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Sample records for adenosine-to-inosine rna editing

  1. Adenosine-to-inosine RNA editing and human disease

    PubMed Central

    2013-01-01

    A-to-I RNA editing is a post-transcriptional modification that converts adenosines to inosines in both coding and noncoding RNA transcripts. It is catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes, which exist throughout the body but are most prevalent in the central nervous system. Inosines exhibit properties that are most similar to those of guanosines. As a result, ADAR-mediated editing can post-transcriptionally alter codons, introduce or remove splice sites, or affect the base pairing of the RNA molecule with itself or with other RNAs. A-to-I editing is a mechanism that regulates and diversifies the transcriptome, but the full biological significance of ADARs is not understood. ADARs are highly conserved across vertebrates and are essential for normal development in mammals. Aberrant ADAR activity has been associated with a wide range of human diseases, including cancer, neurological disorders, metabolic diseases, viral infections and autoimmune disorders. ADARs have been shown to contribute to disease pathologies by editing of glutamate receptors, editing of serotonin receptors, mutations in ADAR genes, and by other mechanisms, including recently identified regulatory roles in microRNA processing. Advances in research into many of these diseases may depend on an improved understanding of the biological functions of ADARs. Here, we review recent studies investigating connections between ADAR-mediated RNA editing and human diseases. PMID:24289319

  2. ADAR-related activation of adenosine-to-inosine RNA editing during regeneration.

    PubMed

    Witman, Nevin M; Behm, Mikaela; Ohman, Marie; Morrison, Jamie I

    2013-08-15

    Urodele amphibians possess an amazing regenerative capacity that requires the activation of cellular plasticity in differentiated cells and progenitor/stem cells. Many aspects of regeneration in Urodele amphibians recapitulate development, making it unlikely that gene regulatory pathways which are essential for development are mutually exclusive from those necessary for regeneration. One such post-transcriptional gene regulatory pathway, which has been previously shown to be essential for functional metazoan development, is RNA editing. RNA editing catalyses discrete nucleotide changes in RNA transcripts, creating a molecular diversity that could create an enticing connection to the activated cellular plasticity found in newts during regeneration. To assess whether RNA editing occurs during regeneration, we demonstrated that GABRA3 and ADAR2 mRNA transcripts are edited in uninjured and regenerating tissues. Full open-reading frame sequences for ADAR1 and ADAR2, two enzymes responsible for adenosine-to-inosine RNA editing, were cloned from newt brain cDNA and exhibited a strong resemblance to ADAR (adenosine deaminase, RNA-specific) enzymes discovered in mammals. We demonstrated that ADAR1 and ADAR2 mRNA expression levels are differentially expressed during different phases of regeneration in multiple tissues, whereas protein expression levels remain unaltered. In addition, we have characterized a fascinating nucleocytoplasmic shuttling of ADAR1 in a variety of different cell types during regeneration, which could provide a mechanism for controlling RNA editing, without altering translational output of the editing enzyme. The link between RNA editing and regeneration provides further insights into how lower organisms, such as the newt, can activate essential molecular pathways via the discrete alteration of RNA sequences. PMID:23534823

  3. Adenosine-to-Inosine RNA Editing Affects Trafficking of the γ-Aminobutyric Acid Type A (GABAA) Receptor*

    PubMed Central

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Öhman, Marie

    2011-01-01

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABAA receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABAA receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  4. Adenosine-to-inosine RNA editing affects trafficking of the gamma-aminobutyric acid type A (GABA(A)) receptor.

    PubMed

    Daniel, Chammiran; Wahlstedt, Helene; Ohlson, Johan; Björk, Petra; Ohman, Marie

    2011-01-21

    Recoding by adenosine-to-inosine RNA editing plays an important role in diversifying proteins involved in neurotransmission. We have previously shown that the Gabra-3 transcript, coding for the α3 subunit of the GABA(A) receptor is edited in mouse, causing an isoleucine to methionine (I/M) change. Here we show that this editing event is evolutionarily conserved from human to chicken. Analyzing recombinant GABA(A) receptor subunits expressed in HEK293 cells, our results suggest that editing at the I/M site in α3 has functional consequences on receptor expression. We demonstrate that I/M editing reduces the cell surface and the total number of α3 subunits. The reduction in cell surface levels is independent of the subunit combination as it is observed for α3 in combination with either the β2 or the β3 subunit. Further, an amino acid substitution at the corresponding I/M site in the α1 subunit has a similar effect on cell surface presentation, indicating the importance of this site for receptor trafficking. We show that the I/M editing during brain development is inversely related to the α3 protein abundance. Our results suggest that editing controls trafficking of α3-containing receptors and may therefore facilitate the switch of subunit compositions during development as well as the subcellular distribution of α subunits in the adult brain. PMID:21030585

