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Summary Most protein-coding sequences (CDSs) are predicted sequences based on criteria such as a size sufficient to encode a product of at least 100 amino acids and with translation starting at an AUG initiation codon. However, recent studies based on ribosome profiling and mass spectrometry have shown that several RNAs annotated as long as noncoding RNAs are actually translated to generate polypeptides of fewer than 100 amino acids and that many proteins are translated from near-cognate initiation codons such as CUG and GUG. Furthermore, studies of genetically engineered mouse models have revealed that such polypeptides and proteins contribute to diverse physiological processes. In this review, we describe the latest methods for the identification of unannotated CDSs and provide examples of their physiological functions.

An anion-exchange electrolyte membrane, QPAF(C6)-4, polymerized with hydrophobic 1,4'-bis(3-chlorophenyl)perfluorohexane and hydrophilic (6,6'-(2,7-dichloro-9

A 2,2′-tert-butyl-5,9-6′,8′-cyclooctameric pyrenylene ([8]CP) was synthesized by a one-pot Suzuki–Miyaura cross-coupling reaction from two kinds of bifunctionalized monomers, as a rare example of a cyclic octamer. The octameric molecular structure of [8]CP was revealed by single-crystal X-ray diffraction analysis.

The versatility of the synthesis of sulfonated aromatic polymers using NiBr2 was investigated. The copolymerization of sulfo-dichlorobenzene protected with a neopentyl group was carried out with ch...

Novel anion conductive aromatic copolymers containing hexafluoroisopropylidene groups as the hydrophobic component and fluorenyl groups substituted with pendant hexyltrimethylammonium groups as the...

Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal differentiation–induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL., Author summary Although, by definition, long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins, recent studies have shown that some lncRNAs actually contain small open reading frames (ORFs) that are translated. Although TINCR (terminal differentiation–induced ncRNA) was originally identified as a lncRNA that contributes to keratinocyte differentiation, recent mass spectrometry–based analysis has suggested that TINCR is translated. Translation of the ORF within the TINCR lncRNA was validated by generating mice that harbor an in-frame insertion of the FLAG epitope tag sequence at the COOH-terminus of the ORF. The TINCR-encoded protein contains a ubiquitin-like (Ubl) domain and was designated TUBL (TINCR-encoded ubiquitin-like protein). TUBL accelerated cell cycle progression in keratinocytes, and TUBL-deficient mice manifested delayed wound healing after injury with a biopsy punch as a result of attenuated keratinocyte proliferation. TUBL plays a key role at the protein level in the maintenance of skin homeostasis after injury through promotion of keratinocyte proliferation.

Substituted acene derivatives are regarded as promising materials for organic electronic devices such as organic light-emitting diodes (OLEDs). In particular, anthracene derivatives are known to exhibit good fluorescence property, with the air stability and solubility in common organic solvents expected to give advantages for solution-processed device fabrication. In this study, a series of bistriisopropylsilyl(TIPS)ethynyl anthracene derivatives with azaacene-containing iptycene wings have been synthesized by using condensation reactions. Effects of size of azaacenes on optical properties and packing structures were investigated. UV/Vis absorption and fluorescence spectra indicate that the π-elongation of iptycene units has small effects on the overall π-system, which is also supported by electrochemical measurements. Secondly, single-crystal X-ray analysis implies that the molecules likely have interactions with the iptycene units of adjacent molecules, while the iptycene wings and TIPSethynyl groups can prevent the central anthracene unit from undesirable non-radiative energy loss. Finally, the most emissive derivative was used as a dopant for solution-processed OLEDs, showing obvious electroluminescence with a luminance of over 920 cd m-2 .

Thermal equilibration between singlet and triplet excited states achieves high-performance pure organic light-emitting diodes., In any complex molecular system, electronic excited states with different spin multiplicities can be described via a simple statistical thermodynamic formalism if the states are in thermal equilibrium. However, this ideal situation has hitherto been infeasible for efficient fluorescent organic molecules. Here, we report a highly emissive metal-free purely organic fluorophore that enables thermal equilibration between singlet and triplet excited states. The key to this unconventional excitonic behavior is the exceptionally fast spin-flipping reverse intersystem crossing from the triplet to singlet excited states with a rate constant exceeding 108 per second, which is considerably higher than that of radiative decay (fluorescence) from the singlet excited state. The present fluorophoric system exhibits an emission lifetime as short as 750 nanoseconds and, therefore, allows organic light-emitting diodes to demonstrate external electroluminescence quantum efficiency exceeding 20% even at a practical high luminance of more than 10,000 candelas per square meter.

