Cell cycle expression of FLJ25439, a cytokinesis-associated protein, is mediated by D-box recognition and APC/C-Cdc20 regulated degradation.

The midbody is a transient structure forming out of the central spindle at late telophase. Both the midbody and central spindle have important functions ensuring completion of cytokinesis and defects in this process may lead to genetic diseases, including cancer. Thus, understanding the mechanisms that control cytokinesis during mitosis can reveal the key components taking part in some of the processes that promote accurate cell division. Our previous study showed that overexpression of FLJ25439 causes cytokinesis defect with midbody arrest and induces tetraploids with prolonged cell growth/cell cycle progression (Pan et al., 2015). Here, we extend our investigation with regard to the expression profile/regulation and cellular localization/function of FLJ25439 during mitosis/cytokinesis. Using a monoclonal antibody 2A4 we found that FLJ25439 expression is cell cycle-dependent and subjected to APC/C complex regulation. Furthermore, it is a novel substrate for the APC/C-Cdc20 complex and its degradation is proteasome-dependent through D-box recognition during mitotic exit. Immunofluorescence microscopy showed it is distributed at the central spindle and midbody, two structures considered important for completion of cell division, in telophase and cytokinesis, respectively, during cell cycle progression. Depletion of FLJ25439 expression revealed defects in chromosome alignment/segregation and delayed mitosis/cytokinesis progression. We thus conclude that FLJ25439 is a hitherto undiscovered factor involved in cytokinesis regulation.

Depletion of UXT, a novel TSG101 interaction protein, leads to enhanced CEP55 attenuation through lysosome degradation.

The expression level of CEP55, a centrosome and midbody-associated protein is pivotal for cell cytokinesis and is significantly correlated with tumor stage. Our previous study demonstrated that ectopic expression of TSG101 can decrease androgen receptor expression level through the lysosome degradation pathway. Here, we further extended the investigation of TSG101 in modulating protein levels through lysosomes, and identified ubiquitously expressed transcript (UXT) to be a novel TSG101 interaction partner associated with TSG101-containing cytoplasmic vesicles. We also demonstrated that CEP55 can be recruited to TSG101 cytoplasmic vesicles resulting in downregulation of CEP55 through lysosome degradation. Moreover, UXT depletion promoted TSG101 vesicle-lysosome association and elevated autophagic carrier flux to enhance CEP55 degradation upon TSG101 overexpression. In summary, we identified a novel CEP55 regulation pathway mediated by TSG101 overexpression via lysosome degradation and revealed that UXT plays a role in the late endosome/autophagosome-lysosome fusion event, engaging in TSG101-mediated lysosome degradation.

Ectopically expressed pNO40 suppresses ribosomal RNA synthesis by inhibiting UBF-dependent transcription activation.

Ribosomal RNA (rRNA) production occurs in the nucleolus and is a critical process for ribosome biogenesis which affects protein synthesis capacity and determines the cell growth. Dysregulation of nucleolar homeostasis elicits a nucleolar stress response and is related to disease etiology, indicating that the regulation of nucleolar activity is crucial and tightly coordinated. We previously reported that nucleolar protein pNO40 overexpression mediates SR family splicing factors into the nucleolus and impairs mRNA metabolism, while the function of pNO40 in nucleolar homeostasis is unclear. Here, we demonstrate that overexpression of pNO40 downregulates RNA polymerase I transcription activity, resulting in pre-rRNA synthesis reduction and induces nucleolar segregation, a hallmark of rRNA synthesis inhibition and nucleolar stress response. Moreover, co-immunoprecipitation experiments revealed that ectopically expressed pNO40 interacts with UBF, a master transcription factor involved in pre-initiation complex (PIC) (containing SL-1 complex and RNA polymerase I complex) to activate and promote RNA polymerase I-mediated transcription, but disturbs its rDNA promoter binding ability. Collectively, our results demonstrate the role of pNO40 in rRNA biosynthesis regulation by compromising UBF function in rDNA transcription activation with subsequent rRNA synthesis inhibition.

TSG101 interacts with the androgen receptor and attenuates its expression through the endosome/lysosome pathway.

