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Browsing by Author "Hundley, Heather A."
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Item Accurate Identification of RNA Editing Events Using Matched RNA and DNA Sequenced Samples Uncovers the Contribution of the Editing Landscape to Disease Progression in Glioblastoma PatientsHashemikhabir, Seyedsasan; Hundley, Heather A.; Janga, Sarath ChandraRNA editing event is increasingly appreciated as an important posttranscriptional regulatory mechanism in mammals. Adenosine deaminases that act on RNA (ADARs) are the enzymes that catalyze adenosine (A) to inosine (I) editing events. Human brain RNA is reported to have highest number of editing events. Many neurotransmitter receptors and ion channels undergo editing within exonic regions which generates a different protein than that encoded by the genome. ALU repeats in introns and untranslated regions of brain mRNAs are often targeted by editing events and result in altered splicing and post-transcriptional gene regulation.Item ADAR-mediated regulation of PQM-1 expression in neurons impacts gene expression throughout C. elegans and regulates survival from hypoxia(Public Library of Science, 2023-09-25) Mahapatra, Ananya; Dhakal, Alfa; Noguchi, Aika; Vadlamani, Pranathi; Hundley, Heather A.; Biology, School of ScienceThe ability to alter gene expression programs in response to changes in environmental conditions is central to the ability of an organism to thrive. For most organisms, the nervous system serves as the master regulator in communicating information about the animal's surroundings to other tissues. The information relay centers on signaling pathways that cue transcription factors in a given cell type to execute a specific gene expression program, but also provide a means to signal between tissues. The transcription factor PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as impacting survival from hypoxia. Herein, we reveal a novel mechanism for regulating PQM-1 expression specifically in neural cells of larval animals. Our studies reveal that the RNA-binding protein (RBP), ADR-1, binds to pqm-1 mRNA in neural cells. This binding is regulated by the presence of a second RBP, ADR-2, which when absent leads to reduced expression of both pqm-1 and downstream PQM-1 activated genes. Interestingly, we find that neural pqm-1 expression is sufficient to impact gene expression throughout the animal and affect survival from hypoxia, phenotypes that we also observe in adr mutant animals. Together, these studies reveal an important posttranscriptional gene regulatory mechanism in Caenorhabditis elegans that allows the nervous system to sense and respond to environmental conditions to promote organismal survival from hypoxia.Item ADAR3 activates NF-κB signaling and promotes glioblastoma cell resistance to temozolomide(Springer Nature, 2022-08-03) Kurup, Reshma Raghava; Oakes, Eimile K.; Vadlamani, Pranathi; Nwosu, Obi; Danthi, Pranav; Hundley, Heather A.; Medicine, School of MedicineThe RNA binding protein ADAR3 is expressed exclusively in the brain and reported to have elevated expression in tumors of patients suffering from glioblastoma compared to adjacent brain tissue. Yet, other studies have indicated that glioblastoma tumors exhibit hemizygous deletions of the genomic region encompassing ADAR3 (10p15.3). As the molecular and cellular consequences of altered ADAR3 expression are largely unknown, here we directly examined the impacts of elevated ADAR3 in a glioblastoma cell line model. Transcriptome-wide sequencing revealed 641 differentially expressed genes between control and ADAR3-expressing U87-MG glioblastoma cells. A vast majority of these genes belong to pathways involved in glioblastoma progression and are regulated by NF-κB signaling. Biochemical and molecular analysis indicated that ADAR3-expressing U87-MG cells exhibit increased NF-κB activation, and treatment with an NF-κB inhibitor abrogated the impacts of ADAR3 on gene expression. Similarly, we found that increased cell survival of ADAR3-expressing cells to temozolomide, the preferred chemotherapeutic for glioblastoma, was due to increased NF-κB activity. Aberrant constitutive NF-κB activation is a common event in glioblastoma and can impact both tumor progression and resistance to treatment. Our results suggest that elevated ADAR3 promotes NF-κB activation and a gene expression program that provides a growth advantage to glioblastoma cells.Item ADARs employ a neural-specific mechanism to regulate PQM-1 expression and survival from hypoxia(bioRxiv, 2023-05-05) Mahapatra, Ananya; Dhakal, Alfa; Noguchi, Aika; Vadlamani, Pranathi; Hundley, Heather A.; Medicine, School of MedicineThe ability to alter gene expression programs in response to changes in environmental conditions is central to the ability of an organism to thrive. For