and 4 h, P < 0.05) and dexamethasone (30 min and 2 h, P < .05). Conclusions: The insensitivity to GC treatment does not appear to be due to an alteration in GR translocationto the nucleus. Neither does the severity of asthma or intolerance to NSAIDs appear to alterGR translocation. The association between atopy and the alteration in GR function deserves furtherinvestigation(AU)

Function of nuclear transport factor 2 and Ran in the 20E signal transduction pathway in the cotton bollworm, Helicoverpa armigera.

BACKGROUND: Nuclear transport factor 2 and small GTPase Ran participate in the nucleo-cytoplasm transport of macromolecules, but their function in the 20-hydroxyecdysone (20E) signal transduction pathway are not well known. RESULTS: A 703 bp encoding Ntf2 and a 1233 bp encoding Ran full-length cDNAs were cloned from Helicoverpa armigera, and named Ha-Ntf2 and Ha-Ran, respectively. Northern blot and immunoblotting revealed that Ha-Ntf2 had an obviously higher expression levels in the head-thorax and integument of the metamorphically committed larvae. In contrast, the expression of Ha-Ran did not show obvious variation at various developmental stages in four tissues by immunoblotting analysis, except in the midgut, which showed increased expression from 5th-36 h (molting) to 6th-48 h. Both expressions of Ha-Ntf2 and Ha-Ran could be upregulated by 20E in vitro. Immunohistochemistry revealed that Ha-Ntf2 and Ha-Ran were primarily localized in the nucleus of various tissues. Protein binding assay and co-immunoprecipitation indicated that Ha-Ntf2 and Ha-Ran can combine with each other in vitro and in vivo. Knock down of Ha-Ntf2 or Ha-Ran by RNAi resulted in the suppression of other 20E regulated genes including EcR-B1, USP1, E75B, BR-CZ2, HHR3 and Ha-eIF5c. In addition, the knockdown of Ha-Ntf2 resulted in Ha-Ran being prevented in the cytoplasm. The nuclear location of the ecdysone receptor b1 (EcR-B1) was also blocked after the knockdown of Ha-Ntf2 and Ha-Ran. CONCLUSION: These evidences suggested that Ha-Ntf2 and Ha-Ran participated in the 20E signal transduction pathway by regulating the location of EcR-B1.

Coassociation of estrogen receptor and p160 proteins predicts resistance to endocrine treatment; SRC-1 is an independent predictor of breast cancer recurrence.

PURPOSE: This study investigates the role of the p160 coactivators AIB1 and SRC-1 independently, and their interactions with the estrogen receptor, in the development of resistance to endocrine treatments. EXPERIMENTAL DESIGN: The expression of the p160s and the estrogen receptor, and their interactions, was analyzed by immunohistochemistry and quantitative coassociation immunofluorescent microscopy, using cell lines, primary breast tumor cell cultures, and a tissue microarray with breast cancer samples from 560 patients. RESULTS: Coassociation of the p160s and estrogen receptor alpha was increased in the LY2 endocrine-resistant cell line following treatment with tamoxifen in comparison with endocrine-sensitive MCF-7 cells. In primary cultures, there was an increase in association of the coactivators with estrogen receptor alpha following estrogen treatment but dissociation was evident with tamoxifen. Immunohistochemical staining of the tissue microarray revealed that SRC-1 was a strong predictor of reduced disease-free survival (DFS), both in patients receiving adjuvant tamoxifen treatment and untreated patients (P < 0.0001 and P = 0.0111, respectively). SRC-1 was assigned a hazard ratio of 2.12 using a Cox proportional hazards model. Endocrine-treated patients who coexpressed AIB1 with human epidermal growth factor receptor 2 had a significantly shorter DFS compared with all other patients (P = 0.03). Quantitative coassociation analysis in the patient tissue microarray revealed significantly stronger colocalization of AIB1 and SRC-1 with estrogen receptor alpha in patients who have relapsed in comparison with those patients who did not recur (P = 0.026 and P = 0.00001, respectively). CONCLUSIONS: SRC-1 is a strong independent predictor of reduced DFS, whereas the interactions of the p160 proteins with estrogen receptor alpha can predict the response of patients to endocrine treatment.

Cohesin subunit SMC1 associates with mitotic microtubules at the spindle pole.

Accurate mitotic chromosome segregation depends on the formation of a microtubule-based bipolar spindle apparatus. We report that the cohesin subunit structural maintenance of chromosomes subunit 1 (SMC1) is recruited to microtubule-bound RNA export factor 1 (Rae1) at the mitotic spindle pole. We locate the Rae1-binding site to a 21-residue-long region, SMC1(947-967) and provide several lines of evidence that phosphorylation of Ser(957) and Ser(966) of SMC1 stimulates binding to Rae1. Imbalances in these assembly pathways caused formation of multipolar spindles. Our data suggest that cohesin's known bundling function for chromatids in mitotic and interphase cells extends to microtubules at the spindle pole.

