Mechanisms of Smaug-mediated Post-transcriptional Regulation in the Early Drosophila Embryo PDF Download

Author: Benjamin Douglas Pinder
Publisher:
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Category :
Languages : en
Pages :

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Author: Meryl Nelson
Publisher:
ISBN:
Category :
Languages : en
Pages :

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Author: Wael Tadros
Publisher:
ISBN: 9780494280232
Category :
Languages : en
Pages : 264

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Early embryonic development is controlled by maternally deposited proteins and transcripts. Egg activation triggers a cascade of posttranscriptional mechanisms that are crucial to the regulation of these maternal mRNAs during this time of transcriptional quiescence. These mechanisms include translational activation, repression and transcript destabilization. Here I show that, in Drosophila, the PAN GU (PNG) kinase complex sits near the top of this cascade that ultimately leads to the destabilization of maternal mRNAs. The genes png, plutonium (plu) and giant nuclei (gnu), which encode the components of this complex, were recovered in a screen for maternal effect lethal mutants which fail to undergo degradation. I show that png's control of transcript destabilization is genetically separable and therefore independent of its well characterized role in the cell cycle. PNG acts following egg activation in promoting the translation of SMAUG (SMG), a major posttranscriptional regulator. Our gene-expression profiling experiments show that SMG is responsible for targeting two thirds of degrading maternal mRNAs. PNG activates smg translation in a poly(A)-independent manner acting through the smg 3'UTR. Finally, I show that PNG also has a SMG-independent mechanism of eliciting transcript decay.

Author: Jacques Bothma
Publisher:
ISBN:
Category :
Languages : en
Pages : 86

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Contemplating how a single cell can turn into the trillions of specialized cells that make a human being staggers the imagination. We still do not fully understand how the information in a genome is interpreted by a cell to orchestrate this incredible process. One thing that we do know is that much of the complexity we see in the natural world comes down to how essentially the same set of proteins are differentially deployed. One of the key places where this is controlled is at the level of transcription which is the first step in protein production. In this thesis we attempt to shed light on this process by looking at how transcription is regulated in the early Drosophila embryo with a focus on mechanisms of transcriptional precision. We developed imaging and segmentation techniques that allowed for the quantitative visualization of the transcriptional state of thousands of nuclei in the embryo. Using this approach we discovered the phenomenon of repression lag, whereby genes containing large introns are not only slow to be switched on (intron delay), but are also slow to be repressed. Many sequence-specific repressors have been implicated in early development, but the mechanisms by which they silence gene expression have remained elusive. We found that elongating Pol II complexes complete transcription after the onset of repression. As a result, moderately sized genes are fully silenced only after tens of minutes of repression. We propose that this "repression lag" imposes a severe constraint on the regulatory dynamics of embryonic patterning. Having laid the foundations for using quantitative imaging in the early Drosophila embryo we next sought to understand the mechanisms underlying developmental timing, the temporal control of gene expression. Previous studies have provided considerable information about the spatial regulation of gene expression, but there is very little information regarding the temporal coordination of expression. Paused RNA Polymerase (pausd Pol II) is a pervasive feature of Drosophila embryos and mammalian stem cells, but its role in development is uncertain. We demonstrate that there is a spectrum of paused Pol II, which determines the "time to synchrony" the time required to achieve coordinate gene expression across the different cells of a tissue. To determine whether synchronous patterns of gene activation are significant in development, we manipulated the timing of snail expression, which controls the coordinated invagination of 1000 mesoderm cells during gastrulation. Replacement of the strongly paused snail promoter with moderately paused or nonpaused promoters resulted in stochastic activation of snail expression and the progressive loss of mesoderm invagination. Computational modeling of the dorsal-ventral patterning network recapitulated these variable and bistable gastrulation profiles, and emphasized the importance of timing of gene activation in development. We concluded that paused Pol II and transcriptional synchrony are essential for coordinating cell behavior during morphogenesis. These studies and others have helped launch a new approach to the well-established problem of differential gene expression in animal development. The quantitative imaging methods developed here have permitted the assessment of temporal dynamics of gene expression and the underlying mechanisms for coordinating gene expression across the different cells of a tissue. The next frontier will be to apply these methods to live embryos, thereby permitting an even deeper analysis of gene dynamics in development.

