Browsing by Subject "RNA"
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Item Cell-Free Formation of RNA Granules(2012-07-16) Han, Tina Wei; McKnight, Steven L.Asymmetric RNA localization is a mechanism by which a cell can spatially and temporally regulate the translation of RNAs. This mechanism is essential for many developmental processes such as germ cell formation in Drosophila embryos, as well as establishment of cell polarity and synaptic plasticity in the brain. In many instances, asymmetric RNA localization is achieved through transport and sequestration by RNA granules. RNA granules are large, non-membrane bound ribonucleoparticles that have been observed in various biological contexts. Unfortunately, because RNA granules are highly heterogeneous and weakly associating aggregates, they can be difficult to study biochemically, which constitutes a major impediment for gaining a more detailed understanding of the mechanisms governing RNA granule assembly. Here we describe two in vitro models for studying RNA granule assembly. The first method is based on the precipitation activity of a 3,5-disubstituted isoxazole compound that can be used as a quick and efficient pharmacological tool to probe the function and regulation of RNA granules. The second method utilizes a three-dimensional protein-retaining hydrogel formed from a recombinant protein. Polypeptides of low amino acid complexity were found to be the sequence determinants of isoxazole precipitation and hydrogel retention. Next generation sequencing was used to identify RNAs that partitioned with granule components in both isoxazole and hydrogel models and were found to be enriched in mRNAs known to be constituents of neuronal transport granules for dendritic localization. The overrepresented gene ontology categories for these RNAs included cell adhesion, extracellular matrix, and synaptic proteins. The average length of the 3’UTR of these RNAs was found to be longer than the 3’UTRs of RNAs excluded from the cell-free RNA granule preparations. These two in vitro models for studying RNA granule assembly offer a novel approach to identify candidate targets recruited to RNA granules by specific RNA-binding proteins.Item Characterization of an Orphan Riboswitch: Identification of a Metal-Sensing Regulatory RNA(2011-09-30) Wakeman, Catherine Ann; Winkler, Wade C.Riboswitches are RNA-based genetic control elements found in untranslated regions of the mRNA transcript that they regulate. These RNA motifs are highly structured and bind metabolites to elicit control of gene expression. Typically, the metabolite sensed by these RNAs is a component of the metabolic pathway in which the regulated gene product resides. The focus of this project has been the identification of the ligand for a riboswitch that was discovered using bioinformatics-based search methods. This riboswitch was designated the ykoK RNA element due to its location in the 5' UTR of the B. subtilis ykoK (mgtE) gene, which appears to be a magnesium transporter. Therefore, the possibility that this RNA senses magnesium levels was explored. The data revealed that the RNA element imparts magnesium-responsive regulation to the ykoK gene. These data also indicated which portions of the RNA are essential for genetic regulation. The results of a battery of biochemical tests demonstrated that magnesium triggers a concerted conformational change in the RNA such that it adopts a compacted tertiary structure. Resolution of the three-dimensional structure of the RNA in the magnesium bound state revealed the basis of this metal-induced tertiary conformation and how this relates to genetic control. Intriguingly, this structure revealed the presence of six magnesium ions, making this the first example of multiple ligands binding to a single riboswitch aptamer. When individual metal-binding sites were eliminated using phosphorothioate substitutions, it became evident that all six of these magnesium-binding sites and up to three additional metal-binding sites are required for function of this RNA. Therefore, these data demonstrate that the ykoK RNA element, now designated the M-box RNA, directly senses intracellular magnesium levels for the purposes of genetic control. These findings should have broad implications given that this RNA element is wide spread among Gram-positive bacteria and appears to regulate many additional gene categories such as ABC transporters, cell division proteins, and proteins of unknown function. The exploration of the connection