Although the mammalian intestinal epithelium manifests robust regenerative capacity after various cytotoxic injuries, the underlying mechanism has remained unclear. Here we identify the cyclin-dependent kinase inhibitor p57 as a specific marker for a quiescent cell population located around the +4 position of intestinal crypts. Lineage tracing reveals that the p57+ cells serve as enteroendocrine/tuft cell precursors under normal conditions but dedifferentiate and act as facultative stem cells to support regeneration after injury. Single-cell transcriptomics analysis shows that the p57+ cells undergo a dynamic reprogramming process after injury that is characterized by fetal-like conversion and metaplasia-like transformation. Population-level analysis also detects such spatiotemporal reprogramming widely in other differentiated cell types. In intestinal adenoma, p57+ cells manifest homeostatic stem cell activity, in the context of constitutively activated spatiotemporal reprogramming. Our results highlight a pronounced plasticity of the intestinal epithelium that supports maintenance of tissue integrity in normal and neoplastic contexts.

An integrative understanding of nuclear events including transcription in normal and cancer cells requires comprehensive and quantitative measurement of protein dynamics that underlie such events. However, the low abundance of most nuclear proteins hampers their detailed functional characterization. We have now comprehensively quantified the abundance of nuclear proteins with the use of proteomics approaches in both normal and transformed human diploid fibroblasts. We found that subunits of the 26S proteasome complex were markedly down-regulated in the nuclear fraction of the transformed cells compared with that of the wild-type cells. The intranuclear proteasome abundance appeared to be inversely related to the rate of cell cycle progression, with restraint of the cell cycle being associated with an increase in the amount of proteasome subunits in the nucleus, suggesting that the nuclear proteasome content is dependent on the cell cycle. Furthermore, chromatin enrichment for proteomics (ChEP) analysis revealed enrichment of the proteasome in the chromatin fraction of quiescent cells and its apparent dissociation from chromatin in transformed cells. Our results thus suggest that translocation of the nuclear proteasome to chromatin may play an important role in control of the cell cycle and oncogenesis through regulation of chromatin-associated transcription factors.

Although the definition of a noncoding RNA (ncRNA) is an RNA molecule that does not encode a protein, recent evidence has revealed that some ncRNAs are indeed translated to give rise to small polypeptides (usually containing fewer than 100 amino acids). Despite their small size, however, these peptides are often biologically relevant in that they are required for a variety of cellular processes. In this review, we summarize the production and functions of peptides that have been recently identified as translation products of putative ncRNAs.Key words: long noncoding RNA (lncRNA), circular RNA (circRNA), primary miRNA (pri-miRNA), translation, peptide.

A large 1,3-phenylene-bridged hexameric naphthalene wheel N6 and a heptameric wheel N7 were synthesized simply by Suzuki–Miyaura coupling via one-pot reaction from monomers. We could control the distribution of N6 and N7 via the reaction conditions. The hexameric wheel structure was revealed by X-ray diffraction analysis. The wheel N6 exhibited C60 encapsulation ability in the solid state, which was also confirmed by single crystal X-ray analysis.

Air-stable organic semiconductors based on tetrathiafuluvalene (TTF) were developed by synthesising a series of dinaphthotetrathiafulvalene bisimides (DNTTF-Im) using electron-donating TTF, π-extended naphthalene, and electron-withdrawing imide. Electron-spin-resonance spectroscopy and X-ray single-crystal structure analysis of aryl-substituted DNTTF-Im radical cations confirmed that localisation of the spin resides on the electron-donating TTF moiety. The organic field-effect transistor properties derived from the use of highly crystalline n-butyl (C4) and n-hexyl(C6)-substituted DNTTF-Im were assessed. The hole carrier mobility of C6-DNTTF-Im was improved from 3.7×10−3 cm2 V−1 s−1 to 0.30 cm2 V−1 s−1 in ambient conditions. This is attributed to the raise of the substrate temperature from 25 °C to 200 °C during sublimation. The XRD and microscopy analysis suggested that increasing the substrate temperature accelerates the end-on packing resulting in larger grains suitable for hole charge transport parallel to the substrate.

