The Nanos3-3′UTR Is Required for Germ Cell Specific NANOS3 Expression in Mouse Embryos

BACKGROUND: The regulation of gene expression via a 3' untranslated region (UTR) plays essential roles in the discrimination of the germ cell lineage from somatic cells during embryogenesis. This is fundamental to the continuation of a species. Mouse NANOS3 is an essential protein required for the germ cell maintenance and is specifically expressed in these cells. However, the regulatory mechanisms that restrict the expression of this gene in the germ cells is largely unknown at present. METHODOLOGY/PRINCIPAL FINDINGS: In our current study, we show that differences in the stability of Nanos3 mRNA between germ cells and somatic cells is brought about in a 3'UTR-dependent manner in mouse embryos. Although Nanos3 is transcribed in both cell lineages, it is efficiently translated only in the germ lineage. We also find that the translational suppression of NANOS3 in somatic cells is caused by a 3'UTR-mediated mRNA destabilizing mechanism. Surprisingly, even when under the control of the CAG promoter which induces strong ubiquitous transcription in both germ cells and somatic cells, the addition of the Nanos3-3'UTR sequence to the coding region of exogenous gene was effective in restricting protein expression in germ cells. CONCLUSIONS/SIGNIFICANCE: Our current study thus suggests that Nanos3-3'UTR has an essential role in translational control in the mouse embryo

The centrosome and spindle as a ribonucleoprotein complex

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Chromosome Research 19 (2011): 367-376, doi:10.1007/s10577-011-9186-7.The presence of nucleic acids in centrosomes and the spindle have been proposed, observed, and reported since the 1950s. Why did the subject remain, perhaps even until today, such a controversial issue? The explanation is manifold, and includes legitimate concern over contamination from other cellular compartments in biochemical preparations. With a typically high background of cytoplasmic ribosomes, even microscopic images of stained intact cells could be difficult to interpret. Also, evidence for RNA and DNA in centrosomes accumulated for approximately 40 years but was interspersed with contradictory studies, primarily regarding the presence of DNA (reviewed in Johnson and Rosenbaum, 1991; Marshall and Rosenbaum, 2000). Perhaps less tangible but still a likely cause for lingering controversy is that the presence of nucleic acids in the spindle or centrosomes will require us to look differently at these structures from a functional, and more to the point, evolutionary standpoint.This work was supported by grants from the NIH (GM088503) and NSF (MCB0843092) to MCA

Sequence and expression pattern of the germ line marker vasa in honey bees and stingless bees

Queens and workers of social insects differ in the rates of egg laying. Using genomic information we determined the sequence of vasa, a highly conserved gene specific to the germ line of metazoans, for the honey bee and four stingless bees. The vasa sequence of social bees differed from that of other insects in two motifs. By RT-PCR we confirmed the germ line specificity of Amvasa expression in honey bees. In situ hybridization on ovarioles showed that Amvasa is expressed throughout the germarium, except for the transition zone beneath the terminal filament. A diffuse vasa signal was also seen in terminal filaments suggesting the presence of germ line cells. Oocytes showed elevated levels of Amvasa transcripts in the lower germarium and after follicles became segregated. In previtellogenic follicles, Amvasa transcription was detected in the trophocytes, which appear to supply its mRNA to the growing oocyte. A similar picture was obtained for ovarioles of the stingless bee Melipona quadrifasciata, except that Amvasa expression was higher in the oocytes of previtellogenic follicles. The social bees differ in this respect from Drosophila, the model system for insect oogenesis, suggesting that changes in the sequence and expression pattern of vasa may have occurred during social evolution

The Phylogenetic Origin of oskar Coincided with the Origin of Maternally Provisioned Germ Plasm and Pole Cells at the Base of the Holometabola

The establishment of the germline is a critical, yet surprisingly evolutionarily labile, event in the development of sexually reproducing animals. In the fly Drosophila, germ cells acquire their fate early during development through the inheritance of the germ plasm, a specialized maternal cytoplasm localized at the posterior pole of the oocyte. The gene oskar (osk) is both necessary and sufficient for assembling this substance. Both maternal germ plasm and oskar are evolutionary novelties within the insects, as the germline is specified by zygotic induction in basally branching insects, and osk has until now only been detected in dipterans. In order to understand the origin of these evolutionary novelties, we used comparative genomics, parental RNAi, and gene expression analyses in multiple insect species. We have found that the origin of osk and its role in specifying the germline coincided with the innovation of maternal germ plasm and pole cells at the base of the holometabolous insects and that losses of osk are correlated with changes in germline determination strategies within the Holometabola. Our results indicate that the invention of the novel gene osk was a key innovation that allowed the transition from the ancestral late zygotic mode of germline induction to a maternally controlled establishment of the germline found in many holometabolous insect species. We propose that the ancestral role of osk was to connect an upstream network ancestrally involved in mRNA localization and translational control to a downstream regulatory network ancestrally involved in executing the germ cell program

