Download or read book RNA binding Protein Mediated Post transcriptional Control of Gene Expression in Eye Development and Disease written by Soma Dash and published by . This book was released on 2018 with total page 156 pages. Available in PDF, EPUB and Kindle. Book excerpt: Eye development in vertebrates is initiated in late gastrulation and involves coordinated morphogenesis between the optic vesicle and the non-neural surface ectoderm resulting in the formation of the neural retina and the lens, respectively. While transcription and signaling events required for eye development are well understood, post-transcriptional control of gene expression, especially mediated by RNA-binding proteins (RBPs) is less clear. This represents a significant knowledge-gap as RBPs are important regulatory molecules in the cell that can control the fate of their target mRNAs by interacting with them throughout the mRNA life-cycle and mediating their processing, intra-cellular transport and localization, stability, translation into protein, and ultimately, their degradation. This is also a significant knowledge gap because there are similar number of RBPs encoded by the human genome as there are transcription factors, but the former class of proteins are not as well understood in the context of organogenesis and birth defects as compared to the latter. ☐ While high-throughput sequencing has identified several RBPs to be expressed in the eye, the functional significance in eye development for the vast majority of these factors is yet to be determined. Recently, the Lachke laboratory has identified two conserved RBPs required for eye development, Tdrd7 and Celf1, whose deficiency in the lens results in cataract in vertebrates. To further investigate the importance of RBP-mediated post-transcriptional gene expression control in eye development, I applied a systems-based bioinformatics tool iSyTE (integrated Systems Tool for Eye gene discovery) to identify two new RBPs, Rbm24 and Caprin2, which are enriched during early mouse lens development, but whose molecular function in eye development had thus far not been determined. In this research dissertation, I have characterized the function of both Rbm24 and Caprin2 using constitutive and conditional targeted gene deletion mouse models. Further, in collaboration with Dr. Diane Slusarski’s laboratory (University of Iowa), zebrafish rbm24a knockout (by CRISPR/Cas9) and knockdown (by morpholino) mutants were generated and characterized. Together, these findings have led to a comprehensive understanding of the function of these RBPs in vertebrate eye development. ☐ Rbm24-targeted deletion in mouse and rbm24a-CRISPR/Cas9-targeted knockout or morpholino-knockdown in zebrafish causes the developmental defects microphthalmia (small eye) or anophthalmia (no eye). Rbm24 deficiency leads to apoptotic defects in the mouse ocular tissue as well as downregulation of eye development markers such as Sox2, Lhx2, Jag1, E-cadherin and g-Crystallins. Further, similar to the observations in the mouse, sox2 expression is also found to be reduced in rbm24a-morphant zebrafish, indicating the conservation of the Rbm24-Sox2 regulatory module in vertebrate eye development. About 20% of human anophthalmia cases are linked to SOX2 mutations alone. Therefore, I focused on investigating the post-transcriptional molecular mechanism of Rbm24-mediated Sox2 regulation. Sox2 is an intronless gene whose encoded mRNA contains AU-rich regions (ARE) in its 3’UTR. Interestingly, Rbm24 is known to bind to ARE sites in target mRNA. Therefore, to test if Rbm24 directly binds to Sox2 mRNA in vitro and in vivo, I performed RNA-Electrophoretic Mobility Shift assay (EMSA) and RNA-Immunoprecipitation (RIP), respectively. RNA-EMSA showed that Rbm24 protein directly