The structure and electronic features of neutral and positively charged pyrene and perylene derivatives were explored. The radical cation of 1,3,6,8-tetraarylpyrene 1 was examined by ESR, UV-vis-NIR spectroscopy and theoretical calculations. The addition of 2 equiv of oxidant to 1 resulted in the formation of dication 12+. The single-crystal X-ray structure of 12+ proved that the aromatic part relocates from biphenyl unit to naphthyl unit upon 2e- oxidation of 1. We have also investigated the oxidation processes of 3,9-diarylperylene 2 and 3,10-diarylperylene 3. The radical cations of 2・+ and 3・+ showed ESR signals and the spin densities were proven to delocalize at 3,4,9,10-positions. In the case of doubly charged 3,9-diarylperylene, we could find the anthracene structure in the core, while the phenanthrene skeleton appeared in two-electron oxidized 3,10-diarylperylene. Finally we validated this phenomenon to apply for the higher analogue terrylene, discovering its large aromaticity relocation upon the 2e- oxidation.

Although long non-coding RNAs (lncRNAs) are non-protein-coding transcripts by definition, recent studies have shown that a fraction of putative small open reading frames within lncRNAs are translated. However, the biological significance of these hidden polypeptides is still unclear. Here we identify and functionally characterize a novel polypeptide encoded by the lncRNA LINC00961. This polypeptide is conserved between human and mouse, is localized to the late endosome/lysosome and interacts with the lysosomal v-ATPase to negatively regulate mTORC1 activation. This regulation of mTORC1 is specific to activation of mTORC1 by amino acid stimulation, rather than by growth factors. Hence, we termed this polypeptide â small regulatory polypeptide of amino acid response' (SPAR). We show that the SPAR-encoding lncRNA is highly expressed in a subset of tissues and use CRISPR/Cas9 engineering to develop a SPAR-polypeptide-specific knockout mouse while maintaining expression of the host lncRNA. We find that the SPAR-encoding lncRNA is downregulated in skeletal muscle upon acute injury, and using this in vivo model we establish that SPAR downregulation enables efficient activation of mTORC1 and promotes muscle regeneration. Our data provide a mechanism by which mTORC1 activation may be finely regulated in a tissue-specific manner in response to injury, and a paradigm by which lncRNAs encoding small polypeptides can modulate general biological pathways and processes to facilitate tissue-specific requirements, consistent with their restricted and highly regulated expression profile.

A new series of partially fluorinated copolymers with varying alkyl side chain length (C3, C6 and C9) and piperidinium head groups have been synthesized and characterized in detail in an effort to improve membrane properties for alkaline fuel cell applications. The copolymers (QPAF4-Cx-pip) provided thin and bendable membranes by solution casting, and achieved high hydroxide ion conductivity up to 97 mS cm−1 in water at 80 °C. Membrane properties such as water absorbability, conductivity, and mechanical properties were tunable with the side chain length. The copolymer main chain and the piperidinium groups were both alkaline stable and the membranes retained high conductivity in 4 M KOH at 80 °C for as long as 1000 h, however, conductivity was lost in 8 M KOH due to Hofmann degradation of the side chain. QPAF4-C3-pip copolymer with the best-balanced properties as anion exchange membrane functioned well in a hydrogen/oxygen alkaline fuel cell to achieve 226 mW cm−2 peak power density at 502 mA cm−2 current density under fully humidified conditions with no back pressure.

Chromodomain helicase DNA-binding protein 8 (CHD8) is an ATP-dependent chromatin-remodeling factor that is encoded by the most frequently mutated gene in individuals with autism spectrum disorder. CHD8 is expressed not only in neural tissues but also in many other organs; however, its functions are largely unknown. Here, we show that CHD8 is highly expressed in and maintains the stemness of hematopoietic stem cells (HSCs). Conditional deletion of Chd8 specifically in mouse bone marrow induces cell cycle arrest, apoptosis, and a differentiation block in HSCs in association with upregulation of the expression of p53 target genes. A colony formation assay and bone marrow transplantation reveal that CHD8 deficiency also compromises the stemness of HSCs. Furthermore, additional ablation of p53 rescues the impaired stem cell function and differentiation block of CHD8-deficient HSCs. Our results thus suggest that the CHD8-p53 axis plays a key role in regulation of the stemness and differentiation of HSCs.

