11 results on '"Christine A Roden"'
Search Results
2. Design considerations for analyzing protein translation regulation by condensates
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Christine A. Roden and Amy S. Gladfelter
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Biomolecular Condensates ,Protein Folding ,Binding Sites ,Eukaryota ,Fluorescent Antibody Technique ,RNA-Binding Proteins ,RNA ,Translation (biology) ,Computational biology ,Biology ,Eukaryotic Cells ,Ribonucleoproteins ,Genes, Reporter ,Translational repression ,Protein Biosynthesis ,Perspective ,Translational regulation ,RNA Sequence ,Native protein ,RNA, Messenger ,Protein translation ,Genetic Engineering ,Ribosomes ,Molecular Biology ,Protein Binding - Abstract
One proposed role for biomolecular condensates that contain RNA is translation regulation. In several specific contexts, translation has been shown to be modulated by the presence of a phase-separating protein and under conditions which promote phase separation, and likely many more await discovery. A powerful tool for determining the rules for condensate-dependent translation is the use of engineered RNA sequences, which can serve as reporters for translation efficiency. This Perspective will discuss design features to consider in engineering RNA reporters to determine the role of phase separation in translational regulation. Specifically, we will cover (i) how to engineer RNA sequence to recapitulate native protein/RNA interactions, (ii) the advantages and disadvantages for commonly used reporter RNA sequences, and (iii) important control experiments to distinguish between binding- and condensation-dependent translational repression. The goal of this review is to promote the design and application of faithful translation reporters to demonstrate a physiological role of biomolecular condensates in translation.
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- 2021
3. An Extensive Network of TET2-Targeting MicroRNAs Regulates Malignant Hematopoiesis
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Jijun Cheng, Shangqin Guo, Suning Chen, Stephen J. Mastriano, Chaochun Liu, Ana C. D’Alessio, Eriona Hysolli, Yanwen Guo, Hong Yao, Cynthia M. Megyola, Dan Li, Jun Liu, Wen Pan, Christine A. Roden, Xiao-Ling Zhou, Kartoosh Heydari, Jianjun Chen, In-Hyun Park, Ye Ding, Yi Zhang, and Jun Lu
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Biology (General) ,QH301-705.5 - Abstract
The Ten-Eleven-Translocation 2 (TET2) gene, which oxidates 5-methylcytosine in DNA to 5-hydroxylmethylcytosine (5hmC), is a key tumor suppressor frequently mutated in hematopoietic malignancies. However, the molecular regulation of TET2 expression is poorly understood. We show that TET2 is under extensive microRNA (miRNA) regulation, and such TET2 targeting is an important pathogenic mechanism in hematopoietic malignancies. Using a high-throughput 3′ UTR activity screen, we identify >30 miRNAs that inhibit TET2 expression and cellular 5hmC. Forced expression of TET2-targeting miRNAs in vivo disrupts normal hematopoiesis, leading to hematopoietic expansion and/or myeloid differentiation bias, whereas coexpression of TET2 corrects these phenotypes. Importantly, several TET2-targeting miRNAs, including miR-125b, miR-29b, miR-29c, miR-101, and miR-7, are preferentially overexpressed in TET2-wild-type acute myeloid leukemia. Our results demonstrate the extensive roles of miRNAs in functionally regulating TET2 and cellular 5hmC and reveal miRNAs with previously unrecognized oncogenic potential. Our work suggests that TET2-targeting miRNAs might be exploited in cancer diagnosis.
