Dans les tumeurs hiérarchiques, les cellules souches du cancer (CSC), au sommet de la hiérarchie tumorale, peuvent s'auto-renouveler et se différencier en progéniteurs amplificateurs transitoires (TAP) avec un potentiel d'auto-renouvellement limité. Au cours du développement, les cellules souches neurales de Drosophile, appelées neuroblastes (NB), expriment en séquence deux protéines antagonistes se liant à l'ARN, Imp et Syncrip (Syp), qui respectivement favorise et réprime l'auto-renouvellement des NB. La perturbation de mécanismes de division asymétrique des NB peut générer leur amplification illimitée induisant de véritables tumeurs. À l’aide d’une analyse clonale et de modélisations mathématiques, nous avons démontré que les progéniteurs Imp+ dans les tumeurs agissent comme des cellules semblables aux CSC, capables de se renouveler indéfiniment et de se différencier en progéniteurs Syp+, qui, à l’instar des TAP, ont un potentiel d’auto-renouvellement limité et une forte tendance à entrer en quiescence. De plus, nous avons démontré que les tumeurs du NB suivent une organisation hiérarchique rigide dans laquelle la transition Imp-Syp est irréversible. Fait intéressant, en utilisant l’analyse transcriptomique, nous avons constaté que la transition Imp à Syp dans les NB de tumeurs induit une régulation négative des gènes glycolytiques et respiratoires, épuisant vraisemblablement la capacité de croissance et d’auto-renouvellement des progéniteurs Syp+. La conservation frappante de ces protéines de liaison à l'ARN ouvre la possibilité passionnante que des hiérarchies analogues puissent exister dans les cancers humain. / In hierarchical tumors, cancer stem cells (CSCs), at the top of the tumor hierarchy, can self-renew and differentiate in transient-amplifying progenitors (TAPs), with a limited self-renewal potential. Understanding the molecular mechanisms that drive tumor hierarchy and heterogeneity is crucial to develop effective therapies to eliminate CSCs. During development, Drosophila asymmetrically-dividing neural stem cells, called neuroblasts (NBs), sequentially express two antagonistic RNA-binding proteins, Imp and Syncrip (Syp), that respectively promote and repress NB self-renewal. Genetic perturbation of NB asymmetric division cause NB amplification and malignant tumors. By using lineage tracing, clonal analysis and stochastic mathematical modeling of tumor growth, we demonstrated that Imp+ progenitors act as CSCs. They are able to self-renew endlessly and differentiate in Syp+ progenitors, that have a limited self-renewal potential and the high tendency to undergo quiescence. NB tumors follow a rigid hierarchical organization, where the Imp-to-Syp transition is irreversible. Hence, Syp+ progenitors cannot revert to an Imp+ malignant state. Transcriptomic analysis revealed that the Imp-to-Syp transition in tumors induces a downregulation of glycolytic and respiratory genes that exhausts the growth and self-renewing potential of Syp+ progenitors. The striking conservation of these RNA-binding proteins opens the exciting possibility that analogous Imp-Syp hierarchies may exist in human cancers.

Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA binding protein that regulates the alternative splicing of a variety of transcripts. In Myotonic Dystrophy (DM) aberrant cellular localization of MBNL1 results in disease-associated mis-splicing of several MBNL1 target pre-mRNAs. Due to its role in DM pathogenesis, MBNL1 has been a topic of intense study for the last decade, however many open mechanistic questions remain regarding how MBNL1 recognizes RNA substrates to mediate splicing. The RNA recognition motif for MBNL1, 5'-YGCY-3', was defined herein. This motif was used to identify novel MBNL1 binding sites within regulated transcripts and create synthetic MBNL1-regulated splicing reporters. MBNL1 contains four zinc finger (ZF) RNA binding domains arranged into two pairs of two ZFs. A comprehensive, combinatorial mutagenic study of MBNL1 was conducted to determine the role of each ZF in RNA binding and splicing activity. Functional analysis of the mutant proteins in cellular splicing assays and assessment of RNA binding activity demonstrated that the ZF pairs (i.e. ZF1-2 or ZF3-4) do not have equivalent activity. The ZF1-2 pair is responsible for MBNL1's high affinity RNA binding and splicing activity, whereas the ZF3-4 pair has reduced affinity for RNA and impaired ability to regulate splicing of some transcripts. Hierarchical clustering analysis revealed that two distinct classes of MBNL1-regulated splicing events exist within the small set of splicing events examined. For Class II splicing events the binding and splicing activity for the ZF mutants correlated well. However, for Class I events there was no significant correlation between RNA binding and splicing activity. For pre-mRNAs in the latter class it appears that MBNL1 exerts surprisingly robust splicing activity in the absence of strong RNA binding, suggesting that MBNL1 may be recruited to some pre-mRNA substrates through protein-protein interactions. This study provides the first demonstration that functionally distinct classes of MBNL1-mediated splicing events exist in terms of requirements for different ZFs and the importance of RNA binding. This dissertation includes previously published and unpublished co-authored material as well as recently co-authored material that has been submitted for publication.