The androgen receptor (AR) signaling pathway plays a vital role in the normal development and function of the male reproductive organs. Dysregulation of the androgen-AR signaling pathway has been linked to prostate cancer. Here, we demonstrate that tumor susceptibility gene 101 (TSG101) regulates AR expression level via the endosome/lysosome degradation pathway. In LNCaP cells, TSG101 overexpression recruits the AR to TSG101-containing cytoplasmic vesicles resulting in reduced AR protein level and AR transactivation activity downregulation. Immunofluorescence microscopy demonstrated that TSG101-decorated cytoplasmic vesicles are associated with late endosomes/lysosomes and the AR could be found within the Lamp2 positive TSG101 vesicles upon lysosomal protease inhibitor treatment. Furthermore, chloroquine or bafilomycin A1 treatment was able to restore TSG101-mediated AR expression reduction. Based on these data, we conclude that the interaction of the AR and TSG101 leads to AR recruitment to TSG101-containing cytoplasmic vesicles and induces AR-attenuated expression through the late endosome/lysosome degradation pathway.

Bmp5 Regulates Neural Crest Cell Survival and Proliferation via Two Different Signaling Pathways.

Neural crest progenitor cells, which give rise to many ectodermal and mesodermal derivatives, must maintain a delicate balance of apoptosis and proliferation for their final tissue contributions. Here we show that zebrafish bmp5 is expressed in neural crest progenitor cells and that it activates the Smad and Erk signaling pathways to regulate cell survival and proliferation, respectively. Loss-of-function analysis showed that Bmp5 was required for cell survival and this response is mediated by the Smad-Msxb signaling cascade. However, the Bmp5-Smad-Msxb signaling pathway had no effect on cell proliferation. In contrast, Bmp5 was sufficient to induce cell proliferation through the Mek-Erk-Id3 signaling cascade, whereas disruption of this signaling cascade had no effect on cell survival. Taken together, our results demonstrate an important regulatory mechanism for bone morphogenic protein-initiated signal transduction underlying the formation of neural crest progenitors. Stem Cells 2017;35:1003-1014.

Target disruption of ribosomal protein pNO40 accelerates aging and impairs osteogenic differentiation of mesenchymal stem cells.

pNO40/PS1D, a novel nucleolar protein, has been characterized as a core protein of eukaryotic 60S ribosome and at least two splicing forms of pNO40 mRNAs with alternative starting sites have been identified. Through production of knockout (ko) mice with either exon 2 (△E2), exon 4 (△E4) or △E2+E4 targeted disruption we identified a cryptic splicing product occurring in the ko tissues examined which in general cannot be observed in regular RT-PCR detection of wild-type (wt) animals. Among ko animals, △E4 null embryos exhibited prominent senescence-associated ß-galactosidase (SA-ß-gal) staining, a marker for senescent cells, in notochord, forelimbs and heart while bone marrow-derived mesenchymal stem cells (MSCs) from △E4 null mice developed accelerated aging and osteogenic differentiation defects compared to those from wt and other isoform mutant mice. Examination of the causal relationship between pNO40 deficiency and MSC-accelerated aging revealed △E4 null disruption in MSCs elicits high levels of ROS and elevated expression levels of p16 and Rb but not p53. Further analysis with iTraq identified CYP1B1, a component of the cytochrome p450 system, as a potential molecule mediating ROS generation in pNO40 deficient MSCs. We herein established a mouse model of MSC aging through pNO40-targeted depletion and demonstrated the effects of loss of pNO40 on bone homeostasis.

Loss of Pnn expression results in mouse early embryonic lethality and cellular apoptosis through SRSF1-mediated alternative expression of Bcl-xS and ICAD.

Pinin (Pnn), a serine/arginine-rich (SR)-related protein, has been shown to play multiple roles within eukaryotic cells including cell-cell adhesion, cell migration, regulation of gene transcription, mRNA export and alternative splicing. In this study, an attempt to generate mice homozygously deficient in Pnn failed because of early embryonic lethality. To evaluate the effects of loss of Pnn expression on cell survival, RNA interference experiments were performed in MCF-7 cells. Depletion of Pnn resulted in cellular apoptosis and nuclear condensation. In addition, nuclear speckles were disrupted, and expression levels of SR proteins were diminished. RT-PCR analysis showed that alternative splicing patterns of SRSF1 as well as of apoptosis-related genes Bcl-x and ICAD were altered, and expression levels of Bim isoforms were modulated in Pnn-depleted cells. Cellular apoptosis induced by Pnn depletion was rescued by overexpression of SRSF1, which also restored generation of Bcl-xL and functionless ICAD. Pnn expression is, therefore, essential for survival of mouse embryos and the breast carcinoma cell line MCF-7. Moreover, Pnn depletion, modulated by SRSF1, determines cellular apoptosis through activation of the expression of pro-apoptotic Bcl-xS transcripts.

