[Show abstract][Hide abstract] ABSTRACT: The piwi-like 2 (piwil2) gene is widely expressed in tumors and protects cells from apoptosis induced by a variety of stress
stimuli. However, the role of Piwil2 in Fas-mediated apoptosis remains unknown. Here, we present evidence that Piwil2 inhibits
Fas-mediated apoptosis. By a bacterial two-hybrid screening, we identify a new Piwil2-interacting partner, keratin 8 (K8),
a major intermediate filament protein protecting the cell from Fas-mediated apoptosis. Our results show that Piwil2 binds
to K8 and p38 through its PIWI domain and forms a Piwil2/K8/P38 triple protein-protein complex. Thus, Piwil2 increases the
phosphorylation level of K8 Ser-73 and then inhibits ubiquitin-mediated degradation of K8. As a result, the knockdown of Piwil2
increases the Fas protein level at the membrane. In addition to our previous finding that Piwil2 inhibits the expression of
p53 through the Src/STAT3 pathway, here we demonstrate that Piwil2 represses p53 phosphorylation through p38. Our present
study indicates that Piwil2 plays a role in Fas-mediated apoptosis for the first time and also can affect p53 phosphorylation
in tumor cells, revealing a novel mechanism of Piwil2 in apoptosis, and supports that Piwil2 plays an active role in tumorigenesis.
Full-text · Article · Aug 2014 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Znf45l, containing classical C2H2 domains, is a novel member of Zinc finger proteins in zebrafish. In vertebrates, TGF-β signaling plays a critical role in hematopoiesis. Here, we showed that Znf45l is expressed both maternally and zygotically throughout early development. Znf45l-depleted Zebrafish embryos display shorter tails and necrosis with reduced expression of hematopoietic maker genes. Furthermore, we revealed that znf45l locates downstream of TGF-β ligands and maintains normal level of TGF-β receptor type II phosphorylation. In brief, our results indicate that znf45l affects initial hematopoietic development through regulation of TGF-β signaling.
[Show abstract][Hide abstract] ABSTRACT: PIWIL2, called HILI in humans, is a member of the PIWI subfamily. This subfamily has highly conserved PAZ and Piwi domains and is implicated in several critical functions, including embryonic development, stem-cell self-renewal, RNA silencing, and translational control. However, the underlying molecular mechanism remains largely unknown. Transforming growth factor-β (TGF-β) is a secreted multifunctional protein that controls several developmental processes and the pathogenesis of many diseases. TGF-β signaling is activated by phosphorylation of transmembrane serine/threonine kinase receptors, TGF-β type II (TβRII), and type I (TβRI), which are stabilized by Hsp90 via specific interactions with this molecular chaperone. Here, we present evidence that HILI suppresses TGF-β signaling by physically associating with Hsp90 in human embryonic kidney cells (HEK-293). Our research shows that HILI mediates the loss of TGF-β-induced Smad2/3 phosphorylation. We also demonstrate that HILI interacts with Hsp90 to prevent formation of Hsp90-TβR heteromeric complexes, and improves ubiquitination and degradation of TβRs dependent on the ubiquitin E3 ligase Smurf2. This work reveals a critical negative regulation level of TGF-β signaling mediated by HILI (human PIWIL2) by its ability to interact with Hsp90 and promote TβR degradation.
[Show abstract][Hide abstract] ABSTRACT: Bone morphogenetic protein (Bmp) signaling plays a pivotal role in dorsal-ventral (DV) patterning in vertebrate embryos. Piwi proteins are required for germline and stem cell development. Our previous study demonstrated that Zili, zebrafish Piwil2, inhibits transforming growth factor (TGF)-βsignaling by interacting with Smad4, suggesting a role for zili in Bmp signaling. In the present study, zili-MO or zili mRNA was microinjected into one-cell embryos to knock down or elevate the expression of zili to study the role of zili during early zebrafish embryogenesis. Knockdown of zili inhibited the expression of dorsal marker genes, and enhanced that of ventral marker genes. In contrast, overexpression of zili promoted expression of dorsal marker genes, while it inhibited ventral marker genes. These results suggest that zili regulates DV patterning. The influence of zili on the Bmp pathway was further explored. Knockdown of zili resulted in higher expression levels of bmp2b, and bmp4, the Bmp signaling ligands, and reduced expression of chordin (chd), noggin (nog1), and follistatin (fst), which encode BMP antagonists. Meanwhile, overexpression of zili produced opposite effects. In conclusion, our results indicate that zili regulates dorsal-ventral patterning by antagonizing Bmp signaling during early embryogenesis in zebrafish.