by Chan Wai Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 170-185). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of Content --- p.vii / Abbreviations --- p.xii / for Nucleotides --- p.xii / for Amino acids --- p.xii / for Standard genetic codes --- p.xiii / for Units --- p.xiii / for Prefixes of units --- p.xiv / for Terms commonly used in the report --- p.xiv / List of Figures --- p.xvii / List of Tables --- p.xxiii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Epidemiology of the Severe Acute Respiratory Syndrome --- p.1 / Chapter 1.2 --- The SARS Coronavirus --- p.3 / Chapter 1.3 --- Cell Biology of Coronavirus Infection and Replication and the Role of Nucleocapsid Protein --- p.9 / Chapter 1.4 --- Recent Advances in the SARS-CoV Nucleocapsid Protein --- p.16 / Chapter 1.5 --- The Sumoylation System --- p.24 / Chapter 1.6 --- Objectives of the Present Study --- p.28 / Chapter Chapter II --- SARS-CoV N protein and Fragment Purification --- p.29 / Chapter 2.1 --- INTRODUCTION --- p.29 / Chapter 2.2 --- METHODOLOGY --- p.31 / Materials --- p.31 / Methods --- p.39 / Chapter 2.2.1 --- Construction of the pMAL-c2P vector --- p.39 / Chapter 2.2.2 --- Sub-cloning of the N protein into expression vectors --- p.42 / Chapter 2.2.2.1 --- Design of primers for the cloning of N protein --- p.43 / Chapter 2.2.2.2 --- DNA amplification using Polymerase Chain Reaction (PCR) --- p.44 / Chapter 2.2.2.3 --- DNA extraction from agarose gel --- p.45 / Chapter 2.2.2.4 --- Restriction digestion of purified PCR product and vectors --- p.46 / Chapter 2.2.2.5 --- Ligation of N protein into expression vectors --- p.47 / Chapter 2.2.2.6 --- Preparation of competent cells --- p.48 / Chapter 2.2.2.7 --- Transformation of plasmids into competent Escherichia coli --- p.49 / Chapter 2.2.2.8 --- Preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.1 --- Mini-preparation of plasmid DNA --- p.49 / Chapter 2.2.2.8.2 --- Midi-preparation of plasmid DNA --- p.51 / Chapter 2.2.3 --- Expression of tagged and untagged N protein --- p.53 / Chapter 2.2.3.1 --- Preparation of E. coli competent cells for protein expression --- p.53 / Chapter 2.2.3.2 --- Expression of N protein --- p.53 / Chapter 2.2.3.3 --- Solubility tests on the fusion proteins expressed --- p.54 / Chapter 2.2.4 --- Purification of N protein Chromatographic methods --- p.55 / Chapter 2.2.4.1 --- Affinity chromatography --- p.55 / Chapter 2.2.4.1.1 --- Ni-NTA affinity chromatography --- p.55 / Chapter 2.2.4.1.2 --- Glutathione affinity chromatography --- p.56 / Chapter 2.2.4.1.3 --- Amylose affinity chromatography --- p.56 / Chapter 2.2.4.2 --- Ion exchange chromatography --- p.57 / Chapter 2.2.4.2.1 --- Cation exchange chromatography --- p.57 / Chapter 2.2.4.2.2 --- Anion exchange chromatography --- p.58 / Chapter 2.2.4.3 --- Heparin affinity chromatography --- p.58 / Chapter 2.2.4.4 --- Size exclusion chromatography Purification strategies --- p.60 / Chapter 2.2.4.5 --- Purification of His6-tagged N proteins --- p.60 / Chapter 2.2.4.6 --- Purification of MBP-tagged N proteins --- p.60 / Chapter 2.2.4.7 --- Purification of GST-tagged N proteins --- p.61 / Chapter 2.2.4.8 --- Purification of untagged N proteins --- p.61 / Chapter 2.2.5 --- Trypsin digestion assay for the design of stable fragment --- p.64 / Chapter 2.2.6 --- Partial purification of the N protein amino acid residue 214-422 fragment --- p.65 / Chapter 2.2.7 --- Sumoylation of the SARS-CoV N protein --- p.67 / Chapter 2.2.7.1 --- In vitro sumoylation assay --- p.67 / Chapter 2.2.7.2 --- Sample preparation for mass spectrometric analysis --- p.68 / Chapter 2.3 --- RESULTS --- p.70 / Chapter 2.3.1 --- Construction of the vector pMAL-c2P --- p.70 / Chapter 2.3.2 --- "Construction of recombinant N protein-pAC28m, N-protein- pGEX-6P-l,N protein-pMAL-c2E and N protein-pMAL-c2P plasmids" --- p.72 / Chapter 2.3.3 --- Optimization of expression conditions --- p.79 / Chapter 2.3.4 --- Screening of purification strategies --- p.82 / Chapter 2.3.4.1 --- Purification of His6-N protein --- p.82 / Chapter 2.3.4.2 --- Purification of MBP-N protein --- p.84 / Chapter 2.3.4.3 --- Purification of GST-N protein --- p.85 / Chapter 2.3.4.4 --- Purification of untagged N protein --- p.87 / Chapter 2.3.5 --- Limited trypsinolysis for the determination of discrete structural unit --- p.91 / Chapter 2.3.6 --- Partial purification of the N protein 214-422 fragment --- p.94 / Chapter 2.3.7 --- Sumoylation of N protein --- p.97 / Chapter 2.2.7.1 --- Sumoylation site prediction --- p.97 / Chapter 2.2.7.2 --- In vitro sumoylation assay --- p.99 / Chapter 2.2.7.3 --- Mass spectrometric identification of sumoylated SARS-CoV N protein --- p.103 / Chapter 2.4 --- DISCUSSION --- p.109 / Chapter Chapter III --- Characterization of the Nucleic Acid Binding Ability of N protein --- p.119 / Chapter 3.1 --- INTRODUCTION --- p.119 / Chapter 3.2 --- METHODOLOGY --- p.120 / Materials --- p.120 / Methods --- p.124 / Chapter 3.2.1 --- Spectrophotometric Measurement of ratio OD260/ OD280 --- p.124 / Chapter 3.2.2 --- Native gel electrophoresis --- p.124 / Chapter 3.2.3 --- Quantitative determination of nucleic acids content --- p.125 / Chapter 3.2.3.1 --- Dische assay - quantitative determination of DNA content --- p.125 / Chapter 3.2.3.2 --- Orcinol assay - quantitative determination of RNA content --- p.126 / Chapter 3.2.4 --- RNase digestion of the N protein-bound RNA --- p.128 / Chapter 3.2.5 --- Isolation of RNA from purified GST-N proteins --- p.128 / Chapter 3.2.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.129 / Chapter 3.2.7 --- Vero E6 cell line maintenance and total RNA extraction --- p.131 / Chapter 3.2.8 --- Electrophoretic mobility shift assay (EMSA) --- p.131 / Chapter 3.3 --- RESULTS --- p.133 / Chapter 3.3.1 --- Detection of nucleic acids in the purified N proteins byspectrophotometric Measurement of ratio OD260/ OD280 --- p.133 / Chapter 3.3.2 --- Native gel electrophoresis --- p.135 / Chapter 3.3.3 --- Quantitative determination of nucleic acids content in purified GST-N proteins --- p.136 / Chapter 3.3.3.1 --- Dische assay for the determination of DNA --- p.136 / Chapter 3.3.3.2 --- Orcinol assay for the determination of RNA --- p.138 / Chapter 3.3.4 --- RNase digestion treatment --- p.139 / Chapter 3.3.5 --- Extraction of RNA from GST-N proteins --- p.140 / Chapter 3.3.6 --- In vitro transcription of SARS-CoV genomic RNA fragment --- p.142 / Chapter 3.3.7 --- Electrophoretic mobility shift assay (EMSA) --- p.144 / Chapter 3.4 --- DISCUSSION --- p.147 / Chapter Chapter IV --- Discussion --- p.154 / Chapter 4.1 --- "Purity, Aggregation and RNA Binding Property of the SARS-CoV Nucleocapsid Protein" --- p.154 / Chapter 4.2 --- Future perspectives --- p.156 / Chapter 4.2.1 --- Structural study of the SARS-CoV N protein through x-ray crystallography --- p.156 / Chapter 4.2.2 --- Mapping the RNA binding domain in the SARS-CoV N protein --- p.156 / Chapter 4.2.3 --- Determination of aggregation state by lateral turbidimetry analysis --- p.156 / Chapter 4.2.4 --- Exploring protein interacting partners that enhance RNA binding specificity --- p.157 / Appendix --- p.159 / Chapter I. --- Sequence of the SARS-CoV N protein --- p.159 / Chapter II. --- Sequence of the SARS-CoV genome fragment used for RNA binding assay in section 3.37.1 --- p.161 / Chapter III. --- Vector maps --- p.161 / Chapter a) --- Vector map of pACYC177 --- p.161 / Chapter b) --- Vector map and MCS of pET28a --- p.163 / Chapter c) --- Vector map and MCS of pAC28 --- p.164 / Chapter d) --- Vector map and MCS of pGEX-6P-1 / Chapter e) --- Vector map of pMAL-c2X and MCS of pMAL-c2E / Chapter IV. --- Electrophoresis markers --- p.166 / Chapter V. --- SDS-PAGE gel parathion protocol --- p.169 / References --- p.170

