Characterization of the N-terminal Region of the RNA-Binding Protein Smaug in Post-transcriptional Regulation During Drosophila Embryogenesis PDF Download
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Author: Matthew Cheng
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Languages : en
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Author: Matthew Cheng
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Languages : en
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Author: Benjamin Douglas Pinder
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Languages : en
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Author: Lucía Bruzzone
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Category :
Languages : en
Pages : 0
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Post-transcriptional regulation of gene expression plays a critical role in a variety of cellular processes during development. RNA binding proteins are fundamental mediators of post-transcriptional regulations that control mRNA expression by recognizing specific cis acting elements within the target transcripts. Smaug is a highly conserved sequence specific RNA-binding protein that is essential during Drosophila early embryogenesis. Smaug binds Smaug Recognition Elements (SRE) in the target mRNA and recruits additional factors, via protein-protein interactions, that regulate the bound mRNA. An emergent concept that signaling pathways can modulate RBP activity by post-translation modifications adds a new layer in the control of gene expression. During my thesis work, I sought to understand how the Hedgehog pathway regulates Smaug by promoting its phosphorylation. My work shows that HH signaling downregulates Smaug protein levels affecting its ability to repress mRNA translation. This negative effect seems to be dependent on the interaction between Smaug and the HH signal transducer Smoothened. Moreover, Smaug is constitutively phosphorylated in its RNA binding domain, which appears to be necessary for cytoplasmic Smaug foci formation.
Author: Alexander John Marsolais
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Languages : en
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The maternal-to-zygotic transition (MZT) is a characteristic phase of early metazoan development where control of embryogenesis transitions from products encoded by the mother to those encoded by the zygotic genome. Post-transcriptional regulation (PTR) plays a critical role in the MZT, particularly in the clearance of maternal mRNAs. Mechanisms of maternal transcript decay that rely exclusively on maternal protein factors and function early during the MZT, as well as mechanisms that require zygotic factors and function later during the MZT, have been characterized. The Drosophila embryo has long-served as a model for the MZT. The RNA-binding protein (RBP) SMAUG (SMG) has been shown to function during the early (maternal) phase of degradation. In contrast, computational methods suggest the RBP PUMILIO (PUM) functions in the late (zygotic) phase of maternal mRNA degradation. Such a role is curious as PUM is maternally-contributed and functional during the maternal (early) phase of embryogenesis. I show here that: 1) PUM is required for the degradation of approximately 500 maternal mRNAs during the late (zygotic) wave of degradation; 2) degradation of PUM target mRNAs is likely delayed to the late (zygotic) phase due to the presence of sub-optimal PUM binding sites within these target mRNAs, 3) degradation of PUM targets is dependent on additional factors such as the RBP BRAIN TUMOUR (BRAT) and a core component of the RNAi machinery, ARGONAUTE 1 (AGO1); and 4) a critical function of PUM appears to be clearance of smg mRNA, since in pum mutant embryos SMG protein persists post-MZT and is associated with an inappropriate down-regulation of SMG target transcripts. Taken together, these data support a multi-factorial view of RBP function, in which the activity of a given RBP is determined by other RBPs associated with a particular mRNA.
Author: Gerrit Martin Daubner
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Languages : en
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Author: Caitlin DeJong
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Category :
Languages : en
Pages : 155
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RNA-binding proteins as regulators of transcription and axial patterning during Xenopus embryogenesis by Caitlin Suzanne DeJong Doctor of Philosophy in Molecular and Cell Biology University of California, Berkeley Professor Richard M. Harland, Chair The over-arching goal of this thesis is to expand our knowledge of the mechanisms by which one cell, a fertilized egg, develops into an organism composed of multiple cell types, each with different functions and behaviors. RNA-binding proteins have been identified as potent regulators of development and embryogenesis. The studies presented in this thesis illustrate the pleiotropic effects of RNA-binding proteins in Xenopus development and will focus specifically on two RNA-binding proteins that are maternally deposited and zygotically transcribed: TAF15 and DGCR8. TATA-binding protein-associated factor 15 (TAF15) belongs to the FET family of atypical RNA-binding proteins, which also includes Fused in sarcoma (Fus) and Ewing’s sarcoma (EWS). FET proteins were originally discovered as components of fusion oncogenes and are most noted for their implication in various cancers and neuromuscular degenerative diseases. However, little is known of the endogenous function of FET proteins. The diverse biological activities of the FET family proteins can be likened to a biological Swiss army knife; as these proteins contain domains for transcriptional activation, RNA-binding, DNA-binding, and function in both RNA Polymerase II-mediated transcription and pre-mRNA splicing. An exciting possibility is that the FET proteins may function to connect transcription and splicing. By employing the bioinformatics approach of RNA-sequencing, I generated a list of significant genes that are