Components Acting in Localization of Bicoid mRNA Are Conserved among Drosophila Species

Substantial insights into basic strategies for embryonic body patterning have been obtained from genetic analyses of Drosophila melanogaster. This knowledge has been used in evolutionary comparisons to ask if genes and functions are conserved. To begin to ask how highly conserved are the mechanisms of mRNA localization, a process crucial to Drosophila body patterning, we have focused on the localization of bcd mRNA to the anterior pole of the embryo. Here we consider two components involved in that process: the exuperantia (exu) gene, required for an early step in localization; and the cis-acting signal that directs bcd mRNA localization. First, we use the cloned D. melanogaster exu gene to identify the exu genes from Drosophila virilis and Drosophila pseudoobscura and to isolate them for comparisons at the structural and functional levels. Surprisingly, D. pseudoobscura has two closely related exu genes, while D. melanogaster and D. virilis have only one each. When expressed in D. melanogaster ovaries, the D. virilis exu gene and one of the D. pseudoobscura exu genes can substitute for the endogenous exu gene in supporting localization of bcd mRNA, demonstrating that function is conserved. Second, we reevaluate the ability of the D. pseudoobscura bcd mRNA localization signal to function in D. melanogaster. In contrast to a previous report, we find that function is retained. Thus, among these Drosophila species there is substantial conservation of components acting in mRNA localization, and presumably the mechanisms underlying this process.     

Piwi Genes Are Dispensable for Normal Hematopoiesis in Mice

Hematopoietic stem cells (HSC) must engage in a life-long balance between self-renewal and differentiation to sustain hematopoiesis. The highly conserved PIWI protein family regulates proliferative states of stem cells and their progeny in diverse organisms. A Human piwi gene (for clarity, the non-italicized “piwi” refers to the gene subfamily), HIWI (PIWIL1), is expressed in CD34⁺ stem/progenitor cells and transient expression of HIWI in a human leukemia cell line drastically reduces cell proliferation, implying the potential function of these proteins in hematopoiesis. Here, we report that one of the three piwi genes in mice, Miwi2 (Piwil4), is expressed in primitive hematopoetic cell types within the bone marrow. Mice with a global deletion of all three piwi genes, Miwi, Mili, and Miwi2, are able to maintain long-term hematopoiesis with no observable effect on the homeostatic HSC compartment in adult mice. The PIWI-deficient hematopoetic cells are capable of normal lineage reconstitution after competitive transplantation. We further show that the three piwi genes are dispensable during hematopoietic recovery after myeloablative stress by 5-FU. Collectively, our data suggest that the function of the piwi gene subfamily is not required for normal adult hematopoiesis.     

Panspecies Small-Molecule Disruptors of Heterochromatin-Mediated Transcriptional Gene Silencing

Heterochromatin underpins gene repression, genome integrity, and chromosome segregation. In the fission yeast Schizosaccharomyces pombe, conserved protein complexes effect heterochromatin formation via RNA interference-mediated recruitment of a histone H3 lysine 9 methyltransferase to cognate chromatin regions. To identify small molecules that inhibit heterochromatin formation, we performed an in vivo screen for loss of silencing of a dominant selectable kanMX reporter gene embedded within fission yeast centromeric heterochromatin. Two structurally unrelated compounds, HMS-I1 and HMS-I2, alleviated kanMX silencing and decreased repressive H3K9 methylation levels at the transgene. The decrease in methylation caused by HMS-I1 and HMS-I2 was observed at all loci regulated by histone methylation, including centromeric repeats, telomeric regions, and the mating-type locus, consistent with inhibition of the histone deacetylases (HDACs) Clr3 and/or Sir2. Chemical-genetic epistasis and expression profiles revealed that both compounds affect the activity of the Clr3-containing Snf2/HDAC repressor complex (SHREC). In vitro HDAC assays revealed that HMS-I1 and HMS-I2 inhibit Clr3 HDAC activity. HMS-I1 also alleviated transgene reporter silencing by heterochromatin in Arabidopsis and a mouse cell line, suggesting a conserved mechanism of action. HMS-I1 and HMS-I2 bear no resemblance to known inhibitors of chromatin-based activities and thus represent novel chemical probes for heterochromatin formation and function.     