Ion channel proteins play important roles in various cell functions, making them attractive drug targets. Artificial lipid bilayer recording is a technique used to measure the ion transport activities of channel proteins with high sensitivity and accuracy. However, the measurement efficiency is low. In order to improve the efficiency, we developed a method that allows us to form bilayers on a hydrogel bead and record channel currents promptly. We tested our system by measuring the activities of various types of channels, including gramicidin, alamethicin, &alpha, hemolysin, a voltage-dependent anion channel 1 (VDAC1), a voltage- and calcium-activated large conductance potassium channel (BK channel), and a potassium channel from Streptomyces lividans (KcsA channel). We confirmed the ability for enhanced measurement efficiency and measurement system miniaturizion.

1. Introduction Polymer electrolyte fuel cells are expected as next-generation energy devices because of their high energy conversion efficiency and no carbon dioxide emission. However, expensive noble metals such as platinum are used for the catalyst in the commercialized proton exchange membrane fuel cells. To solve this problem, anion exchange membrane fuel cells (AEMFCs) operable with non-noble metal catalysts have been attracting attentions. The low ionic conductivity and chemical instability of the anion exchange membranes (AEMs), however, are barriers to the practical use of AEMFCs. In our laboratory, we have been developing new AEMs with fluorinated hydrophobic groups, such as BAF-QAF1) and QPAF(C6)-42,3) (Fig. 1) to overcome the problems. QPAF(C6)-4 showed a high conductivity and a high mechanical strength but the thermal stability was insufficient. We have newly synthesized QPAF(C4)-4 with shorter perfluoroalkylene chains than those of QPAF(C6)-4 to tune the interpolymer interaction between the hydrophobic chains. The membrane structures and the properties of two membranes, QPAF(C6)-4 and QPAF(C4)-4, are compared. 2. Experimental QPAF(C6)-4 ionomers were synthesized, and the membranes were formed as previously reported.2,3) QPAF(C4)-4 ionomers were synthesized in the same manner as QPAF(C6)-42) except PAF(C4) monomers were used instead of PAF(C6) monomers. Transmission electron microscopy (TEM) and small-angle X-ray scattering (SAXS) were used to understand the arrangements of the hydrophobic/hydrophilic clusters in membranes. To observe the surface morphology and visualize the current images on the film surfaces, current-sensing atomic force microscopy (CS-AFM) was used.4,5) The water uptake and conductivity were measured in water, and the dynamic mechanical analysis (DMA) was conducted. The storage and loss elastic modules were also measured at different temperatures. 3. Results and discussion By TEM, hydrophilic clusters with a diameter of ca. 2 nm were confirmed in both membranes in vacuum (Fig. 2). In situ SAXS measurements under humidified nitrogen gas revealed the existence of periodic structures with the distance more than two times larger than those obtained between the hydrophilic clusters observed by TEM. The rate of the increase in periodicity was larger for QPAF(C4)-4 when the relative humidity was increased. The QPAF(C4)-4 membrane showed a higher water uptake than that of QPAF(C6)-4, but the conductivity was lower. This decrease in conductivity for QPAF(C4)-4, caused by an excessive film swelling (Fig. 3) confirmed by SAXS, may be related with the lower hydrophobicity with the shorter perfluoroalkylene chains. In DMA curves, QPAF(C4)-4 film retained high storage and loss modules up to ca. 80 ˚C, 15 ˚C higher than those of QPAF(C6)-4 (Fig. 4). By the stress-strain measurements, QPAF(C4)-4 showed a lower strain and a higher maximum stress than QPAF(C6)-4 (Fig. 5). The mechanical improvements of QPAF(C4)-4 may be explained by the improved packing between benzene rings due to the shorter perfluoroalkylene chains in the hydrophobic structure. Other properties are discussed based on the chemical structures (Fig. 1) and the three-dimensional conformations of the membranes. References Kimura, A. Matsumoto, J. Inukai, and K. Miyatake, ACS Appl. Energy Mater., 3, 469 (2020). Ono, J. Miyake, S. Shimada, M. Uchida, and K. Miyatake J. Mater. Chem. A, 43, 21779 (2015). Miyake and K. Miyatake, Sustain. Energy Fuels, 3, 1916 (2019). Hara, T. Kimura, T. Nakamura, M. Shimada, H. Ono, S. Shimada, K. Miyatake, M. Uchida, J. Inukai, and M. Watanabe, Langmuir, 32, 9557 (2016). Kimura, R. Akiyama, K. Miyatake, and J. Inukai, J. Power Sources, 375, 397 (2018). Figure 1