between magnesium concentration and the expression levels of these proteins might provide insights into previously undefined functional roles.Item Characterization of the Roles of Intrinsically Disordered Regions from RNA-Binding Proteins in Phase Separation(2016-05-27) Lin, Yuan; Ross, Elliott M.; Liu, Qinghua; Tu, Benjamin; Rosen, Michael K.Eukaryotic cells organize complex biochemical reactions through compartmentalization. While many intracellular compartments are enclosed by membranes, others are not. Messenger ribonucleoprotein (mRNP) granules are membrane-less organelles that enrich RNA and RNA-binding proteins containing intrinsically disordered regions (IDRs). I demonstrate that IDRs, coupled with RNA binding domain and RNA, can phase separate in vitro, producing dynamic liquid droplets. Over time, these droplets mature into more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with the formation of fibrous structures. Pathological mutation within IDRs leads to the acceleration of maturation. Different disordered domains can co-assemble into phase-separated droplets. In the case of the IDR from FUS (fused in sarcoma), I show that tyrosine residues are important in promoting phase separation. Either mutation of these aromatic residues or phosphorylation of the IDR disassembles liquid droplets. I further discover that the disassembly is due to the disruption of aromatic interactions mediated by critical tyrosine residues and therefore an increase in the overall solubility of proteins. Taken together, these studies demonstrate a plausible mechanism by which interactions between IDRs, coupled with RNA binding, could contribute to mRNP granule assembly in vivo by promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation of maturation could contribute to diseases that are associated with aberrant mRNP granules. Posttranslational modifications of IDRs could modulate the assembly and disassembly of mRNP granules by altering the solubility of IDRs.Item Heme-Based Oxygen Sensors of Commensal, Symbiotic, and Pathogenic Bacteria(2012-08-15) Tuckerman, Jason Robert; Gilles-Gonzalez, Marie-AldaDirect oxygen sensors are proteins that serve as "on-off switches" to cause reversible and adaptive changes in the activities of other proteins or genes, with great specificity in response to fluctuations in oxygen concentration. The heme-based oxygen sensors are a large class of direct oxygen sensors that feature direct binding of oxygen to a sensory heme-containing domain. This heme-binding region couples to a regulatory domain within the same polypeptide. The types of functionalities controlled by these oxygen-specific switches are diverse, and include the regulation of protein activities, gene expression, and second messenger elaboration. A primary focus of this work was the biochemical characterization of a pair of heme-based oxygen sensors involved in the control of the bacterial second messenger cyclic diguanylic acid (c-di-GMP) in Escherichia coli. We discovered that these enzymes, designated DosC and DosP, serve as a diguanylate cyclase and c-di-GMP phosphodiesterase pair that associate with components of the E. coli RNA degradosome in vivo. Importantly, one member of these degradosomes, PNPase, is a direct, high-affinity target of c-di-GMP. These findings imply that specialized oxygen-sensing degradosomes exist in E. coli. In these oxygen-sensing degradosomes cellular oxygen levels regulate PNPase processing of specific RNA transcripts via c-di-GMP. A secondary focus of this work was the characterization of a novel two-component system in M. tuberculosis involved in the non-replicating persistent phase of this bacterium in a typical TB infection. Here, the activities of two heme-containing histidine kinases, DosT and DevS, were discovered to be inhibited specifically by oxygen. As DosT and DevS are the primary regulators of the dormancy survival regulator (DosR/DevR) transcription factor, these results contributed a molecular explanation for the numerous observations linking oxygen and DevR to the dormancy phenotype of M. tuberculosis seen both in vitro and in vivo.Item MicroRNA: bigger isn't better!