binds to a 20 bp oligomer based on the mouse Sox2 mRNA sequence, and that an intact ARE is necessary for this protein-RNA binding. In turn, RIP assay on E14.5 wildtype mouse ocular tissue suggests that Rbm24 directly binds to Sox2 mRNA in vivo in eye development. To understand the biological significance of this direct Rbm24 protein-Sox2 mRNA molecular interaction, I performed an RNA-decay assay in NIH3T3 cells by co-transfected them with an Rbm24-overexpression vector and a Renilla luciferase reporter vector. In this assay, the Renilla luciferase gene ORF (open reading frame) is fused with the mouse Sox2 mRNA 3’UTR, which contains the three intact ARE sites, and reporter transcripts were quantified after Actinomycin-D treatment to transfected cells. This analysis demonstrates that in conditions of Rbm24 over-expression, the intact Sox2 3’UTR can render increased stability to the reporter transcript. Thus, Rbm24 positively controls Sox2 expression by binding to ARE sites in its 3’UTR and increasing its mRNA stability. Further, mutation analysis in the RNA-decay assay extends the in vitro observation that the binding of Rbm24 to the Sox2 mRNA 3’UTR depends on ARE by providing in vivo evidence that the presence of the ARE sites is necessary for the stability effect rendered by the Sox2 mRNA 3’UTR upon Rbm24 overexpression. Further, because Sox2 is one of the original four Yamanaka pluripotency/cellular reprogramming factor (along with Oct4, Klf4 and c-Myc), I investigated the impact of Rbm24 on the expression of other reprogramming factors such as Oct4, Klf4, c-Myc as well as, Nanog, another established pluripotency factor. I find that over-expression of Rbm24 in several different cell lines such as NIH3T3 (mouse embryo fibroblast cell line), 21EM15 (mouse lens epithelial cell line) and C2C12 (mouse myoblast cell line) results in the up-regulation of Sox2, Oct4 and Klf4. Further, in Rbm24-overexpressed C2C12 cells, Nanog and c-Myc are also upregulated. These data highlight that Rbm24 mediates post-transcriptional control of key transcription and pluripotency factors in vertebrate development. ☐ To gain insight into the function of the other newly identified RBP, Caprin2, in lens biology, I first performed expression analysis of Caprin2 in mouse lens development using in situ hybridization, western blotting and immunostaining. These experiments validate the iSyTE prediction that Caprin2 mRNA and protein are highly expressed and enriched in mouse embryonic and postnatal lens. I generated lens-specific Caprin2 conditional knockout (cKO) mouse mutants using a lens-Cre deleter line Pax6GFPCre. Phenotypic analysis of Caprin2cKO/cKO mice, wherein Caprin2 is expected to be deleted in the lens starting from E9.5 due to Cre-mediated re-arrangement of the Caprin2 alleles, revealed two distinct eye defects at variable penetrance. Wheat germ agglutinin staining and scanning electron microscopy demonstrated that Caprin2cKO/cKO mutants have an abnormally compact “lens nucleus”, which is the core of the lens comprised of centrally located terminally differentiated fiber cells. Further, at a reduced penetrance (8%), I find that Caprin2cKO/cKO mutants exhibit an ocular defect wherein the lens and the cornea remain attached by a persistent stalk, resembling the human developmental defect termed Peters anomaly. These data suggest that a conserved RBP Caprin2 functions in distinct morphological events in mammalian eye development. ☐ Together the findings in this dissertation have demonstrated that conserved RBPs such as Rbm24 and Caprin2 have evolved distinct functions in vertebrate eye development and their deficiency leads to microphthalmia and anophthalmia, and lens defects and Peters anomaly, respectively, thus impacting the study of ocular defects in humans.