most organisms, the nervous system serves as the master regulator in communicating information about the animal's surroundings to other tissues. The information relay centers on signaling pathways that cue transcription factors in a given cell type to execute a specific gene expression program, but also provide a means to signal between tissues. The transcription factor PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as impacting survival from hypoxia. Herein, we reveal a novel mechanism for regulating PQM-1 expression specifically in neural cells of larval animals. Our studies reveal that the RNA binding protein, ADR-1, binds to pqm-1 mRNA in neural cells. This binding is regulated by the presence of a second RNA binding protein, ADR-2, which when absent leads to reduced expression of both pqm-1 and downstream PQM-1 activated genes. Interestingly, we find that neural pqm-1 expression is sufficient to impact gene expression throughout the animal and affect survival from hypoxia; phenotypes that we also observe in adr mutant animals. Together, these studies reveal an important post-transcriptional gene regulatory mechanism that allows the nervous system to sense and respond to environmental conditions to promote organismal survival from hypoxia.Item Caenorhabditis elegans expressing a Vitellogenin::GFP fusion protein show reduced embryo content and brood size(Caltech Library, 2022-03-01) Erdmann, Emily A.; Abraham, Olivia; Hundley, Heather A.; Biology, School of ScienceVitellogenin::GFP fusion proteins have been used in several studies of the synthesis, endocytosis, and function of yolk in Caenorhabditis elegans. Here we report that one commonly used transgenic strain harboring a vit-2::gfp fusion displays defects in reproduction that lead to a significantly decreased embryo content and brood size in adult worms.Item Disruption in A-to-I Editing Levels Affects C. elegans Development More Than a Complete Lack of Editing(Elsevier, 2019-04) Ganem, Nabeel S.; Ben-Asher, Noa; Manning, Aidan C.; Deffit, Sarah N.; Washburn, Michael C.; Wheeler, Emily C.; Yeo, Gene W.; Ben-Naim Zgayer, Orna; Mantsur, Einav; Hundley, Heather A.; Lamm, Ayelet T.A-to-I RNA editing, catalyzed by ADAR proteins, is widespread in eukaryotic transcriptomes. Studies showed that, in C. elegans, ADR-2 can actively deaminate dsRNA, whereas ADR-1 cannot. Therefore, we set out to study the effect of each of the ADAR genes on the RNA editing process. We performed comprehensive phenotypic, transcriptomics, proteomics, and RNA binding screens on worms mutated in a single ADAR gene. We found that ADR-1 mutants exhibit more-severe phenotypes than ADR-2, and some of them are a result of non-editing functions of ADR-1. We also show that ADR-1 significantly binds edited genes and regulates mRNA expression, whereas the effect on protein levels is minor. In addition, ADR-1 primarily promotes editing by ADR-2 at the L4 stage of development. Our results suggest that ADR-1 has a significant role in the RNA editing process and in altering editing levels that affect RNA expression; loss of ADR-1 results in severe phenotypes.Item mRNA Editing, Processing and Quality Control in Caenorhabditis elegans(Oxford University Press, 2020-07) Arribere, Joshua A.; Kuroyanagi, Hidehito; Hundley, Heather A.; Biology, School of ScienceWhile DNA serves as the blueprint of life, the distinct functions of each cell are determined by the dynamic expression of genes from the static genome. The amount and specific sequences of RNAs expressed in a given cell involves a number of regulated processes including RNA synthesis (transcription), processing, splicing, modification, polyadenylation, stability, translation, and degradation. As errors during mRNA production can create gene products that are deleterious to the organism, quality control mechanisms exist to survey and remove errors in mRNA expression and processing. Here, we will provide an overview of mRNA processing and quality control mechanisms that occur in Caenorhabditis elegans, with a focus on those that occur on protein-coding genes after transcription initiation. In addition, we will describe the genetic and technical approaches that have allowed studies in C. elegans to reveal important mechanistic insight into these processes.Item Profiling neural editomes reveals a molecular mechanism to regulate RNA editing during development(Cold Spring Harbor Laboratory Press, 2021-01) Rajendren, Suba; Dhakal, Alfa; Vadlamani, Pranathi; Townsend, Jack; Deffit, Sarah N.; Hundley, Heather A.; Biology, School of ScienceAdenosine (A) to inosine (I) RNA editing contributes to transcript diversity and modulates gene expression in a dynamic, cell type-specific manner. During mammalian brain development, editing of specific adenosines increases, whereas the expression of A-to-I editing enzymes remains unchanged, suggesting