No preview · Article · Jun 2011 · ZOOLOGICAL SCIENCE
[Show abstract][Hide abstract] ABSTRACT: Rbmy gene encodes a germ-cell specific nuclear RNA-binding protein and is involved in spermatogenesis. To further investigate the specific events of spermatogenesis in which Rbmy plays a role, the target mRNAs of human RBMY protein were isolated and identified. Through the isolating specific nucleic acids associated with proteins (SNAAP) technique, we isolated twenty potential target genes of human RBMY protein from the human testis in the present study. Some of these target genes play important roles during spermatogenesis and have alternative transcripts in the testis. In this study, we focused on the human- related (never in mitosis gene a) kinase 10 (Nek10) gene, which belongs to the Nek protein kinase subfamily. Nek10 has two transcripts, and the results of RT-PCR and Electrophoretic Mobility Shift Assays (EMSA) show that hRBMY protein can only bind to transcript variant 2 of Nek10 and that hRbmy may take part in alternative splicing of Nek10. Isolation and identification of target genes of hRBMY will be helpful to further investigate the biological function of RBMY in spermatogenesis.
No preview · Article · Feb 2011 · Journal of Reproduction and Development
[Show abstract][Hide abstract] ABSTRACT: Fertilization promoting peptid (FPP) is essential for capacitation and acrosome reaction. The mouse t-complex protein 11 (Tcp11) gene, which encodes the receptor of FPP, plays an important role in fertilization. We had identified three alternative splicing products of its human homologous gene, TCP11, nominated as TCP11a, TCP11b and TCP11c. Their testis-specific expression had been noted, suggesting that TCP11 may play an important role in spermatogenesis and sperm function. In order to explore the function of TCP11, we investigated its expression, subcellular location and binding protein in the sperm. RT-PCR assay shows that all isoforms of TCP11 are present in both human testis and sperm. However, we could only detect the expression of 56-kDa protein, representing TCP11a and TCP11c, but not TCP11b, by western blot analysis. Furthermore, the expression level of 56-kDa TCP11 protein was lower by about threefold in sperm samples containing over 15% of coiled sperms than the level in sperm samples with normal morphology. The coiled sperm, which shows a coiling or bending back of the tail on itself, is associated with infertility. In addition, several TCP11a-binding proteins were isolated using full-length TCP11a as bait. Among them, we focused on outer dense fiber 1 (ODF1), a component of sperm tail outer dense fibers, because outer dense fibers contribute to the distinct morphology and the function of sperm tail. Co-immunoprecipitation assays of sperm cell extracts confirmed that TCP11 protein interacted with ODF1. These results suggest that TCP11 may be responsible for the sperm tail morphology and motility.
No preview · Article · Jan 2011 · The Tohoku Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: Piwi proteins and Piwi-interacting RNAs (piRNAs) have been implicated in transposon control in germline from Drosophila to mammals. To examine the profile of small RNA expression in human cancer cells and explore difference in small RNA transcriptome, small RNA libraries prepared from wildtype, HILI overexpressed and HILI knockdowned Hela cells were sequenced using Solexa technology. piRNAs and other repeat- associated small RNAs were observed in Hela cells. By using in situ hybridization, piR-49322 was localized in the nucleolus and around the periphery of nuclear membrane in Hela cells. Following the overexpression of HILI, the retrotransposon elements LINE1 was significantly repressed, while LINE1-associated small RNAs decreased in abundance. The present study demonstrated that HILI along with piRNAs plays a role in LINE1 suppression in Hela cancer cell line.
[Show abstract][Hide abstract] ABSTRACT: Dnd (dead end) gene encodes an RNA binding protein and is specifically expressed in primordial germ cells (PGCs) as a vertebrate-specific component of the germ plasma throughout embryogenesis. By utilizing a technique of specific nucleic acids associated with proteins (SNAAP), 13 potential target mRNAs of zebrafish Dnd (ZDnd) protein were identified from 8-cell embryo, and 8 target mRNAs have been confirmed using an RT-PCR analysis. Of the target mRNAs, the present study is focused on the regulation of geminin, which is an inhibitor of DNA replication. Using electrophoretic mobility shift assay (EMSA), we demonstrated that ZDND protein bound the 67-nucleotide region from 864 to 931 in the 3'UTR of geminin mRNA, a sequence containing 60.29% of uridine. Results from a dual-luciferase assay in HEK293 cells showed that ZDND increases the translation of geminin. Taken together, the identification of target mRNA for ZDnd will be helpful to further explore the biological function of Dnd in zebrafish germ-line development as well as in cancer cells.