Download or read book RNA Binding Proteins written by Kathryn Sandberg and published by Springer Science & Business Media. This book was released on 2013-03-09 with total page 318 pages. Available in PDF, EPUB and Kindle. Book excerpt: RNA binding proteins are an exciting area of research in gene regulation. A multitude of RNA-protein interactions are used to regulate gene expression including pre-mRNA splicing, polyadenylation, editing, transport, cytoplasmic targeting, translation and mRNA turnover. In addition to these post-transcriptional processes, RNA-protein interactions play a key role in transcription as illustrated by the life cycle of retroviruses. Unlike DNA, the structure of RNA is highly variable and conformationally flexible, thus creating a number of unique binding sites and the potential for complex regulation by RNA binding proteins. Although there is a wide range of topics included in this volume, general themes have been repeated, highlighting the overall integrative nature of RNA binding proteins. The chapters have been separated into three different sections: Translational Control; mRNA Metabolism; and Hormonal and Homeostatic Regulation. The chapters of this volume were written with the seasoned investigator and student in mind. Summaries of key concepts are reviewed within each chapter as well as guiding questions that can be used to stimulate class discussions. The Editors of this volume hope that this compendium educates, enthralls, and stimulates the readers to look to the future possibilities in this rapidly evolving field.

Download or read book Post transcriptional Control of Gene Expression written by Orna Resnekov and published by Springer Science & Business Media. This book was released on 2013-06-29 with total page 276 pages. Available in PDF, EPUB and Kindle. Book excerpt: Many important cellular processes rely on posttranscriptional control of gene expression. This book describes the mechanisms of gene expression at this level that occur in the cytoplasm of prokaryotes and eukaryotes. Several introductory chapters discuss the general principles of translation and mRNA stability. The interactions of mature mRNA with the translational machinery, the components of mRNA degradation and antisense RNA are surveyed. Subsequent chapters discuss protein folding, transport, modification and degradation. The book is an invaluable source of information for both newcomers and those wishing an overview of the field.

Download or read book RNA Binding Proteins written by Zdravko Lorkovic and published by CRC Press. This book was released on 2012-08-10 with total page 174 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gene expression in eukaryotes is regulated at different levels, which need to be coordinated to implement the information in the genome. Now it is clear that post-transcriptional regulation of gene expression such as pre-mRNA splicing, mRNA transport, editing, turnover and translation are as important as the control of transcription. In all aspects

Download or read book Post Transcriptional Regulation by STAR Proteins written by Talila Volk and published by Springer Science & Business Media. This book was released on 2011-01-11 with total page 176 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book examines the available information on the structure of the RNA binding STAR domain and provides insights into how these proteins discriminate between different RNA targets. It reviews what is known about STAR proteins and human disease.

Download or read book Post Transcriptional Gene Regulation written by Jane Wu and published by John Wiley & Sons. This book was released on 2013-04-24 with total page 400 pages. Available in PDF, EPUB and Kindle. Book excerpt: Reflecting the rapid progress in the field, the book presents the current understanding of molecular mechanisms of post-transcriptional gene regulation thereby focusing on RNA processing mechanisms in eucaryotic cells. With chapters on mechanisms as RNA splicing, RNA interference, MicroRNAs, RNA editing and others, the book also discusses the critical role of RNA processing for the pathogenesis of a wide range of human diseases. The interdisciplinary importance of the topic makes the title a useful resource for a wide reader group in science, clinics as well as pharmaceutical industry.

Download or read book RNA Processing written by Gene W. Yeo and published by Springer. This book was released on 2016-06-02 with total page 335 pages. Available in PDF, EPUB and Kindle. Book excerpt: Ribonucleic acid (RNA) binding proteins currently number in the thousands and defects in their function are at the heart of diseases such as cancer and neurodegeneration. RNA binding proteins have become implicated in the intricate control of surprisingly diverse biological settings, such as circadian rhythm, stem cell self-renewal, oncogenesis and germ cell development. This book surveys a range of genome-wide and systems approaches to studying RNA binding proteins, the importance of RNA binding proteins in development, cancer and circadian rhythm.

Download or read book The Conserved RNA binding Proteins Celf1 and Elavl1 Mediate Post transcriptional Gene Expression Control in Lens Development written by Sandeep Aryal and published by . This book was released on 2022 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Thus, my doctoral dissertation has resulted in the development of new omicslevels resources for gene discovery in the lens and other non-lens tissues, including the retina, and has additionally led to the definition of the molecular basis of lens defects in two different RBP gene-specific mouse knockout models. Finally, this work has opened up new areas of research to pursue an RBP-based combinatorial control of posttranscriptional gene expression involving Celf1 and Elavl1 proteins in lens and eye development.