circRNAs arise from back splicing events during mRNA processing, and when deregulated can play an active role in cancer. Here we characterize a new circRNA (circPOK) encoded by the Zbtb7a gene (also kown as POKEMON, LRF) in the context of mesenchymal tumor progression. circPOK functions as a non-coding proto-oncogenic RNA independently and antithetically to its linear transcript counterpart, which acts as a tumor suppressor by encoding the Pokemon transcription factor. We find that circPOK regulates pro-proliferative and pro-angiogenic factors by co-activation of the ILF2/3 complex. Importantly, the expression of Pokemon protein and circRNA is aberrantly uncoupled in cancer through differential post-transcriptional regulation. Thus, we identify a novel type of genetic unit, the iRegulon, that yields biochemically distinct RNA products, circular and linear, with diverse and antithetical functions. Our findings further expand the cellular repertoire towards the control of normal biological outputs, while aberrant expression of such components may underlie disease pathogenesis including cancer.

An oxidative ring-closure reaction of a tetranaphthylpyrene derivative led to the synthesis of a 56 all-carbon conjugated tetrabenzoperipentacene. In the single-crystal X-ray structure, three molecules make a triple-layered cluster by π-stacking, wherein each layer rotates by 120°, and is thus considered a petit β-graphite. As for the optical properties, the Stokes shift is extremely small (10 cm(-1) ), thus indicating its remarkably rigid framework. The tetrabenzoperipentacene exhibits reversible five-electron oxidation waves in cyclic voltammetry, and is regarded as a counterpart to the fullerene C60 in terms of stable multicharge-storage nanocarbon materials.

Proper development of T cells depends on lineage-specific regulators controlled transcriptionally and posttranslationally to ensure precise levels at appropriate times. Conditional inactivation of F-box protein Fbw7 in mouse T-cell development resulted in reduced thymic CD4 single-positive (SP) and splenic CD4(+) and CD8(+) cell proportions. Fbw7 deficiency skewed CD8 SP lineage differentiation, which exhibited a higher incidence of apoptosis. Similar perturbations during development of CD8-positive cells were reported with transgenic mice, which enforced GATA3 (T-cell differentiation regulator) expression throughout T-cell development. We observed augmented GATA3 in CD4/CD8 double negative (DN) stage 4, CD4 SP, and CD8 SP lineages in Fbw7-deficient thymocytes. Using overexpressed proteins in cultured cells, we demonstrated that Fbw7 bound to, ubiquitylated, and destabilized GATA3. Two Cdc4 phosphodegron (CPD) candidate sequences, consensus Fbw7 recognition domains, were identified in GATA3, and phosphorylation of Thr-156 in CPD was required for Fbw7-mediated ubiquitylation and degradation. Phosphorylation of GATA3 Thr-156 was detected in mouse thymocytes, and cyclin-dependent kinase 2 (CDK2) was identified as a respondent for phosphorylation at Thr-156. These observations suggest that Fbw7-mediated GATA3 regulation with CDK2-mediated phosphorylation of CPD contributes to the precise differentiation of T-cell lineages.

The expansive dimension of non-coding genes is by now a well-recognized feature of eukaryotes genomes. Over the past decades, in vitro functional studies and in vivo manipulation of non-coding genes through Genetically Engineered Mouse Models (GEMMs) have provided compelling evidence that almost every biological phenomenon is regulated, at some level, by non-coding RNA transcripts or by coding RNAs with non-coding functions. In this opinion article, we will discuss how recent discoveries in the field of non-coding RNAs are contributing to advance our understanding of evolution and organismal complexity and its relevance to human diseases.