多效生长因子（Pleiotrophin）基因沉默后的基因表达谱初步分析

多效生长因子（pleiotrophin,PTN指蛋白,Ptn指基因）是一种可同肝素结合的分泌性的生长/分化因子,具有刺激细胞粘附、迁移、存活、生长和分化等功能.我们前期研究发现,Ptn稳定沉默可以显著降低细胞的生长及成瘤能力.为进一步了解Ptn表达沉默后小鼠基因转录谱的变化,用小鼠表达谱芯片比较了对照及Ptn沉默细胞的基因表达差异.在检测的24 000个基因中,Ptn沉默后上调2倍以上的基因有240个,下调2倍以上的基因有129个.值得引起注意的是,在Ptn沉默的MEFs细胞中,同DDK综合症相关的基因家族,schlafen（Slfn）家族的Slfn 2、Slfn 3、Slfn 4以及基质金属蛋白酶（matrix metalloproteinase,MMP）家族的Mmp 3、Mmp 10、Mmp 13表达均显著上调;而可促进内皮细胞运动,参与血管发生的基因angiomotin(Amot)表达显著下调.通过研究,获得了一系列Ptn沉默后表达变化的基因信息.     

Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I _CALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I _CALHM1. Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I _CALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I _CALHM1 and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.     

The Drosophila Midkine/Pleiotrophin Homologues Miple1 and Miple2 Affect Adult Lifespan but Are Dispensable for Alk Signaling during Embryonic Gut Formation

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.     

RNA诱导沉默复合体中的生物大分子及其装配

在RNA干扰机制中,双链RNA诱导同源RNA降解的过程依赖于RNA诱导沉默复合体（RISC）的活性。RISC由Dicer酶,Argonaute蛋白,siRNA等多种生物大分子装配而成,对这些大分子的结构和功能进行深入细致的研究,有助于进一步了解RISC的形成过程、作用方式,以及阐明整个RNAi过程的作用机制。研究表明,RISC中的Dicer具有RNaseIII结构域,在RNAi的起始阶段负责催化siRNA的产生,在RISC装配过程中起稳定RISC中间体结构和功能的作用;Argonaute蛋白是RISC中的核心蛋白,有PAZ和PIWI两个主要的结构域,前者为siRNA的传递提供结合位点,后者是RISC中的酶切割活性中心;siRNA是RISC完成特异性切割作用的向导,在成熟的RISC中虽然只包含siRNA的一条链,但siRNA在RISC形成过程中的双链结构是保证RNAi效应的决定因素。尽管RISC中还存在其他一些功能未知的蛋白质,但在RISC组分结构及功能研究方面取得的进展为建立一个可能的RISC装配模型提供了理论基础。     

ERK1/2 Activities Are Dispensable for Oocyte Growth but Are Required for Meiotic Maturation and Pronuclear Formation in Mouse

Previous studies revealed that extracellular regulated kinase-1 and-2(ERK1/2) cascade plays pivotal roles in regulating oocyte meiotic cell cycle progression. However, most knowledge about the in vivo function of ERK1/2 in mammalian oocytes was indirectly obtained from analyzing the phenotypes of Mos knockout mice. In this study, we knocked out Erk1 and Erk2 in mouse oocytes as early as the primordial follicle stage using the well-characterized Gdf9-Cre mouse model, and for the first time directly investigated the in vivo function of ERK1/2 in mouse oocytes. In this novel mouse model, we observed that ERK1/2 activities in oocyte are dispensable for primordial follicle maintenance,activation and follicle growth. Different from the Mos null oocytes, the ERK1/2-deleted oocytes had well-assembled spindles at metaphase Ⅰ(MⅠ), extruded polar body-1(PB1) with normal sizes, and did not undergo a full parthenogenetic activation characterized for pronuclear formation. However, the ovulated ERK1/2-deleted oocytes had poorly-assembled metaphase Ⅱ(MⅡ) spindles, spontaneously released polar body-2(PB2), and were arrested at another metaphase called metaphase Ⅲ(MⅢ). In addition, ERK1/2 deletion prevented male pronuclear formation after fertilization, and caused female infertility. In conclusion, these results indicate that ERK1/2 activities are required for not only MⅡ-arrest maintenance, but also efficient pronuclear formation in mouse oocytes.