Download or read book RNA binding Proteins as Regulators of Transcription and Axial Patterning During Xenopus Embryogenesis written by Caitlin DeJong and published by . This book was released on 2015 with total page 155 pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.

Download or read book Epigenetics Development and Disease written by Tapas Kumar Kundu and published by Springer Science & Business Media. This book was released on 2012-11-13 with total page 698 pages. Available in PDF, EPUB and Kindle. Book excerpt: Epigenetics fine-tunes the life processes dictated by DNA sequences, but also kick-starts pathophysiological processes including diabetes, AIDS and cancer. This volume tracks the latest research on epigenetics, including work on new-generation therapeutics.

Download or read book The Role of the RNA binding Protein Wig 1 in Post transcriptional Regulation of Gene Expression written by and published by . This book was released on 2014 with total page 50 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Post Transcriptional Regulation of Gene Expression by a Nuclear Polyadenylated RNA Binding Protein written by Ronald Earl Hector and published by . This book was released on 2000 with total page 288 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Regulation of Gene Expression by RNA binding Proteins and MicroRNAs written by Daniel Joseph Hogan and published by . This book was released on 2009 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: Circumstantial evidence suggests vast post-transcriptional regulatory networks in eukaryotes. There are scores of examples of mRNAs with rich post-transcriptional lives, and dozens of RNA-binding proteins and noncoding RNAs are suggested to have regulatory functions. While eukaryotic genomes encode hundreds of RNA-binding proteins and metazoans also encode dozens to hundreds of regulatory noncoding RNAs, few RNA-binding proteins or regulatory noncoding RNAs have been studied systematically, leaving unanswered basic questions about the scope and organization of post-transcriptional regulation in any eukaryote. First, I present studies aimed at uncovering some of the basic principles of post-transcriptional regulation in eukaryotes, via searching for the RNA targets of 40 proteins in the yeast Saccharomyces cerevisiae: a selective sample of the approximately 600 annotated and predicted RBPs as well as several proteins not annotated as RBPs. Second, I present a simple biochemical approach to identify direct targets of specific microRNAs, important noncoding regulatory RNAs, that regulate protein synthesis of most mammalian mRNAs. Third, I present experiments that uncover the magnitude and respective contributions of translational repression and mRNA decay of microRNA-mediated regulation. These studies apply simple, yet powerful approaches to systematically dissect various aspects of post-transcriptional regulation and uncover a wealth of novel information regarding regulation by RNA-binding proteins and microRNAs.

Download or read book Systems Biology of RNA Binding Proteins written by Gene W. Yeo and published by Springer. This book was released on 2016-09-10 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: After transcription in the nucleus, RNA binding proteins (RBPs) recognize cis-regulatory RNA elements within pre-mRNA sequence to form mRNA-protein (mRNP) complexes. Similarly to DNA binding proteins such as transcription factors that regulate gene expression by binding to DNA elements in the promoters of genes, RBPs regulate the fate of target RNAs by interacting with specific sequences or RNA secondary structural features within the transcribed RNA molecule. The set of functional RNA elements recognized by RBPs within target RNAs and which control the temporal, functional and spatial dynamics of the target RNA define a putative “mRNP code”. These cis-regulatory RNA elements can be found in the 5’ and 3’ untranslated regions (UTRs), introns, and exons of all protein-coding genes. RNA elements in 5’ and 3’ UTRs are frequently involved in targeting RNA to specific cellular compartments, affecting 3’ end formation, controlling RNA stability and regulating mRNA translation. RNA elements in introns and exons are known to function as splicing enhancers or silencers during the splicing process from pre-mRNA to mature mRNA. This book provides case studies of RNA binding proteins that regulate aspects of RNA processing that are important for fundamental understanding of diseases and development. Chapters include systems-level perspectives, mechanistic insights into RNA processing and RNA Binding proteins in genetic variation, development and disease. The content focuses on systems biology and genomics of RNA Binding proteins and their relation to human diseases.