We prepared perylene dications 1(2+) and 2(2+) by using "capped" perylene derivatives, and for the first time, successfully obtained single crystals of a perylene dication 1(2+) that enabled us to perform its structural analysis. We realized that the substituted aryl groups on perylene control the positions of positive charges, thus the remaining electronic system satisfies Clar's sextet rule toward the highest number of localized sextets. Experimental and theoretical evidence proved that Clar's aromatic π-sextet rule could be applied even for the dicationic perylenes in a very simple way.

Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G(0) to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in normal development (although it is thought to be a key player in the response to DNA damage).

Induced pluripotent stem cells (iPSCs) share many biological properties with embryonic stem cells (ESCs), and are generated from somatic cells by expression of some transcription factors such as Oct3/4, Sox2, Klf4 and c-Myc. Among these factors, the abundance of c-Myc is strictly regulated by Fbxw7, a subunit of Skp1-Cul1-F-box protein-type ubiquitin ligase. We have now shown that the expression of Fbxw7 was increased as ESCs differentiated. To investigate the role of Fbxw7 in the ESCs/iPSCs, we examined the impact of Fbxw7 ablation in the efficiency in iPSC generation. The frequency of iPSC generation from mouse embryonic fibroblasts (MEFs) lacking Fbxw7 was markedly greater than that from control MEFs. Depletion of Fbxw7 also resulted in promotion of iPSC generation. Morphology of iPSC clonies from Fbxw7-depleted MEFs appeared more undifferentiated than that from MEFs overexpressing c-Myc. Additional depletion of c-Myc did not abrogate the effect of Fbxw7 depletion, suggesting that c-Myc accumulation is not necessarily required for the increased efficiency in iPSC generation by Fbxw7 ablation. Substrates of Fbxw7 other than c-Myc might therefore play a key role in iPSC generation. These results suggest that transient inhibition of Fbxw7 would be a more promising approach to efficient generation of iPSCs than c-Myc over-expression.

SummaryThe NFκB/Rel family of proteins play critical roles in a variety of cellular processes. Thus, their physiological activation is tightly controlled. Recently, the NFκB2/p100 precursor has been characterized as the fourth IκB type of suppressor for NFκB. However, the molecular mechanism(s) underlying regulated destruction of NFκB2 remains largely unknown. Here, we report that, unlike other IκBs, ubiquitination and destruction of NFκB2 are governed by SCFFbw7 in a GSK3-dependent manner. In Fbw7−/− cells, elevated expression of NFκB2/p100 leads to a subsequent reduction in NFκB signaling pathways and elevated sensitivity to TNFα-induced cell death. Reintroducing wild-type Fbw7, but not disease-derived mutant forms of Fbw7, rescues NFκB activity. Furthermore, T cell-specific depletion of Fbw7 also leads to reduced NFκB activity and perturbed T cell differentiation. Therefore, our work identifies Fbw7 as a physiological E3 ligase controlling NFκB2′s stability. It further implicates that Fbw7 might exert its tumor-suppressor function by regulating NFκB activity.

Next generation sequencing methods have revealed that 70 ∼90% of the human genome is transcribed, with the majority of RNA species consisting of long non-coding RNAs (lncRNAs). While early breakthr...

SummaryQuiescence is required for the maintenance of hematopoietic stem cells (HSCs). Members of the Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) have been implicated in HSC quiescence, but loss of p21 or p27 in mice affects HSC quiescence or functionality only under conditions of stress. Although p57 is the most abundant family member in quiescent HSCs, its role has remained uncharacterized. Here we show a severe defect in the self-renewal capacity of p57-deficient HSCs and a reduction of the proportion of the cells in G0 phase. Additional ablation of p21 in a p57-null background resulted in a further decrease in the colony-forming activity of HSCs. Moreover, the HSC abnormalities of p57-deficient mice were corrected by knocking in the p27 gene at the p57 locus. Our results therefore suggest that, among Cip/Kip family CDK inhibitors, p57 plays a predominant role in the quiescence and maintenance of adult HSCs.