Vasa-Like DEAD-Box RNA Helicases of Schistosoma mansoni

Genome sequences are available for the human blood flukes, Schistosoma japonicum, S. mansoni and S. haematobium. Functional genomic approaches could aid in identifying the role and importance of these newly described schistosome genes. Transgenesis is established for functional genomics in model species, which can lead to gain- or loss-of-functions, facilitate vector-based RNA interference, and represents an effective forward genetics tool for insertional mutagenesis screens. Progress toward routine transgenesis in schistosomes might be expedited if germ cells could be reliably localized in cultured schistosomes. Vasa, a member of the ATP-dependent DEAD-box RNA helicase family, is a prototypic marker of primordial germ cells and the germ line in the Metazoa. Using bioinformatics, 33 putative DEAD-box RNA helicases exhibiting conserved motifs that characterize helicases of this family were identified in the S. mansoni genome. Moreover, three of the helicases exhibited vasa-like sequences; phylogenetic analysis confirmed the three vasa-like genes—termed Smvlg1, Smvlg2, and Smvlg3—were members of the Vasa/PL10 DEAD-box subfamily. Transcripts encoding Smvlg1, Smvlg2, and Smvlg3 were cloned from cDNAs from mixed sex adult worms, and quantitative real time PCR revealed their presence in developmental stages of S. mansoni with elevated expression in sporocysts, adult females, eggs, and miracidia, with strikingly high expression in the undeveloped egg. Whole mount in situ hybridization (WISH) analysis revealed that Smvlg1, Smvlg2 and Smvlg3 were transcribed in the posterior ovary where the oocytes mature. Germ cell specific expression of schistosome vasa-like genes should provide an informative landmark for germ line transgenesis of schistosomes, etiologic agents of major neglected tropical diseases

Gene expression patterns associated with blood-feeding in the malaria mosquito Anopheles gambiae

BACKGROUND: Blood feeding, or hematophagy, is a behavior exhibited by female mosquitoes required both for reproduction and for transmission of pathogens. We determined the expression patterns of 3,068 ESTs, representing ~2,000 unique gene transcripts using cDNA microarrays in adult female Anopheles gambiae at selected times during the first two days following blood ingestion, at 5 and 30 min during a 40 minute blood meal and at 0, 1, 3, 5, 12, 16, 24 and 48 hours after completion of the blood meal and compared their expression to transcript levels in mosquitoes with access only to a sugar solution. RESULTS: In blood-fed mosquitoes, 413 unique transcripts, approximately 25% of the total, were expressed at least two-fold above or below their levels in the sugar-fed mosquitoes, at one or more time points. These differentially expressed gene products were clustered using k-means clustering into Early Genes, Middle Genes, and Late Genes, containing 144, 130, and 139 unique transcripts, respectively. Several genes from each group were analyzed by quantitative real-time PCR in order to validate the microarray results. CONCLUSION: The expression patterns and annotation of the genes in these three groups (Early, Middle, and Late genes) are discussed in the context of female mosquitoes' physiological responses to blood feeding, including blood digestion, peritrophic matrix formation, egg development, and immunity

Dynein-Dependent Transport of nanos RNA in Drosophila Sensory Neurons Requires Rumpelstiltskin and the Germ Plasm Organizer Oskar

Intracellular mRNA localization is a conserved mechanismfor spatially regulating protein production in polarized cells, such as neurons. The mRNA encoding the translational repressor Nanos (Nos) forms ribonucleoprotein (RNP) particles that are dendritically localized in Drosophila larval class IV dendritic arborization (da) neurons. In nos mutants, class IV da neurons exhibit reduced dendritic branching complexity, which is rescued by transgenic expression of wild-type nos mRNA but not by a localization-compromised nos derivative. While localization is essential for nos function in dendrite morphogenesis, the mechanism underlying the transport of nos RNP particles was unknown. We investigated the mechanism of dendritic nos mRNA localization by analyzing requirements for nos RNP particle motility in class IV da neuron dendrites through live imaging of fluorescently labeled nos mRNA. We show that dynein motor machinery components mediate transport of nosmRNA in proximal dendrites. Two factors, the RNA-binding protein Rumpelstiltskin and the germ plasm protein Oskar, which are required for diffusion/entrapment-mediated localization of nos during oogenesis, also function in da neurons for formation and transport of nos RNP particles. Additionally, we show that nos regulates neuronal function, most likely independent of its dendritic localization and function in morphogenesis. Our results reveal adaptability of localization factors for regulation of a target transcript in different cellular contexts

Changes in bicoid mRNA Anchoring Highlight Conserved Mechanisms during the Oocyte-to-Embryo Transition

SummaryIntracellular mRNA localization directs protein synthesis to particular subcellular domains to establish embryonic polarity in a variety of organisms. In Drosophila, bicoid (bcd) mRNA is prelocalized at the oocyte anterior. After fertilization, translation of this RNA produces a Bcd protein gradient that determines anterior cell fates [1, 2]. Analysis of bcd mRNA during late stages of oogenesis suggested a model for steady-state bcd localization by continual active transport [3]. However, this mechanism cannot explain maintenance of bcd localization throughout the end of oogenesis, when microtubules disassemble in preparation for embryogenesis [4, 5], or retention of bcd at the anterior in mature oocytes, which can remain dormant for weeks before fertilization [6]. Here, we elucidate the path and mechanism of sustained bcd mRNA transport by direct observation of bcd RNA particle translocation in living oocytes. We show that bcd mRNA shifts from continuous active transport to stable actin-dependent anchoring at the end of oogenesis. Egg activation triggers bcd release from the anterior cortex for proper deployment in the embryo, probably through reorganization of the actin cytoskeleton. These findings uncover a surprising parallel between flies and frogs, as cortically tethered Xenopus Vg1 mRNA undergoes a similar redistribution during oocyte maturation [7]. Our results thus highlight a conserved mechanism for regulating mRNA anchoring and redeployment during the oocyte-to-embryo transition

Ribosome profiling reveals pervasive and regulated stop codon readthrough in Drosophila melanogaster

Ribosomes can read through stop codons in a regulated manner, elongating rather than terminating the nascent peptide. Stop codon readthrough is essential to diverse viruses, and phylogenetically predicted to occur in a few hundred genes in Drosophila mela