[Show abstract][Hide abstract] ABSTRACT: Piwi proteins are required for germ cell proliferation, differentiation, and germ line stem cell maintenance. In normal tissues,
human and mouse Piwil2 are primarily expressed in testis but widely expressed in tumors. However, the underlying mechanism
remains largely unknown. In vertebrates, transforming growth factor (TGF)-β signaling plays an important role in patterning
embryo and control of cell growth and differentiation. A previous study has shown a role for Zili, a Piwil2 gene in zebrafish,
in germ cells in zebrafish. Here we report that zili functions in patterning the early embryo and inhibits TGF-β signaling. Whole mount expression analysis shows that zili expresses not only in PGCs but also in axis. Ectopic expression of zili causes fusion of the eyes and reduction of mesodermal marker genes expression, suggesting that zili functions to inhibit Nodal signaling and mesoderm formation. Genetic interaction shows that zili inhibits Nodal and bone morphogenetic protein signaling. The results of protein interaction assays identify that Zili binds
to Smad4 via its N-terminal domain and prevents the formation of Smad2/3/4 and Smad1/5/9/4 complexes to antagonize TGF-β signaling.
This work shows that zili plays a role in early embryogenesis beyond germ line as a novel negative regulator of TGF-β signaling, extending the function
of Piwi proteins in vertebrates.
No preview · Article · Feb 2010 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Piwi (P-element-induced wimpy testis) proteins have been shown to play important roles in maintenance of germ line stem cells, germ cell proliferation and differentiation, and control of Piwi-interacting RNAs (PiRNAs). PiRNAs comprise a broad class of small noncoding RNAs that function as an endogenous defense system against transposable elements. Fibroblast growth factor (Fgf) signals, mediated partly by no tail gene (ntl), are responsible for patterning embryo and mesoderm formation. To understand the function of Piwi proteins, we used zebrafish as a model system. In zebrafish, piwi-like 2 gene (piwil2) is also required for germ cell differentiation and meiosis. Here we report that piwil2 knockdown is able to inhibit the expression of fibroblast growth factor 8a (fgf8a). In contrast, injection with piwil2 mRNA enhances fgf8a expression. Knockdown of piwil2 reduces the inductive effect of fgf8a on dorsalized phenotype, in which embryos extend to an oval shape at the end of epiboly stage. Coinjection with fgf8a and piwil2 mRNAs led to more seriously dorsalized phenotype than coinjection with fgf8a mRNA and piwil2-cMO. In addition, knockdown of piwil2 inhibits the inductive effect of fgf8a on ntl, whereas overexpression of piwil2 enhances the inductive effect of fgf8a on ntl. We also demonstrate that piwil2 positively regulates ntl expression at bud stage, while piwil2 negatively regulates ntl expression at 24 hours post-fertilization. Thus, the functional consequences of piwil2 expression vary during early development of zebrafish embryo. Taken together, we suggest that zebrafish piwil2 is a mediator of Fgf signals in gastrula period.
No preview · Article · Jan 2010 · The Tohoku Journal of Experimental Medicine
[Show abstract][Hide abstract] ABSTRACT: ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE). The open reading frame (ORF) encodes a polypeptide of 222 amino acids which shares 75.65% identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are small non-coding RNA molecules that have been identified as potent regulators of gene expression. Recent studies indicate that miRNAs are involved in mammalian spermatogenesis but the mechanism of regulation is largely unknown.
miRNA microarray was employed to compare miRNA expression profiles of testis tissues from immature rhesus monkey (Sample IR), mature rhesus monkey (Sample MR), and mature human (Sample MH). Real-time RT-PCR was used to confirm the changed miRNAs.
Twenty-six miRNAs were shared by samples IR/MR and IR/MH with differential expression patterns greater than three-fold difference. PicTar and TargetScan prediction tools predicted a number of target mRNAs, and some of these target genes predicted by miRNAs have been shown to associate with spermatogenesis.
Our results indicate that miRNAs are extensively involved in spermatogenesis and provide additional information for further studies of spermatogenetic mechanisms.
Full-text · Article · Mar 2009 · Journal of Assisted Reproduction and Genetics
[Show abstract][Hide abstract] ABSTRACT: Spermatogenesis is a complex process subject to strict controls at both levels of transcription and translation. It has been proposed that DAZL protein binds to RNA in the cytoplasm of germ cells and controls spermatogenesis. In male mice, loss of Dazl results in numerous defects throughout the mitotic and meiotic process of germ cell development. Tex19.1 also plays an important role during spermatogenesis and Tex19.1(-/-) knockout males exhibit impaired spermatogenesis. Mouse DAZL protein can bind to 3'UTR of mTex19.1 mRNAs and may repress mTex19.1 expression at the translational level. These have been confirmed by both electrophoretic mobility shift assay and translation assay in Zebrafish embryo detecting the luciferase activity. Taken together these data suggest that mDazl may regulate mTex19.1 expression through binding to 3'UTR of mTex19.1 mRNAs in germ cells.