differentially expressed between uninjected and taf15 depleted embryos. From this analysis I found that TAF15 regulates target genes at both the transcriptional and post-transcriptional level. The studies that focus on the role of TAF15 in Xenopus development are described in chapters two and three of this thesis. In the second chapter of this thesis I describe studies that illustrate the novel concept that a protein can regulate the same set of target genes but through different molecular mechanisms. Both maternal and zygotic TAF15 regulate the expression of the transcripts fgfr4, isl1, and pax8. Interestingly, maternal TAF15 is required for the post-transcriptional regulation of fgfr4, isl1, and pax8, regulating the splicing of single introns within these transcripts, whereas zygotic TAF15 is required for the transcriptional regulation of these genes. Therefore, the studies described in chapter two demonstrate, for the first time, that a single protein can utilize a different molecular mechanism to control the same target genes and the use of these different mechanisms of action appears to be dependent on whether the protein is maternally deposited or zygotically transcribed. Single intron retention is a known mechanism to retain transcripts in the nucleus, preventing their translation. In chapter two of this thesis I provide evidence for the following model: in the absence of genome activation, before the zygotic genome is transcribed, maternal TAF15 cooperates with a splicing factor, the RNA-binding protein SRSF4, to regulate the splicing of single introns from transcripts. As a result, TAF15 and SRSF4 control the splicing of target genes and thus control the timing of transcript maturation and subsequent translation. This mechanism is logical as it provides a mechanism by which to spatially and temporally regulate gene expression in the absence of the ability to transcriptionally regulate genes. I further show evidence that following zygotic genome is activation, zygotic TAF15 activates target gene transcription, regulating genes at the transcriptional level, likely associating with the core promoter. The findings described in chapter two of this thesis are the first to show that a single protein can regulate the same gene targets but depending on the milieu of maternal of zygotic cofactors, regulates these targets via different underlying mechanisms. The variety of functional domains intrinsic to TAF15 supports the hypothesis that this atypical RNA-binding protein could operate as part of both a splicing and transcriptional complex. In the third chapter of this thesis I describe studies that illustrate the novel finding that TAF15 is required for dorsoventral patterning via the repression of ventx2.1. Ventx2 and BMP4 function in an autocatalytic positive feedback loop to specify ventral tissue and antagonize organizer function. Following taf15 depletion, ventx2.1 expression is expanded in the neural ectoderm and embryos exhibit a BMP overexpression phenotype: reduction in head, and dorsal, and posterior fin structures, with an increase in ventral tissue. Unlike the findings in chapter two, in this study, both maternal and zygotic TAF15 function to suppress ventx2.1 expression. These findings place TAF15 in the regulatory network of dorsoventral patterning and suggest that maternal and zygotic TAF15 control expression of ventx2.1 in a similar manner but do not rule out differential mechanisms of this control. Currently, it is unknown if TAF15 represses ventx2.1 expression directly or if TAF15 is required to activate a repressor of ventx2.1. In the fourth chapter of this thesis I describe studies that serve as a resource for future investigations into the role of microRNAs (miRNAs) in Xenopus development. DiGeorge syndrome critical region 8 (DGCR8) is a subunit of the microprocessor complex required for miRNA biogenesis. Unlike most members (e.g. Dicer, Argonaute2) of the RNA interference biogenesis pathway, DGCR8 is required specifically for miRNA biogenesis. Furthermore, unlike previous studies in mice and zebrafish that have depleted maternal dgcr8 throughout oogenesis to look at the role of miRNAs during embryogenesis, the antisense oligodeoxynucleotide (ODN) that I have designed can be used in host transfer assays to assess the effects of maternal dgcr8 depletion once oogenesis is complete, specifically during embryogenesis. Additionally, I have designed a splice-blocking morpholino (MO) antisense oligonucleotide that targets zygotic dgcr8 for depletion. Using these two tools (ODN and MO), the first studies can be performed that tease apart the role of maternal versus zygotic DGCR8 during embryogenesis. The work presented in this thesis exemplifies the value of carefully assessing biological functions of genes that are both maternally deposited and zygotically transcribed. The surprising finding that TAF15 utilizes distinct molecular mechanisms to control conserved target genes depending on whether this protein is maternally deposited or zygotically expressed demonstrates a new level of molecular complexity that future studies must address. Additionally, these studies further support the motivation to investigate RNA-binding proteins in development and disease as they continually prove to be multifaceted players in molecular biology.
Author: Kiyoshi Nagai
Publisher: Oxford University Press, USA
ISBN:
Category : Medical
Languages : en
Pages : 302
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The study of RNA-protein interactions is crucial to understanding the mechanisms and control of gene expression and protein synthesis. The realization that RNAs are often far more biologically active than was previously appreciated has stimulated a great deal of new research in this field. Uniquely, in this book, the world's leading researchers have collaborated to produce a comprehensive and current review of RNA-protein interactions for all scientists working in this area. Timely, comprehensive, and authoritative, this new Frontiers title will be invaluable for all researchers in molecular biology, biochemistry and structural biology.
