The Fox Family of RNA Binding Proteins Activate Neuron-specific Pre-mRNA Splicing Through the Intronic Element, UGCAUG PDF Download
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Author: Jason Glenn Underwood
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ISBN:
Category :
Languages : en
Pages : 278
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Author: Jason Glenn Underwood
Publisher:
ISBN:
Category :
Languages : en
Pages : 278
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Author:
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Category :
Languages : en
Pages :
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Activation of protein 4.1R exon 16 (E16) inclusion during erythropoiesis represents a physiologically important splicing switch that increases 4.1R affinity for spectrin and actin. Previous studies showed that negative regulation of E16 splicing is mediated by the binding of hnRNP A/B proteins to silencer elements in the exon and that downregulation of hnRNP A/B proteins in erythroblasts leads to activation of E16 inclusion. This paper demonstrates that positive regulation of E16 splicing can be mediated by Fox-2 or Fox-1, two closely related splicing factors that possess identical RNA recognition motifs. SELEX experiments with human Fox-1 revealed highly selective binding to the hexamer UGCAUG. Both Fox-1 and Fox-2 were able to bind the conserved UGCAUG elements in the proximal intron downstream of E16, and both could activate E16 splicing in HeLa cell co-transfection assays in a UGCAUG-dependent manner. Conversely, knockdown of Fox-2 expression, achieved with two different siRNA sequences resulted in decreased E16 splicing. Moreover, immunoblot experiments demonstrate mouse erythroblasts express Fox-2, but not Fox-1. These findings suggest that Fox-2 is a physiological activator of E16 splicing in differentiating erythroid cells in vivo. Recent experiments show that UGCAUG is present in the proximal intron sequence of many tissue-specific alternative exons, and we propose that the Fox family of splicing enhancers plays an important role in alternative splicing switches during differentiation in metazoan organisms.
Author: Caitlin DeJong
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ISBN:
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:
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Category : Dissertations, Academic
Languages : en
Pages : 848
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Author: Larry R. Squire
Publisher: Academic Press
ISBN: 0080963935
Category : Medical
Languages : en
Pages : 12505
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The Encyclopedia of the Neuroscience explores all areas of the discipline in its focused entries on a wide variety of topics in neurology, neurosurgery, psychiatry and other related areas of neuroscience. Each article is written by an expert in that specific domain and peer reviewed by the advisory board before acceptance into the encyclopedia. Each article contains a glossary, introduction, a reference section, and cross-references to other related encyclopedia articles. Written at a level suitable for university undergraduates, the breadth and depth of coverage will appeal beyond undergraduates to professionals and academics in related fields.
Author: Angela Norie Brooks
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ISBN:
Category :
Languages : en
Pages : 236
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A majority of metazoan genes contain introns in their primary transcripts (pre-mRNA) that require removal by the spliceosome--a cellular complex composed of protein and RNA. Upon removal of introns from the primary transcript, the remaining exonic portion of the transcript is spliced together. It is essential to remove the correct intronic portion of a primary transcript in order to produce the desired product, typically a protein-coding mRNA. Pre-mRNAs are alternatively spliced when different intron boundaries are used by the spliceosome, thus creating different mRNA products. Alternative splicing allows for an additional step of gene regulation by producing transcript isoforms that can be differentially processed in a particular tissue or developmental time point. Alternative splicing is primarily regulated by RNA binding proteins that bind to pre-mRNA and act to recruit or inhibit the spliceosome at specific splice sites. A central aim of this work is to gain a better understanding of splicing regulation by the identification and characterization of protein regulators of splicing and cis-acting splicing regulatory sequences in the model organism, Drosophila melanogaster. To identify splicing regulatory elements, many previous studies in vertebrate genomes have used computational methods. In collaboration with Anna I. Podgornaia, I applied such an approach to predict splicing regulatory elements in Drosophila melanogaster and compared them with elements found in vertebrates. I identified 330 putative splicing enhancer sequences enriched near weak 5' and 3' splice sites of constitutively spliced introns. I found that a significant proportion (58%) of D. melanogaster enhancers were previously reported as splicing enhancers in vertebrates. To provide additional evidence for the function of the intronic splicing enhancers (ISEs), I identified intronic hexamers significantly enriched within sequences phylogenetically conserved among 15 insect species. This analysis uncovered 73 putative ISEs that are also enriched in conserved regions of