  5. Adenosine to Inosine editing frequency controlled by splicing efficiency.

    PubMed

    Licht, Konstantin; Kapoor, Utkarsh; Mayrhofer, Elisa; Jantsch, Michael F

    2016-07-27

    Alternative splicing and adenosine to inosine (A to I) RNA-editing are major factors leading to co- and post-transcriptional modification of genetic information. Both, A to I editing and splicing occur in the nucleus. As editing sites are frequently defined by exon-intron basepairing, mRNA splicing efficiency should affect editing levels. Moreover, splicing rates affect nuclear retention and will therefore also influence the exposure of pre-mRNAs to the editing-competent nuclear environment. Here, we systematically test the influence of splice rates on RNA-editing using reporter genes but also endogenous substrates. We demonstrate for the first time that the extent of editing is controlled by splicing kinetics when editing is guided by intronic elements. In contrast, editing sites that are exclusively defined by exonic structures are almost unaffected by the splicing efficiency of nearby introns. In addition, we show that editing levels in pre- and mature mRNAs do not match. This phenomenon can in part be explained by the editing state of an RNA influencing its splicing rate but also by the binding of the editing enzyme ADAR that interferes with splicing. PMID:27112566

  6. Improved design of hammerhead ribozyme for selective digestion of target RNA through recognition of site-specific adenosine-to-inosine RNA editing

    PubMed Central

    Fukuda, Masatora; Kurihara, Kei; Yamaguchi, Shota; Oyama, Yui; Deshimaru, Masanobu

    2014-01-01

    Adenosine-to-inosine (A-to-I) RNA editing is an endogenous regulatory mechanism involved in various biological processes. Site-specific, editing-state–dependent degradation of target RNA may be a powerful tool both for analyzing the mechanism of RNA editing and for regulating biological processes. Previously, we designed an artificial hammerhead ribozyme (HHR) for selective, site-specific RNA cleavage dependent on the A-to-I RNA editing state. In the present work, we developed an improved strategy for constructing a trans-acting HHR that specifically cleaves target editing sites in the adenosine but not the inosine state. Specificity for unedited sites was achieved by utilizing a sequence encoding the intrinsic cleavage specificity of a natural HHR. We used in vitro selection methods in an HHR library to select for an extended HHR containing a tertiary stabilization motif that facilitates HHR folding into an active conformation. By using this method, we successfully constructed highly active HHRs with unedited-specific cleavage. Moreover, using HHR cleavage followed by direct sequencing, we demonstrated that this ribozyme could cleave serotonin 2C receptor (HTR2C) mRNA extracted from mouse brain, depending on the site-specific editing state. This unedited-specific cleavage also enabled us to analyze the effect of editing state at the E and C sites on editing at other sites by using direct sequencing for the simultaneous quantification of the editing ratio at multiple sites. Our approach has the potential to elucidate the mechanism underlying the interdependencies of different editing states in substrate RNA with multiple editing sites. PMID:24448449

  7. Editor meets silencer: crosstalk between RNA editing and RNA interference

    PubMed Central

    Nishikura, Kazuko

    2010-01-01

    The most prevalent type of RNA editing is mediated by ADAR (adenosine deaminase acting on RNA) enzymes, which convert adenosines to inosines (a process known as A→I RNA editing) in double-stranded (ds)RNA substrates. A→I RNA editing was long thought to affect only selected transcripts by altering the proteins they encode. However, genome-wide screening has revealed numerous editing sites within inverted Alu repeats in introns and untranslated regions. Also, recent evidence indicates that A→I RNA editing crosstalks with RNA-interference pathways, which, like A→I RNA editing, involve dsRNAs. A→I RNA editing therefore seems to have additional functions, including the regulation of retrotransposons and gene silencing, which adds a new urgency to the challenges of fully understanding ADAR functions. PMID:17139332

  8. The Genomic Landscape and Clinical Relevance of A-to-I RNA Editing in Human Cancers | Office of Cancer Genomics

    Cancer.gov

    Adenosine-to-inosine (A-to-I) RNA editing is a widespread post-transcriptional mechanism, but its genomic landscape and clinical relevance in cancer have not been investigated systematically. We characterized the global A-to-I RNA editing profiles of 6,236 patient samples of 17 cancer types from The Cancer Genome Atlas and revealed a striking diversity of altered RNA-editing patterns in tumors relative to normal tissues. We identified an appreciable number of clinically relevant editing events, many of which are in noncoding regions.