Author: John Laver
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Languages : en
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Post-transcriptional regulation of gene expression, through the control of mRNA splicing, polyadenylation, nuclear export, localization, translation, and stability, is essential for achieving appropriate temporal and spatial patterns of protein expression. This regulation is mediated by trans-acting factors, such as RNA-binding proteins (RBPs) and non-coding RNAs, which associate with specific mRNA targets through the recognition of sequence- or structure-based cis-elements present in the transcripts. The genomes of most organisms encode hundreds of RBPs, each of which likely associates with hundreds of mRNAs. Thus, a genome-wide view of the regulation being mediated by all trans-factors is essential for a complete understanding of post-transcriptional control. While post-transcriptional regulation is crucial in all biological systems, it has a particularly prominent role during early embryo development, as during this time there is no transcription from the zygotic genome of the embryo, and, thus, gene expression and development is controlled entirely post-transcriptionally. In this thesis, I describe my efforts towards obtaining a global understanding of post-transcriptional regulation in early Drosophila melanogaster embryos, through the development and use of synthetic antibodies as tools to identify, genome-wide, RBP-mRNA interactions. First, I demonstrated that synthetic antibodies generated against RBPs can be used as tools to identify RBP-associated mRNAs through immunoprecipitation-based approaches, or, conversely, to disrupt RBP-mRNA interactions. I then used synthetic antibodies to identify the entire complement of mRNAs associated with 3 developmentally-important RBPs: the double-stranded RBP Staufen, the TRIM-NHL protein Brain Tumor, and the PUF protein Pumilio. Computational analyses of these mRNAs revealed: (1) novel cis-elements likely mediating the mRNA-binding activity of Staufen and Brain Tumor; (2) that, unexpectedly, Brain Tumor and Pumilio function largely independently of each other in early embryos; and, (3) a novel role for Brain Tumor in promoting mRNA decay, which was demonstrated through a transcriptome-wide analysis of mRNA levels in brain tumor mutant embryos. To facilitate a truly genome-wide analysis of RBP-mRNA interactions, we developed a high-throughput pipeline for production of synthetic antibodies, and used this pipeline to generate 279 antibodies against 61 RBPs. In future this pipeline and the antibodies generated will allow for global studies of post-transcriptional regulation.
Author: Jason Dumelie
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Category :
Languages : en
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Author: Piyada Juntawong
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Category : Arabidopsis thaliana
Languages : en
Pages : 221
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Light and temperature are two of the most important factors that regulate plant growth and development. The adaptation to alterations in light and temperature involves programed changes in gene expression that are necessary for physiological and morphological adaptations. Transcript abundance is frequently used to monitor changes in gene expression in response to sub-optimal growth conditions. However, transcript accumulation may not accurately mirror gene expression due to extensive post-transcriptional and post-translational regulation. In this dissertation, the significance in post-transcriptional regulation in response to unanticipated alterations in light availability was evaluated in seedlings of Arabidopsis thaliana. Early darkness resulted in translational inhibition and sequestration of a subset of cellular mRNAs. The translationally regulated mRNAs were enriched in transcripts encoding chloroplastic and protein synthesis machinery. The reduced engagement of the majority of these transcripts with ribosomes was rapidly reversed upon re-illumination. These results suggest that regulation of the translational status of chloroplastic and protein synthesis mRNAs may aid in energy conservation during unanticipated darkness. Towards the elucidation of RNA binding proteins that control selective mRNA translation, the complexes of Arabidopsis Cold Shock Proteins (CSPs 1-4) were characterized. Transgenic lines overexpressing epitope-tagged CSPs were established and used for cellular fractionation and mass spectrometric protein identification of immunopurified CSP complexes. Fluorescently-tagged CSPs and associated proteins were localized in transiently transformed cells by confocal microscopy. Together, the results of this survey indicate Arabidopsis CSPs are involved in multiple processes of post-transcriptional regulation, including pre-mRNA/rRNA processing and mRNA translation. Of the four CSPs, CSP1 co-fractionated with ribosome. A mild RNase A treatment of ribosome complexes combined with sucrose gradient fractionation confirmed that CSP1 is a polysomeassociated RNA binding protein. CSP1 accumulated under normal growth condition and was induced by low-temperature. A polyclonal antibody prepared to specifically recognize CSP1 protein was used to co-immunopurify native CSP1 complexes. DNA microarray hybridization was used to compare total (transcriptome), polysomal (translatome), and CSP1-associated mRNAs from plants grown under normal or lowtemperature conditions. The results demonstrate that CSP1 preferentially associated with mRNAs involved in RNA processing and protein synthesis. Many of the CSP1-associated mRNA also have high 5'-UTR with a high G+C content. The transcriptome and translatome adjustments during low-temperature stress were highly correlated, in contrast to the findings with early darkness. This work provides new perspectives on post-transcriptional gene regulation in response to environmental cues in Arabidopsis, as well as a foundation for future in-depth characterization of mRNA-RNP control networks in plants.