Borrelia burgdorferi, the causative agent of Lyme disease, exists in a defined enzootic cycle involving Ixodes scapularis ticks and various vertebrates. Humans can serve as an accidental host, if a tick colonized with B. burgdorferi happens to feed on a human. B. burgdorferi are also accidental pathogens: they do not make toxins, or destroy host tissue by other mechanisms. They merely transmit between vector and host to survive. In order to do this, they must effectively sense their current environment, and appropriately alter cellular processes. Understanding the regulatory mechanisms of how B. burgdorferi manages to do this has been a focus of the Stevenson lab for many years. Previous work identified SpoVG as a DNA-binding protein. Although a homologue of this protein had been implicated to serve a regulatory role in other bacteria, the Stevenson lab was the first to demonstrate a function for the protein, both for B. burgdorferi and two other bacteria. Studies contained in this body of work aim to provide insight into regulation of SpoVG by B. burgdorferi as well the impact that it has on gene regulation. By using genetic mutants, we determined that SpoVG is regulated at the levels of transcription and translation in culture by growth rate, temperature, and other regulatory factors. Additionally, we provide evidence that SpoVG regulates its own expression. Numerous genes are under control of SpoVG. Biochemical analyses revealed that SpoVG specifically interacts with DNAs and RNAs associated with genes found to be under its regulatory control. Finally, we provide evidence for SpoVG acting in concert with other known regulatory factors such as other DNA-binding proteins and the cyclic di-nucleotide second messengers cyclic-di-GMP and cyclic-di-AMP. All together, these studies provide insight into how B. burgdorferi broadly regulates cellular processes during different stages of the enzootic cycle. We hypothesize that SpoVG does this through globally manipulating the three-dimensional structure of the bacterial chromosome, and that exactly how SpoVG acts at any given point will be dependent on the other regulatory factors that are also present in the cell.

Identification and annotation of RNA Binding Proteins (RBPs) and RNA Binding residues from sequence information alone is one of the most challenging problems in computational biology. RBPs play crucial roles in several fundamental biological functions including transcriptional regulation of RNAs and RNA metabolism splicing. Existing experimental techniques are time-consuming and costly. Thus, efficient computational identification of RBPs directly from the sequence can be useful to annotate RBP and assist the experimental design. Here, we introduce AIRBP, a computational sequence-based method, which utilizes features extracted from evolutionary information, physiochemical properties, and disordered properties to train a machine learning method designed using stacking, an advanced machine learning technique, for effective prediction of RBPs. Furthermore, it makes use of efficient machine learning algorithms like Support Vector Machine, Logistic Regression, K-Nearest Neighbor and XGBoost (Extreme Gradient Boosting Algorithm). In this research work, we also propose another predictor for efficient annotation of RBP residues. This RBP residue predictor also uses stacking and evolutionary algorithms for efficient annotation of RBPs and RNA Binding residue. The RNA-binding residue predictor also utilizes various evolutionary, physicochemical and disordered properties to train a robust model. This thesis presents a possible solution to the RBP and RNA binding residue prediction problem through two independent predictors, both of which outperform existing state-of-the-art approaches.