Imaging and characterizing influenza A virus mRNA transport in living cells.

The mechanisms of influenza A virus mRNA intracellular transport are still not clearly understood. Here, we visualized the distribution and transport of influenza A virus mRNA in living cells using molecular beacon (MB) technology. Confocal-FRAP measurements determined that the transport of influenza A virus intronless mRNA, in both nucleus and cytoplasm, was energy dependent, being similar to that of Poly(A)(+) RNA. Drug inhibition studies in living cells revealed that the export of influenza A virus mRNA is independent of the CRM1 pathway, while the function of RNA polymerase II (RNAP-II) may be needed. In addition, viral NS1 protein and cellular TAP protein were found associated with influenza A virus mRNA in the cell nucleus. These findings characterize influenza A virus mRNA transport in living cells and suggest that influenza A virus mRNA may be exported from the nucleus by the cellular TAP/p15 pathway with NS1 protein and RNAP-II participation.

Nuclear RNA export factor 7 is localized in processing bodies and neuronal RNA granules through interactions with shuttling hnRNPs.

The nuclear RNA export factor (NXF) family proteins have been implicated in various aspects of post-transcriptional gene expression. This study shows that mouse NXF7 exhibits heterologous localization, i.e. NXF7 associates with translating ribosomes, stress granules (SGs) and processing bodies (P-bodies), the latter two of which are believed to be cytoplasmic sites of storage, degradation and/or sorting of mRNAs. By yeast two-hybrid screening, a series of heterogeneous nuclear ribonucleoproteins (hnRNPs) were identified as possible binding partners for NXF7. Among them, hnRNP A3, which is believed to be involved in translational control and/or cytoplasmic localization of certain mRNAs, formed a stable complex with NXF7 in vitro. Although hnRNP A3 was not associated with translating ribosomes, it was co-localized with NXF7 in P-bodies. After exposing to oxidative stress, NXF7 trans-localized to SGs, whereas hnRNP A3 did not. In differentiated neuroblastoma Neuro2a cells, NXF7 was co-localized with hnRNP A3 in cell body and neurites. The amino terminal half of NXF7, which was required for stable complex formation with hnRNP A3, coincided with the region required for localization in both P-bodies and neuronal RNA granules. These findings suggest that NXF7 plays a role in sorting, transport and/or storage of mRNAs through interactions with hnRNP A3.

Comprehensive analysis of diverse ribonucleoprotein complexes.

The study of the dynamic interactome of cellular ribonucleoprotein (RNP) particles has been hampered by severe methodological limitations. In particular, the affinity purification of intact RNP complexes from cell lysates suffers from RNA degradation, loss of interacting macromolecules and poor overall yields. Here we describe a rapid affinity-purification method for efficient isolation of the subcomplexes that dynamically organize different RNP biogenesis pathways in Saccharomyces cerevisiae. Our method overcomes many of the previous limitations to produce large RNP interactomes with almost no contamination.

Identification and characterization of the mouse nuclear export factor (Nxf) family members.

TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm.

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake.

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)(+) mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)(+) mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.

ICP27 recruits Aly/REF but not TAP/NXF1 to herpes simplex virus type 1 transcription sites although TAP/NXF1 is required for ICP27 export.

Herpes simplex virus type 1 (HSV-1) protein ICP27 interacts with the cellular export adaptor protein Aly/REF, which is part of the exon junction complex implicated in cellular mRNA export. We previously reported that Aly/REF was no longer associated with splicing factor SC35 sites during infection but instead colocalized with ICP27 in distinct structures. Here we show that these structures colocalize with ICP4 and are sites of HSV-1 transcription. ICP27 mutants with lesions in the region required for the interaction with Aly/REF failed to recruit Aly/REF to viral transcription sites; however, ICP27 export to the cytoplasm was unimpaired, indicating that the interaction of ICP27 with Aly/REF is not required for ICP27 shuttling. ICP27 has also been shown to interact with the cellular mRNA export receptor TAP/NXF1. We report that ICP27 interacts directly with TAP/NXF1 and does not require Aly/REF to bridge the interaction. The C terminus of ICP27 is required; however, the N-terminal leucine-rich region also contributes to the interaction of ICP27 with TAP/NXF1. In contrast to the results found for Aly/REF, mutants that failed to interact with TAP/NXF1 were not exported to the cytoplasm, and TAP/NXF1 was not recruited to sites of HSV-1 transcription. Therefore, the interaction of ICP27 with TAP/NXF1 occurs after ICP27 leaves viral transcription sites. We conclude that ICP27 and the viral RNAs to which it binds are exported via the TAP/NXF1 export receptor.