Pin Ouyang

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

Summary 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.

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. J. Cell. Biochem. 109: 1274–1285, 2010.

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 β-galactosidase (SA-β-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.

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.

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.

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

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.

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.