  9. Novel modes of RNA editing in mitochondria.

    PubMed

    Moreira, Sandrine; Valach, Matus; Aoulad-Aissa, Mohamed; Otto, Christian; Burger, Gertraud

    2016-06-01

    Gene structure and expression in diplonemid mitochondria are unparalleled. Genes are fragmented in pieces (modules) that are separately transcribed, followed by the joining of module transcripts to contiguous RNAs. Some instances of unique uridine insertion RNA editing at module boundaries were noted, but the extent and potential occurrence of other editing types remained unknown. Comparative analysis of deep transcriptome and genome data from Diplonema papillatum mitochondria reveals ∼220 post-transcriptional insertions of uridines, but no insertions of other nucleotides nor deletions. In addition, we detect in total 114 substitutions of cytosine by uridine and adenosine by inosine, amassed into unusually compact clusters. Inosines in transcripts were confirmed experimentally. This is the first report of adenosine-to-inosine editing of mRNAs and ribosomal RNAs in mitochondria. In mRNAs, editing causes mostly amino-acid additions and non-synonymous substitutions; in ribosomal RNAs, it permits formation of canonical secondary structures. Two extensively edited transcripts were compared across four diplonemids. The pattern of uridine-insertion editing is strictly conserved, whereas substitution editing has diverged dramatically, but still rendering diplonemid proteins more similar to other eukaryotic orthologs. We posit that RNA editing not only compensates but also sustains, or even accelerates, ultra-rapid evolution of genome structure and sequence in diplonemid mitochondria. PMID:27001515

  10. Novel modes of RNA editing in mitochondria

    PubMed Central

    Moreira, Sandrine; Valach, Matus; Aoulad-Aissa, Mohamed; Otto, Christian; Burger, Gertraud

    2016-01-01

    Gene structure and expression in diplonemid mitochondria are unparalleled. Genes are fragmented in pieces (modules) that are separately transcribed, followed by the joining of module transcripts to contiguous RNAs. Some instances of unique uridine insertion RNA editing at module boundaries were noted, but the extent and potential occurrence of other editing types remained unknown. Comparative analysis of deep transcriptome and genome data from Diplonema papillatum mitochondria reveals ∼220 post-transcriptional insertions of uridines, but no insertions of other nucleotides nor deletions. In addition, we detect in total 114 substitutions of cytosine by uridine and adenosine by inosine, amassed into unusually compact clusters. Inosines in transcripts were confirmed experimentally. This is the first report of adenosine-to-inosine editing of mRNAs and ribosomal RNAs in mitochondria. In mRNAs, editing causes mostly amino-acid additions and non-synonymous substitutions; in ribosomal RNAs, it permits formation of canonical secondary structures. Two extensively edited transcripts were compared across four diplonemids. The pattern of uridine-insertion editing is strictly conserved, whereas substitution editing has diverged dramatically, but still rendering diplonemid proteins more similar to other eukaryotic orthologs. We posit that RNA editing not only compensates but also sustains, or even accelerates, ultra-rapid evolution of genome structure and sequence in diplonemid mitochondria. PMID:27001515

  11. Functions and Regulation of RNA Editing by ADAR Deaminases

    PubMed Central

    Nishikura, Kazuko

    2010-01-01

    One type of RNA editing converts adenosines to inosines (A→I editing) in double-stranded RNA (dsRNA) substrates. A→I RNA editing is mediated by adenosine deaminase acting on RNA (ADAR) enzymes. A→I RNA editing of protein-coding sequences of a limited number of mammalian genes results in recoding and subsequent alterations of their functions. However, A→I RNA editing most frequently targets repetitive RNA sequences located within introns and 5′ and 3′ untranslated regions (UTRs). Although the biological significance of noncoding RNA editing remains largely unknown, several possibilities, including its role in the control of endogenous short interfering RNAs (esiRNAs), have been proposed. Furthermore, recent studies have revealed that the biogenesis and functions of certain microRNAs (miRNAs) are regulated by the editing of their precursors. Here, I review the recent findings that indicate new functions for A→I editing in the regulation of noncoding RNAs and for interactions between RNA editing and RNA interference mechanisms. PMID:20192758

  12. RNA editing in Drosophila melanogaster: new targets and functionalconsequences

    SciTech Connect

    Stapleton, Mark; Carlson, Joseph W.; Celniker, Susan E.

    2006-09-05

    Adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine in primary mRNA transcripts. These re-coding events affect coding potential, splice-sites, and stability of mature mRNAs. ADAR is an essential gene and studies in mouse, C. elegans, and Drosophila suggest its primary function is to modify adult behavior by altering signaling components in the nervous system. By comparing the sequence of isogenic cDNAs to genomic DNA, we have identified and experimentally verified 27 new targets of Drosophila ADAR. Our analyses lead us to identify new classes of genes whose transcripts are targets of ADAR including components of the actin cytoskeleton, and genes involved in ion homeostasis and signal transduction. Our results indicate that editing in Drosophila increases the diversity of the proteome, and does so in a manner that has direct functional consequences on protein function.