Transgenic mice expressing mutant Pinin exhibit muscular dystrophy, nebulin deficiency and elevated expression of slow-type muscle fiber genes.

Pinin (Pnn) is a nuclear speckle-associated SR-like protein. The N-terminal region of the Pnn protein sequence is highly conserved from mammals to insects, but the C-terminal RS domain-containing region is absent in lower species. The N-terminal coiled-coil domain (CCD) is, therefore, of interest not only from a functional point of view, but also from an evolutionarily standpoint. To explore the biological role of the Pnn CCD in a physiological context, we generated transgenic mice overexpressing Pnn mutant in skeletal muscle. We found that overexpression of the CCD reduces endogenous Pnn expression in cultured cell lines as well as in transgenic skeletal muscle fibers. Pnn mutant mice exhibited reduced body mass and impaired muscle function during development. Mutant skeletal muscles show dystrophic histological features with muscle fibers heavily loaded with centrally located myonuclei. Expression profiling and pathway analysis identified over-representation of genes in gene categories associated with muscle contraction, specifically those related to slow type fiber. In addition nebulin (NEB) expression level is repressed in Pnn mutant skeletal muscle. We conclude that Pnn downregulation in skeletal muscle causes a muscular dystrophic phenotype associated with NEB deficiency and the CCD domain is incapable of replacing full length Pnn in terms of functional capacity.

Centrosomal protein 55 (Cep55) stability is negatively regulated by p53 protein through Polo-like kinase 1 (Plk1).

Centrosomal protein 55 (Cep55), which is localized to the centrosome in interphase cells and recruited to the midbody during cytokinesis, is a regulator required for the completion of cell abscission. Up-regulation of Cep55 and inactivation of p53 occur in the majority of human cancers, raising the possibility of a link between these two genes. In this study we evaluated the role of p53 in Cep55 regulation. We demonstrated that Cep55 expression levels are well correlated with cancer cell growth rate and that p53 is able to negatively regulate Cep55 protein and promoter activity. Down-regulation of expression of Cep55 was accompanied by repression of polo-like kinase 1 (Plk1) levels due to p53 induction. Overexpression of Plk1 and knockdown of p53 expression both enhanced the post-translational protein stability of Cep55. BI 2356, a selective Plk1 inhibitor, however, prevented Cep55 accumulation in p53 knockdown cells while persistently keeping Plk1 levels elevated. Our results, therefore, indicate the existence of a p53-Plk1-Cep55 axis in which p53 negatively regulates expression of Cep55, through Plk1 which, in turn, is a positive regulator of Cep55 protein stability.

Dissection of the role of Pinin in the development of zebrafish posterior pharyngeal cartilages.

Pinin (pnn), a nuclear and desmosome-associated SR-like protein, has been shown to play multiple roles in cell adhesion, transcriptional regulation, pre-mRNA splicing and mRNA export. Because of the embryonic lethality of pnn-deficient mice, here we used the zebrafish system to investigate the functions of pnn. Injection of morpholinos into zebrafish to knockdown pnn resulted in several obvious defective phenotypes, such as short body, bent tail, and an abnormal pigment distribution pattern. Moreover, aberrant blood vessels were formed, and most of the cartilages of pharyngeal arches 3-7 were reduced or absent in pnn morphants. Because most of the defects manifested by pnn morphants were reminiscent of those caused by neural crest-derived malformation, we investigated the effects of pnn deficiency in the development of neural crest cells. Neural crest induction and specification were not hindered in pnn morphants, as revealed by normal expression of early crest gene, sox10. However, the morphants failed to express the pre-chondrogenic gene, sox9a, in cells populating the posterior pharyngeal arches. The reduction of chondrogenic precursors resulted from inhibition of proliferation of neural crest cells, but not from cellular apoptosis or premature differentiation in pnn morphants. These data demonstrate that pnn is essential for the maintenance of subsets of neural crest cells, and that in zebrafish proper cranial neural crest proliferation and differentiation are dependent on pnn expression.

Pnn and SR family proteins are differentially expressed in mouse central nervous system.