It is well-established that the organization of nuclear components influences gene expression processes, yet little is known about the mechanisms that contribute to the spatial co-ordination of nuclear activities. The salivary gland cells of Chironomus tentans provide a suitable model system for studying gene expression in situ, as they allow for direct visualization of the synthesis, processing and export of a specific protein-coding transcript, the Balbiani ring (BR) pre-mRNA, in a nuclear environment in which chromatin and non-chromatin structures can easily be distinguished. The RNAbinding protein Hrp65 has been identified in this model system as a protein associated with non-chromatin nucleoplasmic fibers, referred to as connecting fibers (CFs). The CFs associate with BR RNP particles in the nucleoplasm, suggesting that Hrp65 is involved in mRNA biogenesis at the post-transcriptional level. However, the function of Hrp65 is not known, nor is the function or the composition of CFs. In the work described in this thesis, we have identified by yeast two-hybrid screening and characterized different proteins that bind to Hrp65. These proteins include a novel hnRNP protein in C. tentans named Hrp59, various isoforms of Hrp65, the splicing- and mRNA export factor HEL/UAP56, and a RING-domain protein of unknown function. Immuno-electron microscopy experiments showed that Hrp59 and HEL are present in CFs, and in larger structures in the nucleoplasm of C. tentans salivary gland cells. Hrp59 is a C. tentans homologue of human hnRNP M, and it associates cotranscriptionally with a subset of pre-mRNAs, including its own transcript, in a manner that does not depend quantitatively on the amount of synthesized RNA. Hrp59 accompanies the BR pre-mRNA from the gene to the nuclear envelope, and is released from the BR mRNA at the nuclear pore complex. We have identified the preferred RNA targets of Hrp59 in Drosophila cells, and we have shown that Hrp59 binds preferentially to exonic splicing enhancer sequences. Hrp65 self-associates through an evolutionarily conserved domain that can also mediate heterodimerization of Hrp65 homologues. Different isoforms of Hrp65 interact with each other in all possible combinations, and Hrp65 can oligomerize into complexes of at least six molecules. The interaction between different Hrp65 isoforms is crucial for their intracellular localization, and we have discovered a mechanism by which Hrp65-2 is imported into the nucleus through binding to Hrp65-1. Hrp65 binds to HEL/UAP56 in C. tentans cells. We have analyzed the distribution of the two proteins on polytene chromosomes and in the nucleoplasm of salivary gland cells, and our results suggest that Hrp65 and HEL become associated during posttranscriptional gene expression events. HEL binds to the BR pre-mRNP cotranscriptionally, and incorporation of HEL into the pre-mRNP does not depend on the location of introns along the BR pre-mRNA. HEL accompanies the BR mRNP to the nuclear pore and is released from the BR mRNP during translocation into the cytoplasm.

Thesis (Ph. D.)--Case Western Reserve University, 2010. / [School of Medicine] Department of Pharmacology. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 117-127).

Muscle tissue development (myogenesis) involves the formation of specific fibers (myotubes) from muscle cells (myoblasts). For this to occur, the sequential expression of Myogenic Regulatory Factors (MRFs), such as MyoD and myogenin, is required. The expression of these MRFs is regulated posttranscriptionally by the RNA-binding protein HuR, whereby HuR associates with the 3'-untranslated regions of MyoD and myogenin mRNA, leading to a significant increase in their half-lives. Here we show that the cleavage of HuR by caspases at the aspartate (D) 226 residue is one of the main regulators of its pro-myogenic function. This proteolytic activity generates two cleavage products (CPs), HuR-CP1 and HuR-CP2, that differentially affect the myogenic process. Myoblasts overexpressing HuR-CP1 or the non-cleavable mutant of HuR, HuRD226A, are not able to engage myogenesis, while overexpressing HuR-CP2 enhances myotube formation. HuR-CP2 but not -CP1 promotes myogenesis by stabilizing the MyoD and myogenin mRNAs to the same levels as wt-HuR. Conversely, the inhibitory effects of HuR-CP1 and HuRD226A depend on their abilities to associate during myogenesis with the HuR import receptor, Trn2, leading to HuR accumulation in the cytoplasm. Therefore, we propose a model whereby the caspase-mediated cleavage of HuR generates two CPs that collaborate to regulate myogenesis; HuR-CP1 by interfering with the Trn2-mediated import of HuR and HuR-CP2 by participating in the stabilization of mRNAs encoding key MRFs.