(2012-05-25) Igarashi, PeterItem Molecular Underpinnings of Human Brain Evolution and Cognition at Cellular Resolution(December 2023) Caglayan, Emre; Chahrour, Maria; Hon, Gary C.; Madabhushi, Ram; Sun, Lu O.; Konopka, GenevieveMolecular and functional characterization of the human brain is challenging due to its experimental inaccessibility. Most of our understanding about human brain function relies on the assumption that biological processes uncovered in model organisms are conserved in humans. Comparisons of the humanii brain with non-human primate brains offer to both uncover the novelties in human brain evolution and better evaluate the insights obtained from model organisms about human brain function. To achieve this, highthroughput sequencing methods on post-mortem brain tissues provide a rewarding readout to understand human brain evolution at the molecular level. In addition to their use in comparative studies, these technologies were also utilized with a hope to understand molecular underpinnings of measurable human brain activity metrics. During my dissertation, I read relevant literature extensively (Chapter 1) and sought to understand human-specific epigenomic and transcriptomic changes at cellular resolution in the cortical brain (Chapter 2). Additionally, after in-depth analysis of many human brain single-nuclei RNA-seq datasets, I found a pervasive ambient RNA contamination problem, and devised in silico solutions to tackle this problem. My efforts improved the analytical approach in the field as well as in my research (Chapter 3). I have also been involved in efforts to identify transcriptomic correlates of brain activity in human subjects (Chapters 4-5). After detailing these efforts, I discuss the implications of these findings, weigh their impact on our understanding of human brain function and offer ideas for further research (Chapter 6).Item Rbfox1 Regulates mRNA Translation to Promote Germ Cell Differentiation(2016-07-12) Carreira-Rosario, Arnaldo; Zinn, Andrew R.; Buszczak, Michael; Conrad, Nicholas; Olson, Eric N.; Rothenfluh, AdrianGerm cells are the only cells that can give rise to an embryo. During differentiation, female germ cells that will give rise to oocytes form a syncytium called a germline cyst. The mechanisms that regulate germline cyst development remain poorly understood. In Drosophila, germline stem cells (GSCs) undergo an asymmetric division, giving rise to a stem cell and a cystoblast that then divides four times to produce a 16-cell germline cyst. This 16-cell cyst will then continue differentiation until it forms a mature oocyte. Drosophila RNA-binding Fox-1 (Rbfox1), also known as Ataxin-2 Binding Protein 1 (A2BP1), mutant females exhibit a germ cell differentiation defect that results in germline cystic tumors. The Rbfox genes encode several isoforms, many of which contain a highly conserved RNA recognition motif (RRM). Disruption of human RBFOX homologs have been linked with a number of different neurological disorders and cancers. Some of these isoforms localize to the nucleus while others localize to the cytoplasm. Nuclear forms have well-established roles in regulating alternative splicing. However the function of Rbfox in the cytoplasm remains unclear. Here, we demonstrate that cytoplasmic Drosophila Rbfox1 regulates germline cyst development. We further show that Rbfox1 represses the translation of mRNAs that contain (U)GCAUG elements within their 3’ UTRs. We have identified pumilio (pum) as a critical Rbfox1 target gene. Pum is an RNA-binding protein essential for germline maintenance across species. During germline cyst differentiation, Rbfox1 silences pum mRNA translation thereby promoting germ cell development. Mis-expression of pum results in the formation of germline cystic tumors that resemble Rbfox1 mutant phenotype. In addition, these cysts breakdown and dedifferentiate back to single, mitotically active cells. Together these results reveal that cytoplasmic Rbfox family members regulate the translation of specific target mRNAs to promote differentiation. In the Drosophila ovary, this activity provides a genetic barrier that prevents germ cells from reverting back to an earlier developmental state. These findings have thus advanced our understanding of germline development and the molecular function of Rbfox proteins, with implications in cellular differentiation and Rbfox-related disorders.Item Structures and Regulation of the N6-Methyladenosine Methyltransferase METTL16(2020-05-01T05:00:00.000Z) Doxtader, Katelyn Ashley; Chook, Yuh Min; Nam, Yunsun; Kraus, W. Lee; Conrad, NicholasChemical modifications of RNA control the abundance and function of both coding and non-coding RNA molecules. The most abundant mRNA modification is N6 methyladenosine (m6A), which has been shown to regulate RNA splicing, translation and decay. The m6A modification is deposited