Author: Guoqing Chen
Publisher:
ISBN:
Category :
Languages : en
Pages : 342

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Author: George M. Malacinski
Publisher: Springer Science & Business Media
ISBN: 1468446282
Category : Science
Languages : en
Pages : 377

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The early embryo has emerged as the focal point for analysis of the regulation of gene expression for several reasons. First, the fact that embryogenesis is under genetic control has been appreciated from the earliest days of classical embryology. When experimental techniques became available it was therefore logical that they should be applied to the embryo. With each new advance in methodology, interest in embryonic gene expression studies has increased. Second, many embryos offer unique opportunities for the investigation of specific aspects of the regulation of gene expression. Several phenomena--eg. , control of translation--can be very conveniently studied in a variety of marine invertebrate embryos. Those embryos contain large stores of maternally inherited mRNA which are translated in a highly ordered fashion during specific stages of post fertilization development. Marine invertebrate eggs can be conveniently artifically inseminated and labeled with radioactive precursors. Their analysis is leading to important insights into the mechanisms which regulate gene expression at post-transcriptional levels. Third, recent advances in both transmission and recombinant DNA genetics, especially in organisms such as Drosophila, are providing special opportunities for the analysis of regulatory mechanisms which operate at the level of the genome. Specific genes have been identified, isolated, and--in some instances--sequenced. The opportunity is now available to study the regulation of the expression of single genes in a vertical fashion--from the primary sequence of the gene to the tissues and organs which are the products of morphogenesis.

Author: Ramona L. (Ramona Leigh) Cooperstock
Publisher: National Library of Canada = Bibliothèque nationale du Canada
ISBN: 9780612918771
Category :
Languages : en
Pages : 414

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Author: Mandy Jeske
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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During early embryogenesis of Drosophila, transcription does not contribute to the regulation of gene expression, and translational control becomes eminently important. The nanos mRNA specifies the development of posterior structures in the Drosophila embryo. Translation of nanos mRNA is restricted to the posterior pole of the embryo, and the vast majority of nanos mRNA within the embryonic cytoplasm is silenced. Translational repression of nanos is mediated by the protein Smaug, which can bind specific sequences, Smaug recognition elements (SREs), in the nanos 3' UTR. At the posterior pole, translation of nanos mRNA is derepressed by the action of Oskar protein. In this doctoral study, a cell-free system derived from Drosophila embryos was established that recapitulates the SRE-dependent translational regulation. Both SRE-dependent deadenylation and translation repression were biochemically investigated. Addition of recombinant Oskar to the extracts prevents SRE-dependent repression.

Author: Zhiyong Yang
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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Smaug is a RNA-binding protein that regulates translational repression and/or degradation of its targets, and is required for the elimination of a large number of maternal transcripts in early Drosophila embryos. Here, I explored a novel role of Smaug in regulation of Smaug in early embryos. Previous experiments identified mRNAs encoding glycolytic enzymes as associated with Smaug. I found that mRNAs encoding these enzymes have predicted Smaug recognition elements (SREs) and are destabilized by Smaug. Furthermore, I performed enzyme activity assays and showed increased activity of several glycolytic enzymes in smaug mutants. Experiments comparing the metabolome of wild-type and smaug mutant embryos implicated Smaug in an array of metabolic pathways including glycolysis.

Author: Elizabeth Danielle Larson
Publisher:
ISBN:
Category :
Languages : en
Pages : 0

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During the early stages of development, the fertilized germ cells are rapidly reprogrammed to form a pluripotent embryo. This transition in cell fate is coordinated by pioneer transcription factors that have the ability to open inaccessible chromatin to allow other factors to bind and drive gene expression. As chromatin is known to pose a barrier to transcription factor binding, these unique properties of pioneer factors make them instrumental in driving gene-regulatory networks that control critical developmental transitions. Despite the ability to access closed chromatin, pioneer factors do not function the same throughout development, so it is crucial that we understand how specific cellular environments influence pioneer factor binding and activity. The pioneer transcription factor Zelda (Zld) is essential for early embryonic reprogramming in Drosophila melanogaster. Research has shown that Zld shapes the chromatin and transcriptional landscape in the early embryo, but Zld's role later in development and the mechanisms by which Zld was regulated remained unclear. Our data has demonstrated that Zld functions to maintain the undifferentiated state of a neural stem cell population in the developing larval brain. Additionally, the ability of Zld to reprogram is conserved as Zld can also reprogram in the larval neural stem cell lineage. However, Zld binding is redistributed in the larval neuroblasts from the early embryo indicating that developmental context shapes where this transcription factor can bind. We show that Zld levels have to be precisely regulated in both the brain and the early embryo as misexpression of Zld at either stage is detrimental to the animal. The protein Brain Tumor (Brat) regulates Zld levels at both stages of development and we demonstrate that in embryos lacking functional Brat, Zld is prematurely expressed. However, early Zld expression is not sufficient to precociously activate the zygotic genome. Thus, expression of a genomic activator must be coordinated with timing of the division cycles in order to properly activate the genome. Together, our data demonstrate the Zld must be tightly regulated throughout development in order to allow for rapid transitions in cell fate. Together, our studies will help us better understand the transcriptional and post-transcriptional mechanisms regulating pioneer transcription factors.