Control of the growth and differentiation of neural stem cells is fundamental to brain development and is largely dependent on the Notch signaling pathway. The mechanism by which the activity of Notch is regulated during brain development has remained unclear, however. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box protein (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of Notch family members. We now show that mice with brain-specific deletion of Fbxw7 (Nestin-Cre/Fbxw7(F/F) mice) die shortly after birth with morphological abnormalities of the brain and the absence of suckling behavior. The maintenance of neural stem cells was sustained in association with the accumulation of Notch1 and Notch3, as well as up-regulation of Notch target genes in the mutant mice. Astrogenesis was also enhanced in the mutant mice in vivo, and the differentiation of neural progenitor cells was skewed toward astrocytes rather than neurons in vitro, with the latter effect being reversed by treatment of the cells with a pharmacological inhibitor of the Notch signaling pathway. Our results thus implicate Fbxw7 as a key regulator of the maintenance and differentiation of neural stem cells in the brain.

Oxidative stress has been implicated in cancer initiation and progression. Fbxw7 (also known as Fbw7, SEL-10, hCdc4, or hAgo) is the F-box protein subunit of an Skp1-Cul1-F-box (SCF)-type ubiquitin ligase complex that plays a central role in the degradation of oncoproteins such as c-Myc, c-Jun, Notch, and cyclin E. Fbxw7 is therefore thought to function as a tumor suppressor, and indeed the Fbxw7 gene is frequently mutated in many human malignancies. The Fbxw7 gene locus encodes three protein isoforms: Fbxw7α, Fbxw7β, and Fbxw7γ. Whereas Fbxw7α and Fbxw7γ are resident in the nucleus, Fbxw7β shows a cytoplasmic distribution suggestive of localization to the endoplasmic reticulum (ER). The specific function of Fbxw7β has remained unknown, however. We now show that Fbxw7β contains a putative transmembrane domain near its NH(2) -terminus, and topological analysis revealed that Fbxw7β is inserted in the ER membrane. Fbxw7β assembled with Skp1, Cul1, and Rbx1 to form an SCF complex, although the efficiency of this process appeared lower than that for Fbxw7α or Fbxw7γ. To explore the physiological role of Fbxw7β, we generated mice specifically lacking this isoform of Fbxw7. Although these animals did not exhibit any apparent abnormalities in development, primary cultures of neurons prepared from the mutant mice were more vulnerable to oxidative stress than were those prepared from wild-type mice. Conversely, overexpression of Fbxw7β rendered cells resistant to oxidative stress, without affecting sensitivity to ER stress or other apoptosis-inducing agents. Our results thus suggest that Fbxw7β contributes to the protection of cells from oxidative stress.

E3 ubiquitin ligase complexes of the SCF type consist of ring-box 1 (Rbx1), cullin 1 (Cul1), S-phase kinase-associated protein 1 (Skp1), and a member of the F-box family of proteins. The identity of the F-box protein determines the substrate specificity of the complex. The F-box family member F-box– and WD repeat domain–containing 7 (Fbxw7; also known as Fbw7, SEL-10, hCdc4, and hAgo) targets for degradation proteins with wide-ranging functions, and uncovering its in vivo role has been difficult, because Fbxw7–/– embryos die in utero. Using two different Cre-loxP systems (Mx1-Cre and Alb-Cre), we generated mice with liver-specific null mutations of Fbxw7. Hepatic ablation of Fbxw7 resulted in hepatomegaly and steatohepatitis, with massive deposition of triglyceride, a phenotype similar to that observed in humans with nonalcoholic steatohepatitis. Both cell proliferation and the abundance of Fbxw7 substrates were increased in the Fbxw7-deficient liver. Long-term Fbxw7 deficiency resulted in marked proliferation of the biliary system and the development of hamartomas. Fbxw7 deficiency also skewed the differentiation of liver stem cells toward the cholangiocyte lineage rather than the hepatocyte lineage in vitro. This bias was corrected by additional loss of the Notch cofactor RBP-J, suggesting that Notch accumulation triggered the abnormal proliferation of the biliary system. Together, our results suggest that Fbxw7 plays key roles, regulating lipogenesis and cell proliferation and differentiation in the liver.