by methyltransferases using S-adenosylmethionine (SAM) as the methyl donor. Determining the specificity and mechanism of action of m6A methyltransfersaes is an essential step in dissecting the role of the m6A modification in RNA metabolism. Two enzymes that are responsible for creating the m6A mark on mRNA are the METTL3/METTL14 heterodimer and METTL16. METTL3/METTL14 are responsible for most of the m6A modifications found on RNAs, whereas the specific activity of METTL16 on the SAM synthetase gene, MAT2A, modulates SAM homeostasis, implicating METTL16 as a global regulator of methylation in the cell. Here, I present structural and biochemical data towards determining the specificity, function and regulation of both the METTL16 and METTL3/METTL14 methyltransferases. METTL16 uses a consensus RNA sequence and structure to specifically recognize and methylate its targets. METTL16 is also auto-inhibited, and its activity can be increased through perturbations in the protein and RNA structures. These perturbations potentially increase METTL16 activity through enhanced release of both the methylated RNA and cofactor reaction products, which allows for complex regulation of SAM homeostasis. In contrast, METTL3 mediated m6A modification requires METTL14 for structural support and RNA specificity. These data provide the foundation for understanding the function and regulation of two important m6A methyltransferases, which have implications for both SAM homeostasis and RNA metabolism.Item Transcriptional and Cytokine Profiles Identify CXCL9 as a Biomarker of Disease Activity in Morphea(2017-03-31) O'Brien, Jack Christopher; Jacobe, Heidi; Chong, Benjamin F.; Sendelbach, DorothyInterferon-related pathways have not been studied in morphea and biomarkers are needed. We sought to characterize morphea serum cytokine imbalance and interferon-related gene expression in blood and skin to address this gap by performing a case-control study of 87 participants with morphea and 26 healthy controls. We used multiplexed immunoassays to determine serum cytokine concentrations, performed transcriptional profiling of whole blood and lesional morphea skin, and employed double-staining immunohistochemistry to determine the cutaneous cellular source of CXCL9. We found that CXCL9 was present at increased concentrations in morphea serum (p<0.0001) as were other T-helper 1 cytokines. CXCL9 serum concentration correlated with the modified localized scleroderma skin severity index (r=0.44, p=0.0001), a validated measure of disease activity. CXCL9 gene expression was also increased in inflammatory lesional morphea skin (Fold change 30.6, p=0.006), while preliminary transcriptional profiling showed little evidence for interferon signature in whole blood. Double-staining immunohistochemistry revealed CXCL9 co-localized with CD68+ dermal macrophages. In summary, inflammatory morphea is characterized by T-helper 1 cytokine imbalance in serum, particularly CXCL9, which is associated with disease activity. CXCL9 expression in lesional macrophages implicates the skin as the source of circulating cytokines. CXCL9 is a promising biomarker of disease activity in morphea.Item Understanding RNA Regulation Through Analysis of CLIP-Seq Data(2015-11-18) Wang, Tao; Mendell, Joshua T.; Xie, Yang; Xiao, Guanghua; Mangelsdorf, David J.; Zhang, Michael Q.The past decades have witnessed a surge of discoveries revealing RNA regulation as a central player in cellular processes. The advent of cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) technology has recently enabled the investigation of genome-wide RNA binding protein-RNA interactions, which is a very important component of RNA-regulation. However, proper and systematic bioinformatics analysis of CLIP-Seq data is still lacking and challenging. For the past few years, I have been devoting my research to methodological developments of CLIP-Seq data analysis, and developed MiClip and dCLIP for peak calling and differential analysis of CLLIP-Seq data, respectively. I have also applied my CLIP-Seq analysis pipelines in on-campus collaborating projects, in which I identified ORF57 and nuclear AGO2 binding sites. Finally, I conducted analysis of public CLIP-Seq datasets to systematically characterize RNA binding protein targeting sites on circular RNAs.Item [UT Southwestern Medical Center News](2007-03-22) McKenzie, AlineItem [UT Southwestern Medical Center News](2007-01-28) Siegfried, AmandaItem [UT Southwestern Medical Center News](2008-07-06) Siegfried, Amanda