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Author: Alexander John Marsolais Publisher: ISBN: Category : Languages : en Pages :
Book Description
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: Alexander John Marsolais Publisher: ISBN: Category : Languages : en Pages :
Book Description
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: John Laver Publisher: ISBN: Category : Languages : en Pages :
Book Description
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: Lucía Bruzzone Publisher: ISBN: Category : Languages : en Pages : 0
Book Description
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: Jennifer L. Semotok Publisher: ISBN: 9780494399767 Category : Languages : en Pages : 468
Book Description
Early embryonic development in all metazoa is directed by maternally provided transcripts. Precise spatial and temporal control of maternal mRNA expression is essential for the establishment of the embryonic axes and in the specification of cell fates. In early Drosophila development, a subset of maternal transcripts is eliminated from the bulk cytoplasm within the first few hours of embryogenesis. Prior to this research, how and why these maternal mRNAs were specifically eliminated during early Drosophila development was unclear. Understanding the mechanisms of maternal transcript instability has been the primary focus of my Ph. D. dissertation. Here, I have uncovered both a trans-acting factor and a cis-acting element that are major players in specifying maternal transcript instability. With respect to understanding how SMG specifically recognizes its target transcripts, I present a detailed examination of one of SMG's target transcripts, Hsp83. Through cis-mapping approaches, I show that Hsp83 messages are regulated via redundant degradation cis-elements. One of these destabilization elements, the Hsp83 mRNA instability element (HIE), is a 615 nt fragment which resides in the coding region and functions in a SMG-dependent, but translation-independent manner. Future delineation of the minimal functional unit of the HIE as well as the exploration of the molecular interactions between SMG and the HIE would provide additional clues as to how both an mRNA degradation signal and its cognate RNA-binding factor can coordinate the spatial and temporal control of maternal mRNA expression during Drosophila embryogenesis. In my genetic search for trans-acting factors, I have identified SMAUG (SMG) as an RNA-binding protein that triggers maternal mRNA turnover through the removal of the 3'-polyadenylated tail. Both genetic and biochemical analyses indicate that SMG interacts with multiple components of the CCR4-NOT deadenylase complex. Thus, I have uncovered a mechanism of SMG's post-transcriptional function, namely, that SMG recruits the deadenylase complex to its target mRNAs which is sufficient to mediate transcript deadenylation, and consequently, degradation of the mRNA body.
Author: Alexander John Marsolais
Author: Alexander John Marsolais
Author: Mariana Kekis
Author: Jason Dumelie
Author: Matthew Cheng
Author: John Laver
Author: Benjamin Douglas Pinder
Author: Vasily Sysoev
Author: Meryl Nelson
Author: Lucía Bruzzone
Author: Jennifer L. Semotok