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- 2013
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4. Double-stranded RNA drives SARS-CoV-2 nucleocapsid protein to undergo phase separation at specific temperatures
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Christine A Roden, Yifan Dai, Catherine A Giannetti, Ian Seim, Myungwoon Lee, Rachel Sealfon, Grace A McLaughlin, Mark A Boerneke, Christiane Iserman, Samuel A Wey, Joanne L Ekena, Olga G Troyanskaya, Kevin M Weeks, Lingchong You, Ashutosh Chilkoti, and Amy S Gladfelter
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Binding Sites ,biology ,Coronavirus disease 2019 (COVID-19) ,SARS-CoV-2 ,Chemistry ,viruses ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,fungi ,Temperature ,RNA-Binding Proteins ,RNA ,Translation (biology) ,Phosphoproteins ,biology.organism_classification ,Article ,In vitro ,Cell biology ,RNA silencing ,Genetics ,Coronavirus Nucleocapsid Proteins ,RNA, Viral ,Nucleic acid structure ,Betacoronavirus ,RNA, Double-Stranded - Abstract
Nucleocapsid protein (N-protein) is required for multiple steps in betacoronaviruses replication. SARS-CoV-2-N-protein condenses with specific viral RNAs at particular temperatures making it a powerful model for deciphering RNA sequence specificity in condensates. We identify two separate and distinct double-stranded, RNA motifs (dsRNA stickers) that promote N-protein condensation. These dsRNA stickers are separately recognized by N-protein's two RNA binding domains (RBDs). RBD1 prefers structured RNA with sequences like the transcription-regulatory sequence (TRS). RBD2 prefers long stretches of dsRNA, independent of sequence. Thus, the two N-protein RBDs interact with distinct dsRNA stickers, and these interactions impart specific droplet physical properties that could support varied viral functions. Specifically, we find that addition of dsRNA lowers the condensation temperature dependent on RBD2 interactions and tunes translational repression. In contrast RBD1 sites are sequences critical for sub-genomic (sg) RNA generation and promote gRNA compression. The density of RBD1 binding motifs in proximity to TRS-L/B sequences is associated with levels of sub-genomic RNA generation. The switch to packaging is likely mediated by RBD1 interactions which generate particles that recapitulate the packaging unit of the virion. Thus, SARS-CoV-2 can achieve biochemical complexity, performing multiple functions in the same cytoplasm, with minimal protein components based on utilizing multiple distinct RNA motifs that control N-protein interactions.
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- 2021
5. Role of spatial patterning of N-protein interactions in SARS-CoV-2 genome packaging
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Christine A. Roden, Ian Seim, and Amy S. Gladfelter
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Order (biology) ,Biophysics ,Nucleic acid ,RNA ,Computational biology ,Biology ,Genome ,DNA-binding protein ,Paraspeckles ,Article ,Subgenomic mRNA ,Conserved sequence ,Protein–protein interaction - Abstract
Viruses must efficiently and specifically package their genomes while excluding cellular nucleic acids and viral sub-genomic fragments. Some viruses use specific packaging signals, which are conserved sequence/structure motifs present only in the full-length genome. Recent work has shown that viral proteins important for packaging can undergo liquid-liquid phase separation (LLPS), where one or two viral nucleic acid binding proteins condense with the genome. The compositional simplicity of viral components lends itself well to theoretical modeling compared to more complex cellular organelles. Viral LLPS can be limited to one or two viral proteins and a single genome that is enriched in LLPS-promoting features. In our previous study, we observed that LLPS-promoting sequences of SARS-CoV-2 are located at the 5ʹ and 3ʹ ends of the genome, whereas the middle of the genome is predicted to consist mostly of solubilizing elements. Is this arrangement sufficient to drive single genome packaging, genome compaction, and genome cyclization? We addressed these questions using a coarse-grained polymer model, LASSI, to study the LLPS of nucleocapsid protein with RNA sequences that either promote LLPS or solubilization. With respect to genome cyclization, we find the most optimal arrangement restricts LLPS-promoting elements to the 5ʹ and 3ʹ ends of the genome, consistent with the native spatial patterning. Genome compaction is enhanced by clustered LLPS-promoting binding sites, while single genome packaging is most efficient when binding sites are distributed throughout the genome. These results suggest that many and variably positioned LLPS-promoting signals can support packaging in the absence of a singular packaging signal which argues against necessity of such a feature. We hypothesize that this model should be generalizable to multiple viruses as well as cellular organelles like paraspeckles, which enrich specific, long RNA sequences in a defined arrangement.Statement of significanceThe COVID-19 pandemic has motivated research of the basic mechanisms of coronavirus replication. A major challenge faced by viruses such as SARS-CoV-2 is the selective packaging of a large genome in a relatively small capsid while excluding host and sub-genomic nucleic acids. Genomic RNA of SARS-CoV-2 can condense with the Nucleocapsid (N-protein), a structural protein component critical for packaging of many viruses. Notably, certain regions of the genomic RNA drive condensation of N-protein while other regions solubilize it. Here, we explore how the spatial patterning of these opposing elements promotes single genome compaction, packaging, and cyclization. This model informs futurein silicoexperiments addressing spatial patterning of genomic features that are experimentally intractable because of the length of the genome.