the D. melanogaster genome. The functions of nine enhancer sequences were verified in a heterologous splicing reporter by Julie L. Aspden, demonstrating that these sequences are sufficient to enhance splicing in vivo. Taken together, these data identify a set of predicted positive-acting splicing regulatory motifs in the Drosophila genome and highlight those regulatory sequences that are present in distant metazoan genomes. To identify and characterize splicing regulators, collaborators and I have combined RNAi and RNA-Seq to identify exons that are regulated by 58 known or putative splicing regulators. To identify and quantify alternative splicing events from RNA-Seq data, I developed the JuncBASE (Junction Based Analysis of Splicing Events) software package. For a pilot study, I identified 404 splicing events significantly affected upon depletion of pasilla. Preliminary analysis showed 879 splicing events affected by at least one of the 57 other proteins. The sequence regions upstream and within Pasilla-repressed exons and downstream of Pasilla-activated exons are enriched for YCAY repeats, which is consistent with the location of these motifs near regulated exons of the mammalian ortholog, Nova. Thus, the RNA regulatory map of Pasilla and Nova is highly conserved between insects and mammals despite the fact that the pre-mRNAs that are regulated by Pasilla and Nova are almost entirely non-overlapping. This observation strongly suggests that the regulatory codes of individual RNA binding proteins are nearly immutable, yet the regulatory modules controlled by these proteins are highly evolvable. I also present RNA regulatory maps for the four hnRNP proteins: hrp36, hrp38, hrp40, and hrp48. Lastly, I examine splicing regulation throughout the life cycle of D. melanogaster. Using transcriptome data from 30 developmental time points produced by collaborators from the modENCODE Consortium, I identified a total of 23,859 alternative splicing events in Drosophila, taking into account all transcript information from D. melanogaster annotations, short sequenced reads (Illumina RNA-Seq), sequenced cDNA, long RNA-Seq reads (454 RNA-Seq) from adult flies, and short read sequences of rRNA-depleted RNA from embryonic time points. I observed that 60.7% of intron-containing genes in D. melanogaster are alternatively spliced. Using only the Illumina RNA-Seq reads throughout development, 21,216 splicing events were expressed and 13,951 events were differentially spliced in at least one time point. I also observed exons with similar patterns of splicing changes throughout development as well as sex-biased alternative splicing. Integrating information from our pasilla study, I also observed correlations of pasilla gene expression with alternative splicing changes of its target exons throughout development.
Author: Md. Faruk Hossain
Publisher:
ISBN:
Category :
Languages : en
Pages : 188
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Gene expression is regulated at multiple levels, including transcription, RNA editing, pre-mRNA splicing, mRNA export, translation, and posttranslational modifications. Alternative splicing is a process by which exons can be included or excluded, giving rise to multiple mRNA isoforms from the same transcript. Alternative splicing is an important mechanism in developmental, tissue- and cell-specific control of gene expression, and it is key for expanding proteomic diversity and complexity from a limited number of genes. Moreover, more than 95% of multiexon genes undergo alternative splicing in humans, and about half of all disease-causing point mutations in humans affect pre-mRNA splicing, including neurological disorders and cancer. The central nervous system comprises the tissues and cells with the highest rate of alternative splicing in the body, and RNA-binding proteins play a major functional role in neurons. However, the regulatory mechanisms of splicing are still poorly understood. This dissertation specifically aims to advance the understanding of regulatory mechanism of pre-mRNA splicing. To this end, we collaboratively performed two projects. In the first project, we investigated how NOVA, a neuron-specific splicing factor, regulates nerve cell-specific alternative splicing of Z+ Agrin — a molecule that is the master architect of nerve-muscle synapses at the neuromuscular junction (NMJ). We cloned the Ciona ortholog of NOVA, which is present as a single copy gene in tunicates, and that of Agrin, and dissected the regulatory mechanism of alternative splicing of Z+ Agrin by Nova. Moreover, we characterized their function and expression pattern during larval development, which we will discuss in detail in Chapter 2 of this dissertation. The second project was a case study where we investigated how mutations in the SLC25A10 gene cause epileptic encephalopathy by disrupting pre-mRNA splicing. SLC25A10 codes for a solute carrier protein and is a part of complex I in mitochondria. The patient inherited 3 mutations: 1 from the mother and 2 from the father. The maternalderived mutation introduces a stop codon in exon 3. Mutations from the paternal allele are located in exon 9 and intron 10. Although the exonic mutation is a synonymous mutation, the patient had very low levels of SLC25A10 mRNA and lacked protein at detectable levels. Using minigene splicing assay we investigated the molecular mechanism underlying disease pathology in the patient. In Chapter 3 of this dissertation, we will discuss how paternal-derived mutations lead to aberrant splicing.