Pinin (pnn) is an SR-related protein that is ubiquitously expressed in most cell types and functions in regulating pre-mRNA splicing and mRNA export. Previously, we demonstrated that pnn is expressed in all tissues during mouse embryonic development with highest levels of expression in the central nervous system (CNS). Here we show that pnn and other SR proteins including SC35 are differentially expressed in the adult mouse CNS, displaying cell type-specific distribution patterns. Immunohistochemical analysis of whole-brain sections showed that levels of pnn and SR proteins expression were very low or nonexistent in the corpus callosum and white matter of cerebellum and spinal cord. Double-immunostaining with antibodies specific to neuron or glial cells showed that most astrocytes and microglia expressed neither pnn nor SR proteins. In contrast, oligodendrocytes and neurons expressed moderate and high levels, respectively, of both pnn and SR proteins. These results suggest that astrocytes are unique among cell types of neuroblast origin in terms of expression SR family proteins. Our results pave the way for future studies of the functional roles of pnn and SR family proteins in adults.

Characterization of centrosomal proteins Cep55 and pericentrin in intercellular bridges of mouse testes.

Centrosomal protein 55 (Cep55), located in the centrosome in interphase cells and recruited to the midbody during cytokinesis, is essential for completion of cell abscission. Northern blot previously showed that a high level of Cep55 is predominantly expressed in the testis. In the present study, we examined the spatial and temporal expression patterns of Cep55 during mouse testis maturation. We found that Cep55, together with pericentrin, another centrosomal protein, were localized to the intercellular bridges (IBs) interconnecting spermatogenic cells in a syncytium. The IBs were elaborated as a double ring structure formed by an inner ring decorated by Cep55 or pericentrin and an outer ring of mitotic kinesin-like protein 1 (MKLP1) in the male germ cell in early postnatal stages and adulthood. In addition, Cep55 and pericentrin were also localized to the acrosome region and flagellum neck and middle piece in elongated spermatids, respectively. These results suggest that Cep55 and pericentrin are required for the stable bridge between germ cells during spermatogenesis and spermiogenesis.

SRrp37, a novel splicing regulator located in the nuclear speckles and nucleoli, interacts with SC35 and modulates alternative pre-mRNA splicing in vivo.

We report here the identification and characterization of a novel SR-related protein, referred to as SRrp37, based on its apparent molecular weight and subcellular location. SRrp37 was identified through a yeast two-hybrid screen during the course of searching for proteins interacting with pNO40, a ribosomal 60S core subunit. SRrp37 exhibited two alternative spliced isoforms generated by differential usage of the translation start site with the longer one, SRrp37, initiating at first exon and the shorter, SRrp37-2, starting from exon 2. Three distinct motifs can be discerned in the SRrp37 protein: (1) a serine-arginine (SR) dipeptide enriched domain, (2) a polyserine stretch, and (3) a potential nucleolar localization signal comprising a long array of basic amino acids. SRrp37's message was translated in tissue-specific patterns with both isoforms expressed at comparable levels in tissues showing expression. Indirect immunofluorescence analysis with an anti-SRrp37 antibody, as well as an experiment using myc-tagged proteins, demonstrated that SRrp37 was localized in nucleoli and nuclear speckles. GST pull-down assay showed that SRrp37 interacted physically with SC35. Using adenovirus E1A and chimeric calcitonin/dhfr constructs as splicing reporter minigenes, we found that SRrp37 modulated alternative 5' and 3' splicing in vivo. Together, SRrp37 may participate directly in splicing regulation or indirectly through interaction with SC35. Studies on this novel splicing regulator may provide new information on the intricate splicing machinery as related to the RNA metabolism involving processing of mRNA and rRNA.

Polyglutamine-expanded ataxin-3 causes cerebellar dysfunction of SCA3 transgenic mice by inducing transcriptional dysregulation.