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- 2021
6. Genomic RNA Elements Drive Phase Separation of the SARS-CoV-2 Nucleocapsid
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Ethan J. Fritch, Christine A. Roden, Manolis Kellis, Amy S. Gladfelter, Mark A. Boerneke, Ralph S. Baric, Timothy P. Sheahan, Kevin M. Weeks, Grace A. McLaughlin, Chandra L. Theesfeld, Joanne Ekena, Yixuan J. Hou, Christiane Iserman, Chase A. Weidmann, Rachel Sealfon, Olga G. Troyanskaya, and Irwin Jungreis
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Drug Evaluation, Preclinical ,Genome, Viral ,Biology ,medicine.disease_cause ,Antiviral Agents ,Genome ,Article ,03 medical and health sciences ,0302 clinical medicine ,Chlorocebus aethiops ,medicine ,Animals ,Coronavirus Nucleocapsid Proteins ,Humans ,Nucleic acid structure ,Binding site ,Nucleocapsid ,Vero Cells ,Molecular Biology ,030304 developmental biology ,Coronavirus ,0303 health sciences ,SARS-CoV-2 ,HEK 293 cells ,COVID-19 ,RNA ,Cell Biology ,Phosphoproteins ,Small molecule ,COVID-19 Drug Treatment ,HEK293 Cells ,Biophysics ,Vero cell ,RNA, Viral ,030217 neurology & neurosurgery - Abstract
We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with viral RNA. N-protein condenses with specific RNA genomic elements under physiological buffer conditions and condensation is enhanced at human body temperatures (33°C and 37°C) and reduced at room temperature (22°C). RNA sequence and structure in specific genomic regions regulate N-protein condensation while other genomic regions promote condensate dissolution, potentially preventing aggregation of the large genome. At low concentrations, N-protein preferentially crosslinks to specific regions with single-stranded RNA flanked by structure and these features specify the location, number, and strength of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is RNA sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules, and presents a screenable process for identifying antiviral compounds effective against SARS-CoV-2., Graphical Abstract, Highlights Phase separation occurs with the viral genome (gRNA) and at human body temperature. Phase separation is driven by specific elements in gRNA. RBD mutant N-protein fails to undergo LLPS; exhibits altered RNA crosslinking. N-protein forms liquid-like droplets in cells., Iserman and Roden et al. demonstrate phase separation (LLPS) of SARS-CoV-2 nucleocapsid (N-protein) with viral RNA. Viral RNA sequences promote or oppose phase separation depending on binding patterns of N-protein and genomic RNA. LLPS-promoting sequences occur at 5′ and 3′-Ends of the genome, suggestive of a genome packaging role.
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- 2020
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7. RNA contributions to the form and function of biomolecular condensates
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Amy S. Gladfelter and Christine A. Roden
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Chemical Phenomena ,Macromolecular Substances ,Cell ,Cellular functions ,RNA transport ,Article ,Cell Physiological Phenomena ,03 medical and health sciences ,Protein Aggregates ,0302 clinical medicine ,Form and function ,medicine ,Animals ,Humans ,Molecular Biology ,030304 developmental biology ,0303 health sciences ,Chemistry ,RNA ,RNA-Binding Proteins ,Cell Biology ,Compartmentalization (psychology) ,medicine.anatomical_structure ,Cytoplasm ,Multiprotein Complexes ,Biophysics ,030217 neurology & neurosurgery ,Function (biology) - Abstract
Biomolecular condensation partitions cellular contents and has important roles in stress responses, maintaining homeostasis, development and disease. Many nuclear and cytoplasmic condensates are rich in RNA and RNA-binding proteins (RBPs), which undergo liquid-liquid phase separation (LLPS). Whereas the role of RBPs in condensates has been well studied, less attention has been paid to the contribution of RNA to LLPS. In this Review, we discuss the role of RNA in biomolecular condensation and highlight considerations for designing condensate reconstitution experiments. We focus on RNA properties such as composition, length, structure, modifications and expression level. These properties can modulate the biophysical features of native condensates, including their size, shape, viscosity, liquidity, surface tension and composition. We also discuss the role of RNA-protein condensates in development, disease and homeostasis, emphasizing how their properties and function can be determined by RNA. Finally, we discuss the multifaceted cellular functions of biomolecular condensates, including cell compartmentalization through RNA transport and localization, supporting catalytic processes, storage and inheritance of specific molecules, and buffering noise and responding to stress.
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- 2020
8. Specific viral RNA drives the SARS CoV-2 nucleocapsid to phase separate
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Chandra L. Theesfeld, Avinash Boppana, Amy S. Gladfelter, Christine A. Roden, Mark A. Boerneke, Kevin M. Weeks, Rachel Sealfon, Timothy P. Sheahan, Yixuan J. Hou, Grace A. McLaughlin, Ralph S. Baric, Olga G. Troyanskaya, Christopher Y. Park, Ethan J. Fritch, Irwin Jungreis, and Christiane Iserman
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Coronavirus disease 2019 (COVID-19) ,Chemistry ,gRNA packaging ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,nucleocapsid ,SARS CoV-2 ,RNA ,Genome ,Small molecule ,Article ,Cell biology ,Viral replication ,Viral rna ,phase separation ,Sequence (medicine) - Abstract
A mechanistic understanding of the SARS-CoV-2 viral replication cycle is essential to develop new therapies for the COVID-19 global health crisis. In this study, we show that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome, and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5’-End) under physiological conditions and is enhanced at human upper airway temperatures. N-protein condensates exclude non-packaged RNA sequences. We comprehensively map sites bound by N-protein in the 5’-End and find preferences for single-stranded RNA flanked by stable structured elements. Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.