molecular mechanisms that mediate spatiotemporal regulation of RNA editing exist. Herein, by using a combination of biochemical and genomic approaches, we uncover a molecular mechanism that regulates RNA editing in a neural- and development-specific manner. Comparing editomes during development led to the identification of neural transcripts that were edited only in one life stage. The stage-specific editing is largely regulated by differential gene expression during neural development. Proper expression of nearly one-third of the neurodevelopmentally regulated genes is dependent on adr-2, the sole A-to-I editing enzyme in C. elegans However, we also identified a subset of neural transcripts that are edited and expressed throughout development. Despite a neural-specific down-regulation of adr-2 during development, the majority of these sites show increased editing in adult neural cells. Biochemical data suggest that ADR-1, a deaminase-deficient member of the adenosine deaminase acting on RNA (ADAR) family, is competing with ADR-2 for binding to specific transcripts early in development. Our data suggest a model in which during neural development, ADR-2 levels overcome ADR-1 repression, resulting in increased ADR-2 binding and editing of specific transcripts. Together, our findings reveal tissue- and development-specific regulation of RNA editing and identify a molecular mechanism that regulates ADAR substrate recognition and editing efficiency.Item A protein-protein interaction underlies the molecular basis for substrate recognition by an adenosine-to-inosine RNA-editing enzyme(Oxford University Press, 2018-10-12) Rajendren, Suba; Manning, Aidan C.; Al-Awadi, Haider; Yamada, Kentaro; Takagi, Yuichiro; Hundley, Heather A.; Biochemistry and Molecular Biology, School of MedicineAdenosine deaminases that act on RNA (ADARs) convert adenosine to inosine within double-stranded regions of RNA, resulting in increased transcriptomic diversity, as well as protection of cellular double-stranded RNA (dsRNA) from silencing and improper immune activation. The presence of dsRNA-binding domains (dsRBDs) in all ADARs suggests these domains are important for substrate recognition; however, the role of dsRBDs in vivo remains largely unknown. Herein, our studies indicate the Caenorhabditis elegans ADAR enzyme, ADR-2, has low affinity for dsRNA, but interacts with ADR-1, an editing-deficient member of the ADAR family, which has a 100-fold higher affinity for dsRNA. ADR-1 uses one dsRBD to physically interact with ADR-2 and a second dsRBD to bind to dsRNAs, thereby tethering ADR-2 to substrates. ADR-2 interacts with >1200 transcripts in vivo, and ADR-1 is required for 80% of these interactions. Our results identify a novel mode of substrate recognition for ADAR enzymes and indicate that protein-protein interactions can guide substrate recognition for RNA editors.Item RNA binding by ADAR3 inhibits adenosine-to-inosine editing and promotes expression of immune response protein MAVS(American Society for Biochemistry and Molecular Biology, 2022) Kurup, Reshma Raghava; Oakes, Eimile K.; Manning, Aidan C.; Mukherjee, Priyanka; Vadlamani, Pranathi; Hundley, Heather A.; Medicine, School of MedicineMembers of the ADAR family of double-stranded RNA-binding proteins regulate one of the most abundant RNA modifications in humans, the deamination of adenosine to inosine. Several transcriptome-wide studies have been carried out to identify RNA targets of the active deaminases ADAR1 and ADAR2. However, our understanding of ADAR3, the brain-specific deaminase-deficient ADAR family member, is limited to a few transcripts. In this study, we identified over 3300 transcripts bound by ADAR3 and observed that binding of ADAR3 correlated with reduced editing of over 400 sites in the glioblastoma transcriptome. We further investigated the impact of ADAR3 on gene regulation of the transcript that encodes MAVS, an essential protein in the innate immune response pathway. We observed reduced editing in the MAVS 3' UTR in cells expressing increased ADAR3 or reduced ADAR1 suggesting ADAR3 acts as a negative regulator of ADAR1-mediated editing. While neither ADAR1 knockdown or ADAR3 overexpression affected MAVS mRNA expression, we demonstrate increased ADAR3 expression resulted in upregulation of MAVS protein expression. In addition, we created a novel genetic mutant of ADAR3 that exhibited enhanced RNA binding and MAVS upregulation compared with wildtype ADAR3. Interestingly, this ADAR3 mutant no longer repressed RNA editing, suggesting ADAR3 has a unique regulatory role beyond altering editing levels. Altogether, this study provides the first global view of ADAR3-bound RNAs in glioblastoma cells and identifies both a role for ADAR3 in repressing ADAR1-mediated editing and an RNA-binding dependent function of ADAR3 in regulating MAVS expression.