In the present study, we prepared a SCA3 animal model by generating transgenic mice expressing polyglutamine-expanded ataxin-3-Q79. Ataxin-3-Q79 was expressed in brain areas implicated in SCA3 neurodegeneration, including cerebellum, pontine nucleus and substantia nigra. Ataxin-3-Q79 transgenic mice displayed motor dysfunction with an onset age of 5-6 months, and neurological symptoms deteriorated in the following months. A prominent neuronal loss was not found in the cerebellum of 10 to 11-month-old ataxin-3-Q79 mice displaying pronounced ataxic symptoms, suggesting that instead of neuronal demise, ataxin-3-Q79 causes neuronal dysfunction of the cerebellum and resulting ataxia. To test the involvement of transcriptional dysregulation in ataxin-3-Q79-induced cerebellar malfunction, microarray analysis and real-time RT-PCR assays were performed to identify altered cerebellar mRNA expressions of ataxin-3-Q79 mice. Compared to non-transgenic mice or mice expressing wild-type ataxin-3-Q22, 10 to 11-month-old ataxin-3-Q79 mice exhibited downregulated mRNA expressions of proteins involved in glutamatergic neurotransmission, intracellular calcium signaling/mobilization or MAP kinase pathways, GABA(A/B) receptor subunits, heat shock proteins and transcription factor regulating neuronal survival and differentiation. Upregulated expressions of Bax, cyclin D1 and CDK5-p39, which may mediate neuronal death, were also observed in ataxin-3-Q79 transgenic mice. The involvement of transcriptional abnormality in initiating the pathological process of SCA3 was indicated by the finding that 4 to 5-month-old ataxin-3-Q79 mice, which did not display neurological phenotype, exhibited downregulated mRNA levels of genes involved in glutamatergic signaling and signal transduction. Our study suggests that polyglutamine-expanded ataxin-3 causes cerebellar dysfunction and ataxia by disrupting the normal pattern of gene transcriptions.

Ablation of Bax and Bak protects skeletal muscle against pressure-induced injury.

Pressure-induced injury (PI), such as a pressure ulcer, in patients with limited mobility is a healthcare issue worldwide. PI is an injury to skin and its underlying tissue such as skeletal muscle. Muscle compression, composed of mechanical deformation of muscle and external load, leads to localized ischemia and subsequent unloading reperfusion and, hence, a pressure ulcer in bed-bound patients. Although the gross factors involved in PI have been identified, little is known about the exact disease mechanism or its links to apoptosis, autophagy and inflammation. Here, we report that PI is mediated by intrinsic apoptosis and exacerbated by autophagy. Conditional ablation of Bax and Bak activates the Akt-mTOR pathway and Bnip3-mediated mitophagy and preserves mitochondrial contents in compressed muscle. Moreover, we find that the presence/absence of Bax and Bak alters the roles and functions of autophagy in PI. Our results suggest that manipulating apoptosis and autophagy are potential therapeutic targets for treatment and prevention of PI.

Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1.

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.

Ribosomal protein pNO40 mediates nucleolar sequestration of SR family splicing factors and its overexpression impairs mRNA metabolism.

The nucleolus acts as a key stress sensor and responds to changes in cellular growth rate and metabolic activity. In addition to its major role as the site of ribosome biogenesis, high-throughput proteomic analyses of purified nucleoli have highlighted the multi-functional nature of these organelles, and several SR family splicing factors, including SRSF1 and SRSF2, have been detected in human nucleolar proteome analysis. Here we provide evidence that pNO40, a 60s ribosomal protein associated with nucleoli, acts as a mediator for recruitment of SR family splicing factors into nucleoli. As a nucleolar protein, pNO40 was originally identified by yeast two-hybrid analysis as interacting with pnn, an SR-like protein involved in pre-mRNA splicing. To explore its functional interaction with pnn, we characterized the interplay between pNO40 and SR family proteins and demonstrated that pNO40 plays a role in recruiting SR splicing factors into the nucleoli. The targeting of pNO40 to the nucleoli is dependent on its extreme-carboxyl-terminus nuclear localization signals while the sequence at the amino-terminus of pNO40 enables its interaction with pnn. Nucleolar association of SR proteins results in defects in mRNA metabolism leading to global nuclear accumulation of poly(A)+ RNA and splicing defects. Animal studies confirmed aberrant mRNA splicing in transgenic muscles overexpressing pNO40 which displayed histological features of muscular dystrophy. Thus it appears that by pNO40 overexpression, we created mimics of nucleolar association of SR proteins occurring in the presence of transcription inhibitors which induce nucleolar segregation and redistribute SR proteins to the periphery of the nucleolar region. We therefore provide an extra-ribosomal function for pNO40 and, based on our data, it is conceivable that pNO40 may function as a general recruiter for nucleolar association of SR proteins and regulation of its expression may be crucial in cellular homeostasis.

Identification and characterization of a ß-defensin gene involved in the immune defense response of channel catfish, Ictalurus punctatus.