No preview · Article · Mar 2009 · Molecular Biology Reports
[Show abstract][Hide abstract] ABSTRACT: Rbmy gene encodes a RNA-binding protein and its expression is limited to the nuclei of germ cells. Previous studies indicate that
RBMY may function in pre-mRNA processing during spermatogenesis, although its precise target mRNAs remain unclear. By using
specific nucleic acids associated with proteins and immunoprecipitation techniques, we have identified 12 potential target
mRNAs bound by mouse RBMY protein from testis. We detect that both mRbmy-1 and mRbmy-2 transcripts co-exist in mouse testis
and they differ mainly in the 5′UTR. Importantly, our result shows that mRBMY protein can bind to one of its own transcripts,
mRbmy-2, suggesting that mRBMY may affect alternative splicing or regulate the expression of its own gene. Using electrophoretic
mobility shift assay, we demonstrated that mRBMY protein can bind to the testis and sperm-specific spa17 mRNA and that the binding domain contains rich oligo(A), suggesting that mRBMY protein may have high affinity to oligo(A)
rich sequences. In conclusion, the identification of RBMY target mRNAs will be helpful to further explore the biological function
of RBMY in spermatogenesis.
Preview · Article · Jul 2008 · Molecular Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: The Dazl gene encodes a germ-cell-specific RNA-binding protein which is essential for spermatogenesis. It has been proposed that this protein (DAZL) binds to RNA in the cytoplasm of germ cells and controls spermatogenesis. Using the specific nucleic acids associated with proteins (SNAAP) technique, we identified 17 target mRNAs bound by mDAZL. Among these transcripts, we focused on TSSK2, which encodes a testis-specific serine/threonine kinase. To date, five TSSK family members have been cloned, and all are exclusively expressed in the testis. We demonstrated that in addition to the TSSK1 3'UTR , the 3'UTRs of TSSKs 2 and 4 were bound by human and mouse DAZL, and that human DAZL (hDAZL) bound to the 3'UTR of human TSSK5 (hTSSK5). Our results suggest that the Dazl gene may play different roles in human and mouse spermatogenesis by regulating different members of the downstream gene family.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) are short non-coding RNA molecules playing regulatory roles by repressing translation or cleaving RNA transcripts. Recent studies indicate that miRNAs are mechanistically involved in the development of mammalian spermatogenesis. However, little work has been done to compare the miRNA expression patterns between immature and mature mouse testes. Here, we employed a miRNA microarray to detect 892 miRNAs in order to evaluate the expression patterns of miRNA. The expression of 19 miRNAs was significantly different between immature and mature individuals. Fourteen miRNAs were significantly upregulated and five miRNAs were downregulated in immature mice and this result was further confirmed by a quantitative real-time RT-PCR assay. Many target genes involved in spermatogenesis are predicted by MiRscan performing miRNA target scanning. Our data indicated specific miRNAs expression in immature mouse testis and suggested that miRNAs have a role in regulating spermatogenesis.
Full-text · Article · Aug 2007 · Reproduction (Cambridge, England)
[Show abstract][Hide abstract] ABSTRACT: We have cloned a novel mouse zinc finger protein gene Znf313 by rapid amplification of cDNA ends (RACE) according to the homologue of human ZNF313 gene. The cDNA is 2,163 base pairs (bp) in length and encodes a 229 amino acids (aa) protein with a C(3)HC(4) ring finger domain and three C(2)H(2) domains. 89% and 93% nucleotide (nt) and aa sequence identity is observed with its human homologue. Revealed by Northern blot and RT-PCR, full mRNA consists of 2.16 kb and widely expresses in tissues as a single transcript, most abundantly in heart, liver, kidney and testis. The expression of Znf313 in testis is detected in all development stages. Western blot analysis also reveals that Znf313 is expressed in the tissues. Immunohistochemical staining and subcellular localization demonstrate that Znf313 is expressed both in the cytoplasm and nucleus whereas predominantly localized in the nucleus. Present data suggests that Znf313 gene might play a fundamental role in gene transcription and regulation in organism and relates to spermatogenesis.
Preview · Article · Mar 2007 · Journal of biochemistry and molecular biology