Huaqin Sun

A microarray for microRNA profiling in mouse testis tissues

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.

Microarray profiling of microRNAs expressed in testis tissues of developing primates

PurposeMicroRNAs (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.MethodsmiRNA 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 uesd to confirm the changed miRNAs.ResultsTwenty-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.ConclusionsOur results indicate that miRNAs are extensively involved in spermatogenesis and provide additional information for further studies of spermatogenetic mechanisms.

Identification of piRNAs in Hela cells by massive parallel sequencing

Piwi proteins and Piwi-interacting RNAs (piRNAs) have been implicated in transposon control in germ line from Drosophila to mammals. To examine the profile of small RNA transcriptome and explore the potential roles of Human Piwi-like 2 gene (alias HILI) and its associated piRNAs in human cancer cells, small RNA libraries prepared from HILI-overexpressed, HILI-knockdown, and control HeLa cells were respectively sequenced using Solexa, a next-generation massive parallel sequencing technology. A set of 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 element 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.

Zili Inhibits Transforming Growth Factor-β Signaling by Interacting with Smad4

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.

DAZL binds to 3′UTR of Tex19.1 mRNAs and regulates Tex19.1 expression

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.

Zili Antagonizes Bmp Signaling to Regulate Dorsal-Ventral Patterning during Zebrafish Early Embryogenesis

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)-&bgr;signaling 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.

Znf45l affects primitive hematopoiesis by regulating transforming growth factor-β signaling.

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. [BMB Reports 2014; 47(1): 21-26]

Molecular cloning and expression analysis of a zebrafish novel zinc finger protein gene rnf141.

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.

Identification of messenger RNA substrates for mouse T-STAR

Using the method of isolation of specific nucleic acids associated with proteins (SNAAP), we have identified 10 candidate target mRNA substrates bound by mT-STAR (mouse T-STAR protein) from testis extract. Among them, our study focused on Fabp9, a gene that is essential for male gametogenesis, and showed that mT-STAR could directly bind to Fabp9 mRNAs. The binding sites are in a short sequence of the coding region and 3′ untranslated region of Fabp9 mRNA. These suggest that mT-STAR can regulate the metabolism and expression of Fabp9. In conclusion, identification of mT-STAR-bound mRNA substrates might help to illustrate the potential spectrum of the process and provide valuable insight into the biological function of this RNA-binding protein in spermatogenesis.

Clock1a affects mesoderm development and primitive hematopoiesis by regulating Nodal-Smad3 signalings in the zebrafish embryo

Circadian clock and Smad2/3/4-mediated Nodal signaling regulate multiple physiological and pathological processes. However, it remains unknown whether Clock directly cross-talks with Nodal signaling and how this would regulate embryonic development. Here we show that Clock1a coordinated mesoderm development and primitive hematopoiesis in zebrafish embryos by directly up-regulating Nodal-Smad3 signaling. We found that Clock1a is expressed both maternally and zygotically throughout early zebrafish development. We also noted that Clock1a alterations produce embryonic defects with shortened body length, lack of the ventral tail fin, or partial defect of the eyes. Clock1a regulates the expression of the mesodermal markers ntl, gsc, and eve1 and of the hematopoietic markers scl, lmo2, and fli1a. Biochemical analyses revealed that Clock1a stimulates Nodal signaling by increasing expression of Smad2/3/4. Mechanistically, Clock1a activates the smad3a promoter via its E-box1 element (CAGATG). Taken together, these findings provide mechanistic insight into the role of Clock1a in the regulation of mesoderm development and primitive hematopoiesis via modulation of Nodal-Smad3 signaling and indicate that Smad3a is directly controlled by the circadian clock in zebrafish.