<p>Posttranscriptional gene expression is controlled and coordinated by RNA binding proteins (RBPs), many of which recognize specific RNAs through cis-regulatory RNA elements. One of the most highly studied classes of cis-regulatory RNA elements is the AU-rich elements (AREs). AREs are bound by a class of RBPs called ARE binding proteins (ARE-BPs), of which there are over a dozen in humans including HuR, tristetraprolin (TTP) and butyrate response factors 1 and 2 (BRF1 and BRF2). TTP, BRF1 and BRF2 belong to a family of tandem C3H zinc finger proteins that destabilize ARE-containing mRNAs. HuR acts to enhance the stability and translation of ARE-containing mRNAs, a function that is rare among ARE-BPs. While each of these ARE-BPs regulates the expression of ARE-containing mRNAs, some ARE-BPs themselves are also encoded by ARE-containing mRNAs, raising the possibility that each of these ARE-BPs may regulate one another's expression. In order to determine how these ARE-BPs influence each others expression and how this affects the regulation of global gene expression programs we have focused on three different aspects of these ARE-BP networks: control, response to stimuli, and global effects.</p><p>To address of network control of ARE-BPs we have focused on how HuR regulates a network of mRNAs including TTP, BRF1 and HuR's own mRNA. We demonstrate that HuR can bind to TTP's, BRF1's and its own mRNA. Furthermore, by employing overexpression and siRNA knockdown approaches we demonstrate that these mRNAs and their corresponding 3'UTR luciferase reporters are resilient to fluctuations in HuR levels and that the degree of this resiliency is cell type and condition specific.</p><p>To address the temporal responses within an ARE-BP network we focused on how each of the members of the TTP family of ARE-BPs reacts following the induction of the other family members by using epidermal growth factor (EGF) stimulation. Here we show that induction of TTP family member mRNAs during EGF stimulation is partially attributable to changes in mRNA stability. Furthermore, we also show that TTP and BRF1 are able to bind each of the TTP family member mRNAs and subsequently affect their expression by altering their mRNA degradation rates. In addition, we demonstrate that the unique temporal induction patterns of the TTP family member RBPs is correlated with the EGF stimulated induction of TTP-bound mRNAs, suggesting that a network comprised of TTP family members is able to influence the timing of complex gene expression patterns. </p><p>Finally, to address the influence of these networks on regulation of global gene expression programs we have focused on how HuR recognizes AREs and whether it can globally recognize multiple classes of ARE-containing mRNAs, including the canonical class of AREs recognized by the TTP family members. To investigate how the three RNA recognition motifs (RRMs) of HuR contribute to ARE recognition we generated a series of RRM point mutants and test their ability to disrupt RNA recognition of each of the RRMs. To identify different classes of ARE-containing mRNAs we examined these mutants with a global RNA binding site detection method called photoactivatable ribonucleoside crosslinking immunoprecipitation (PAR-CLIP). Together these techniques suggest that the RRMs of HuR cooperate to recognize mRNA targets and that HuR's ability to bind RNA is coupled to the cellular distribution of HuR, and thus, are important in its role for regulating expression of bound mRNAs. </p><p>Together these studies indicate that ARE-BP posttranscriptional networks are highly interconnected and display complex regulatory interactions depending on cell type and stimuli. Furthermore, these networks can create complex behaviors such as timing of expression events or resiliency to fluctuations in protein levels. Finally, the components of these ARE-BP networks target partially overlapping sets of mRNAs to impact global gene expression patterns that ultimately coordinate the cellular responses to external stimuli.</p> / Dissertation