Author: Serge Gueroussov
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Category :
Languages : en
Pages :
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Alternative splicing (AS) greatly increases transcriptome diversity by allowing individual transcripts to be processed into multiple mature RNAs. Advances in RNA sequencing technology have revealed that transcripts from nearly all multi-exon genes in vertebrates are subject to AS, and that patterns of AS change during development and in response to environmental stimuli and disease. Transcriptomic analyses of diverse vertebrate species have provided global insight into AS evolution, revealing an expanded role for this process in mammals and showing that AS patterns have diverged more rapidly than changes in gene expression. While these studies uncovered an extensive number of species- and lineage-specific splice variants, the molecular function of these isoforms and their contribution to phenotypic change remain largely unexplored. In this thesis, I describe a detailed investigation of the functional consequences of splice isoform evolution in multiple RNA binding proteins. I show that mammalian-specific skipping of exon 9 in the PTBP1 splicing factor alters its regulatory activity, and engineered skipping of the orthologous exon in chicken is sufficient to induce a large number of mammalian-like changes in the transcriptome. The inclusion of this exon is regulated during neurogenesis, and significantly impacts the kinetics of activation of neural-specific splicing programs. Subsequent analysis of all mammalian-specific AS events revealed that they are enriched among glycine- and tyrosine-rich, intrinsically disordered protein domains. Such exons affect nearly all members of the heterogeneous nuclear ribonucleoprotein (hnRNP) A and D protein family members, which have diverse functions in RNA biology. At the transcript level, their regulation requires formation of long-range, intramolecular, mammalian-specific RNA duplexes. At the protein level, their inclusion facilitates higher-order hnRNP assemblies on substrate pre-mRNAs that are required for regulation of target AS events. Together, my thesis work demonstrates how splice isoform evolution in RNA binding proteins can expand the regulatory capabilities of mammalian cells, and reveals how these changes can influence evolution of developmental processes.
Author: William Francis Mueller
Publisher:
ISBN: 9781303001017
Category :
Languages : en
Pages : 176
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Human genes are split into regions that code for protein, exons, and regions that don't, introns. Upon transcription, the removal of these intervening introns is necessary if a usable mRNA molecule is to be translated. The process of intron removal and subsequent ligation of exons is called splicing and is carried out by a large complex called the spliceosome. This process is driven by sequence elements within the pre-mRNA itself and is the major contributor of diversity to the human transcriptome. Due to the ubiquitous nature of alternative splicing in almost every multi-exon gene, the regulation pathways of exon inclusion are a subject of wide study The different lengths of introns and exons as well as location of splice sites in a pre-mRNA molecule have been shown to have differing affects on the spliceosomes ability to recognize them. Using \emph{in vitro} splicing and complex formation assays in parallel with cell transfection experiments, we determined that the distance between two splice sites across the intron or across the exon are strong predictors of splice site usage. Additionally, we found that two splice sites interact differently when placed at different lengths apart. Our findings suggest a mechanism for observed selection of specific intron/exon architectures. Splice site recognition is also influenced by the presence of protein binding sequence elements in the pre-mRNA that alter spliceosomal recruitment. Previously, these proteins and sequence elements had been rigidly classified into splice enhancing or inhibiting categories. We show that this rigid classification is incorrect. We found that the location of these elements relative to the splice site determines their enhancing or silencing effect. That is, an enhancing element found upstream of a splice site imposes a silencing effect when relocated downstream of the splice site (and vice versa). Spliceosomal proteins are conserved from yeast to humans. The sequence elements used in pre-mRNA sequences have been evolving over time but under pressure from multiple cellular processes, including splicing. To observe the effect of splicing on evolution, we took advantage of the synonymous mutation positions that are under the least amount of selective pressure from the genetic code. We mutated these positions and found that some caused a large decrease in exon inclusion. When we analyzed the comparative alignment data, we found that these specific nucleotide mutations were selected against across species in order to maintain exon inclusion. SNP analysis showed that this pattern of selection was broadly observable at synonymous positions throughout the human genome.
Author:
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ISBN:
Category : DNA polymerases
Languages : en
Pages : 770
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