Sterically congested tetraarylpyrenes exhibited emission in both solution and the solid state. The monocationic species of pyrene 1 could be isolated because of the reasonably protected system. The aromaticity of 1 relocates from the biphenyl part to the naphthalene unit upon two-electron oxidation.

Cell proliferation is strictly controlled during differentiation. In T cell development, the cell cycle is normally arrested at the CD4+CD8+ stage, but the mechanism underlying such differentiation-specific exit from the cell cycle has been unclear. Fbxw7 (also known as Fbw7, Sel-10, hCdc4, or hAgo), an F-box protein subunit of an SCF-type ubiquitin ligase complex, induces the degradation of positive regulators of the cell cycle, such as c-Myc, c-Jun, cyclin E, and Notch. FBXW7 is often mutated in a subset of human cancers. We have now achieved conditional inactivation of Fbxw7 in the T cell lineage of mice and found that the cell cycle is not arrested at the CD4+CD8+ stage in the homozygous mutant animals. The mutant mice manifested thymic hyperplasia as a result of c-Myc accumulation and eventually developed thymic lymphoma. In contrast, mature T cells of the mutant mice failed to proliferate in response to mitogenic stimulation and underwent apoptosis in association with accumulation of c-Myc and p53. These latter abnormalities were corrected by deletion of p53. Our results suggest that Fbxw7 regulates the cell cycle in a differentiation-dependent manner, with its loss resulting in c-Myc accumulation that leads to hyperproliferation in immature T cells but to p53-dependent cell-cycle arrest and apoptosis in mature T cells.

Fbxw7 is the F-box protein component of an SCF-type ubiquitin ligase that contributes to the ubiquitin-dependent degradation of cell cycle activators and oncoproteins. Three isoforms (alpha, beta, and gamma) of Fbxw7 are produced from mRNAs with distinct 5' exons. We have now investigated regulation of Fbxw7 expression in mouse tissues. Fbxw7alpha mRNA was present in all tissues examined, whereas Fbxw7beta mRNA was detected only in brain and testis, and Fbxw7gamma mRNA in heart and skeletal muscle. The amount of Fbxw7alpha mRNA was high during quiescence (G0 phase) in mouse embryonic fibroblasts (MEFs) and T cells, but it decreased markedly as these cells entered the cell cycle. The abundance of Fbxw7alpha mRNA was unaffected by cell irradiation or p53 status. In contrast, X-irradiation increased the amount of Fbxw7beta mRNA in wild-type MEFs but not in those from p53-deficient mice, suggesting that radiation-induced up-regulation of p53 leads to production of Fbxw7beta mRNA. Our results thus indicate that expression of Fbxw7 isoforms is differentially regulated in a cell cycle- or p53-dependent manner.

Fbxw7 (also known as Sel-10, hCdc4 or hAgo) is the F-box protein component of a Skp1-Cul1-F-box protein (SCF) ubiquitin ligase. Fbxw7 contributes to the ubiquitin-mediated degradation of cyclin E, c-Myc, Aurora-A, Notch and c-Jun, all of which appear to function as cell-cycle promoters and oncogenic proteins. Loss of Fbxw7 results in elevated expression of its substrates, which may lead to oncogenesis. However, it remains largely unclear which accumulating substrate is most related to cancer development in Fbxw7-mutant cancer cells. In the present study, we examined the abundance of cyclin E, c-Myc and Aurora-A in seven cancer cell lines, which harbor wild-type (three lines) or mutant (four lines) Fbxw7. Although these three substrates accumulated in the Fbxw7-mutant cells, the extent of increase in the expression of these proteins varied in each line. Forced expression of Fbxw7 reduced the levels of cyclin E, c-Myc and Aurora-A in the Fbxw7-mutant cells. In contrast, a decrease in the expression of cyclin E, c-Myc or Aurora-A by RNA interference significantly suppressed the rate of proliferation and anchorage-independent growth of the Fbxw7-mutant cells. These findings thus suggest that the loss of Fbxw7 results in accumulation of cyclin E, c-Myc and Aurora-A, all of which appear to be required for growth promotion of cancer cells. Fbxw7 seems to regulate the levels of multiple targets to suppress cancer development.