Download or read book Post transcriptional Regulation by the Pluripotency Associated RNA binding Protein LIN28 written by Melissa L. Wilbert and published by . This book was released on 2014 with total page 195 pages. Available in PDF, EPUB and Kindle. Book excerpt: The field of stem cell biology is moving forward at an unprecedented rate in part due to the discovery that adult somatic cells can be reprogrammed to a pluripotent stem cell like state. The factors first used in reprogramming were transcription factors such as OCT4, SOX2 and NANOG, and the RNA-binding protein LIN28. Like transcription factors, RNA-binding proteins (RBPs) control vast networks of gene targets to direct pathways in the cell; however, for RBPs this is accomplished through post-transcriptional binding to RNA transcripts. Only recently has it been possible to survey the transcripome-wide RNA binding interactions of a protein, through isolation of endogenous RBP-RNA complexes paired with high-throughput sequencing technologies. Using cross-linking followed by immunoprecipitation of protein-RNA complexes and sequencing of isolated transcripts (CLIP-seq) we have identified LIN28 binding sites throughout the human transcriptome. The resolution of our data enabled us to define characteristic LIN28 mRNA interactions at GGAGA rich motifs within unpaired regions of hairpin loops. This binding pattern mimics interactions described for LIN28 binding within let-7 family microRNA precursors. The ability to consider LIN28 targets on a global scale enabled the identification of RNA processing factors, in particular splicing factors, as prevalent functions encoded by LIN28 bound RNAs. This information helped to accurately predict which of the thousands of LIN28 targets would be functionally regulated. We found evidence that LIN28 increases the protein production of splicing factors resulting in massive rearrangement of RNA transcripts through downstream splicing changes. Subsequent transcriptome-wide studies of LIN28 have confirmed these findings despite differences in the pool of direct targets defined by individual reports. Taken together, we understand that LIN28 can bind to a wide network of transcripts, influencing development through these direct RNA interactions and via downstream effects. Combinatorial approaches in the study of LIN28 using changes in RNA-levels, protein production, strength of CLIP-seq binding, and ontological classification of gene targets have extracted meaningful information about mechanisms of LIN28 regulation. We expect that application of similar methods will enable studies of additional RBPs. For example, in the study of other stem cell enriched proteins like the IGFII-mRNA binding proteins (IG2BP or IMP). Furthermore, the overlap of other regulatory networks hold promise of highlighting novel hubs of regulation that may be exploited in reprogramming or directed differentiation. The next step is to use these connections to explain how genetic changes within an individual can affect RBP function and result in disease. We can apply in vitro modeling of development using directed differentiation to iteratively test how the connection of LIN28 to its target transcripts impacts its role in development and disease.

Download or read book The Roles of RNA binding Proteins in the Developing Nervous System written by Jie Quan and published by . This book was released on 2014 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: RNA-binding proteins are key players in post-transcriptional regulation of gene expression by orchestrating RNA fate from synthesis to decay. Hundreds of proteins with RNA-binding capacity have been identified so far, yet only a small fraction has been functionally characterized and presumably many more RNA-binding proteins await discovery. The roles of RNA-binding proteins in the nervous system are of particular interest because accumulative evidence has linked RNA-based mechanisms to neural development, maintenance and repair. Here, the three RNA-binding proteins under study are IGF-II mRNA binding proteins IMP-1 and IMP-2, known to be involved in mRNA localization, translational control and stability, and adenomatous polyposis coli (APC), identified as a novel RNA-binding protein.

Download or read book Systematic Analysis of Protein RNA Interactions in Drosophila written by John Laver and published by . This book was released on 2016 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: 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.