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- 2020
9. FXR1 splicing is important for muscle development and biomolecular condensates in muscle cells
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John M. Crutchley, Xiaomin Chen, Shawn M. Lyons, Anthony Rodríguez-Vargas, Amy S. Gladfelter, Christine A. Roden, Danielle C. Jordan, Ennessa G. Curry, Paul A. Anderson, Marko E. Horb, Jean A. Smith, R. Eric Blue, Jimena Giudice, and Samantha E. R. Dundon
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Adult ,Male ,Gene isoform ,RNA biology ,Xenopus ,Mice, Inbred Strains ,Development ,Xenopus Proteins ,Biology ,Muscle Development ,Article ,Mice ,Young Adult ,Exon ,medicine ,Animals ,Humans ,Myocyte ,Myopathy ,Zebrafish ,Cells, Cultured ,Aged ,Muscle Cells ,Muscles ,Alternative splicing ,Infant ,RNA-Binding Proteins ,Cell Biology ,Middle Aged ,biology.organism_classification ,Cell biology ,Mice, Inbred C57BL ,Alternative Splicing ,RNA splicing ,Female ,medicine.symptom - Abstract
This study shows that FXR1 forms condensates in muscle cells. FXR1 alternative splicing during myogenesis alters the properties of an intrinsically disordered domain and influences the properties of condensates. This study links developmental control of RNA splicing to regulation of biomolecular condensation for muscle formation., Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease., Graphical Abstract
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- 2020
10. Genomic Rna Elements Drive Phase Separation of the Sars-Cov-2 Nucleocapsid
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Mark A. Boerneke, Kevin M. Weeks, Rachel Sealfon, Ethan J. Fritch, Avinash Boppana, Amy S. Gladfelter, Christine A. Roden, Grace A. McLaughlin, Irwin Jungreis, Yixuan J. Hou, Olga G. Troyanskaya, Christiane Iserman, Christopher Y. Park, Chandra L. Theesfeld, Ralph S. Baric, and Timothy P. Sheahan
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Chemistry ,Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ,RNA ,Sequence (biology) ,Guide RNA ,Binding site ,Genome ,Small molecule ,Genomic rna ,Cell biology - Abstract
We report that the SARS-CoV-2 nucleocapsid protein (N-protein) undergoes liquid-liquid phase separation (LLPS) with the viral genome and propose a model of viral packaging through LLPS. N-protein condenses with specific RNA sequences in the first 1000 nts (5'-End) under physiological conditions and condensation is enhanced at human body temperatures. Other regions of gRNA promote dissolution, counteracting aggregation of the large genome. This combination of elements ensures condensates of both specific molecular and physical identity, leading to exclusion of non-packaged RNA sequences. N-protein binds single-stranded RNA flanked by stable structured elements and these features specify the number and location of N-protein binding sites (valency). Liquid-like N-protein condensates form in mammalian cells in a concentration-dependent manner and can be altered by small molecules. Condensation of N-protein is sequence and structure specific, sensitive to human body temperature, and manipulatable with small molecules thus presenting screenable processes for identifying antiviral compounds effective against SARS-CoV-2.
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- 2020
11. Regulation of FXR1 by alternative splicing is required for muscle development and controls liquid-like condensates in muscle cells
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Jimena Giudice, Danielle C. Jordan, John M. Crutchley, Xiaomin Chen, Paul A. Anderson, Ennessa G. Curry, Christine A. Roden, Marko E. Horb, Amy S. Gladfelter, Samantha E. R. Dundon, Shawn M. Lyons, Jean A. Smith, R. Eric Blue, and Anthony Rodríguez-Vargas
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Gene isoform ,0303 health sciences ,biology ,Chemistry ,Alternative splicing ,Xenopus ,biology.organism_classification ,Cell biology ,03 medical and health sciences ,Exon ,0302 clinical medicine ,RNA splicing ,Myocyte ,Zebrafish ,Gene ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
SUMMARYFragile-X mental retardation autosomal homolog-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine and arginine-rich intrinsically-disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.HIGHLIGHTSThe muscle-specific exon 15 impacts FXR1 functionsAlternative splicing of FXR1 is tissue- and developmental stage specificFXR1 forms RNA-dependent condensatesSplicing regulation changes FXR1 condensate properties
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- 2019
- Full Text
- View/download PDF
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