Antimicrobial peptides are small peptides that play important roles in a host's innate immune response. As an important antimicrobial peptide, ß-defensin widely distribute in mammals, insects and plants with broad-spectrum antimicrobial activity. In this study, the ß-defensin gene of the channel catfish, Ictalurus punctatus, was cloned, sequenced, and subjected to a bioinformatic analysis. The ß-defensin gene of the channel catfish contains three exons and two introns, and encodes a precursor peptide consisting of two domains: a signal peptide of 24 amino acid residues and a mature peptide of 43 amino acid residues. The mature peptide is estimated to have a molecular mass of 7.1kDa and a theoretical isoelectric point of 8.21. Channel catfish ß-defensin (ccBD) has six conserved cysteine residues, forming three disulfide bridges at C1-C5, C2-C4, and C3-C6, and a ß-sheet in the predicted three-dimensional structure. A phylogenetic analysis suggests that ccBD belongs to the type 1 ß-defensins. Real-time quantitative PCR showed that channel catfish ß-defensin transcripts are constitutively expressed in various tissues in healthy fish, with highest expression in the skin. The expression of ccBD in vivo increased significantly in the head kidney (2.9-fold), gill (2.2-fold), and skin (6.6-fold) at 48h after bacterial (Edwardsiella ictaluri) challenge. In vitro, lipopolysaccharide (LPS), a bacterial mimic, induced significant changes in ccBD expression in leukocytes from the spleen (3.4-fold) and head kidney (3.9-fold) 24h after stimulation. Chemically synthesized ccBD displayed marked inhibitory activity against a broad range of bacteria. These results suggest that ccBD is involved in the innate antibacterial defenses of the channel catfish.

Spatial and temporal expression profile of pinin during mouse development.

SR and SR-related proteins are splicing regulators involved in embryo development in higher eukaryotes. Pinin (pnn) is a SR-related protein localized both within nucleus (nuclear pnn, N-pnn) and at desmosome of cell-cell adhesion (desmosomal pnn, D-pnn). To investigate the role of N-pnn during mouse embryo development, we examined its expression using Northern blot, real-time RT-PCR, immunostaining, and mRNA in situ hybridization (ISH). On Northern analysis, we found that pnn transcripts display two isoforms due to differential utilization of a polyadenylation site and exhibit tissue variable expression with thymus expressing the highest level of transcript. Analysis of pnn expression in mouse embryos revealed N-pnn expression starts from the two-cell fertilized egg stage and is ubiquitous at all stages of mouse embryo development. ISH and immunofluorescent staining of embryo cryosections showed that during mouse organogenesis N-pnn is highly expressed in the central nervous system. In addition, N-pnn was found to be highly expressed in the cortex region of thymus of E16.5 mouse fetus, while in the hepatic primordium the strongest signals were noted at E13.5 to E14.5 rather than at later developmental stages. Finally, we also determined the subcellular location of N-pnn in photoreceptors of developing retinas by nuclear fractionation and Western blot, because N-pnn displayed a staining pattern reminiscent of cytoplasmic proteins at the microscopic level in developing mouse photoreceptors. Altogether these data provide us with a better understanding of the tissue distribution pattern of N-pnn during mouse development.

FLJ25439, a novel cytokinesis-associated protein, induces tetraploidization and maintains chromosomal stability via enhancing expression of endoplasmic reticulum stress chaperones.

Investigation of the mechanisms leading to aneuploidy and polyploidy is critical to cancer research. Previous studies have provided strong evidence of the importance of tetraploidization as an early step in tumorigenesis. In cancer cells, tetraploid cells may contribute to abnormal mitotic progression, which may be associated with cytokinesis failure. Tetraploidy leads to genomic instability due to centrosome and chromosome over-replication. Until now, the mechanism by which cells maintain tetraploid status has been unknown. Here, we identified a novel D box-containing protein, FLJ25439, which displays a dynamic expression profile during mitosis/cytokinesis with the midbody as the most prominent associated structure. To understand the function of FLJ25439, we established stable cell lines overexpressing FLJ25439. FLJ25439-overexpression cells grew slower and displayed a tetraploid DNA content in comparison with diploid parental cells. They also showed aberrant mitosis and dysregulated expression of p53, pRb and p21, suggesting a defect in cell cycle progression. To explore the molecular mechanisms responsible for FLJ25439-induced tetraploidization, we conducted a comparative analysis of the global protein expression patterns of wild type and overexpressors using proteomics and bioinformatics approaches. Protein category profiling indicated that FLJ25439 is involved in pathways related to anti-apoptosis, protein folding, the cell cycle, and cytoskeleton regulation. Specifically, genotoxic-stress- and ER stress-related chaperone proteins greatly contributed to the FLJ25439 overexpression phenotypes. The results of this study pave the way to our further understanding of the role of this novel cytokinesis-related protein in protecting cells from environmental stress and tetraploid formation.