  13. A strategy for developing a hammerhead ribozyme for selective RNA cleavage depending on substitutional RNA editing

    PubMed Central

    Fukuda, Masatora; Kurihara, Kei; Tanaka, Yasuyoshi; Deshimaru, Masanobu

    2012-01-01

    Substitutional RNA editing plays a crucial role in the regulation of biological processes. Cleavage of target RNA that depends on the specific site of substitutional RNA editing is a useful tool for analyzing and regulating intracellular processes related to RNA editing. Hammerhead ribozymes have been utilized as small catalytic RNAs for cleaving target RNA at a specific site and may be used for RNA-editing-specific RNA cleavage. Here we reveal a design strategy for a hammerhead ribozyme that specifically recognizes adenosine to inosine (A-to-I) and cytosine to uracil (C-to-U) substitutional RNA-editing sites and cleaves target RNA. Because the hammerhead ribozyme cleaves one base upstream of the target-editing site, the base that pairs with the target-editing site was utilized for recognition. RNA-editing-specific ribozymes were designed such that the recognition base paired only with the edited base. These ribozymes showed A-to-I and C-to-U editing-specific cleavage activity against synthetic serotonin receptor 2C and apolipoprotein B mRNA fragments in vitro, respectively. Additionally, the ribozyme designed for recognizing A-to-I RNA editing at the Q/R site on filamin A (FLNA) showed editing-specific cleavage activity against physiologically edited FLNA mRNA extracted from cells. We demonstrated that our strategy is effective for cleaving target RNA in an editing-dependent manner. The data in this study provided an experimental basis for the RNA-editing-dependent degradation of specific target RNA in vivo. PMID:22798264

  14. New Insights into the Biological Role of Mammalian ADARs; the RNA Editing Proteins

    PubMed Central

    Mannion, Niamh; Arieti, Fabiana; Gallo, Angela; Keegan, Liam P.; O’Connell, Mary A.

    2015-01-01

    The ADAR proteins deaminate adenosine to inosine in double-stranded RNA which is one of the most abundant modifications present in mammalian RNA. Inosine can have a profound effect on the RNAs that are edited, not only changing the base-pairing properties, but can also result in recoding, as inosine behaves as if it were guanosine. In mammals there are three ADAR proteins and two ADAR-related proteins (ADAD) expressed. All have a very similar modular structure; however, both their expression and biological function differ significantly. Only two of the ADAR proteins have enzymatic activity. However, both ADAR and ADAD proteins possess the ability to bind double-strand RNA. Mutations in ADARs have been associated with many diseases ranging from cancer, innate immunity to neurological disorders. Here, we will discuss in detail the domain structure of mammalian ADARs, the effects of RNA editing, and the role of ADARs in human diseases. PMID:26437436

  15. ADAR2 affects mRNA coding sequence edits with only modest effects on gene expression or splicing in vivo.

    PubMed

    Dillman, Allissa A; Cookson, Mark R; Galter, Dagmar

    2016-01-01

    Adenosine deaminases bind double stranded RNA and convert adenosine to inosine. Editing creates multiple isoforms of neurotransmitter receptors, such as with Gria2. Adar2 KO mice die of seizures shortly after birth, but if the Gria2 Q/R editing site is mutated to mimic the edited version then the animals are viable. We performed RNA-Seq on frontal cortices of Adar2(-/-) Gria2(R/R) mice and littermates. We found 56 editing sites with significantly diminished editing levels in Adar2 deficient animals with the majority in coding regions. Only two genes and 3 exons showed statistically significant differences in expression levels. This work illustrates that ADAR2 is important in site-specific changes of protein coding sequences but has relatively modest effects on gene expression and splicing in the adult mouse frontal cortex. PMID:26669816

  16. Reduced levels of protein recoding by A-to-I RNA editing in Alzheimer's disease

    PubMed Central

    Khermesh, Khen; D'Erchia, Anna Maria; Barak, Michal; Annese, Anita; Wachtel, Chaim; Levanon, Erez Y.; Picardi, Ernesto; Eisenberg, Eli

    2016-01-01

    Adenosine to inosine (A-to-I) RNA editing, catalyzed by the ADAR enzyme family, acts on dsRNA structures within pre-mRNA molecules. Editing of the coding part of the mRNA may lead to recoding, amino acid substitution in the resulting protein, possibly modifying its biochemical and biophysical properties. Altered RNA editing patterns have been observed in various neurological pathologies. Here, we present a comprehensive study of recoding by RNA editing in Alzheimer's disease (AD), the most common cause of irreversible dementia. We have used a targeted resequencing approach supplemented by a microfluidic-based high-throughput PCR coupled with next-generation sequencing to accurately quantify A-to-I RNA editing levels in a preselected set of target sites, mostly located within the coding sequence of synaptic genes. Overall, editing levels decreased in AD patients’ brain tissues, mainly in the hippocampus and to a lesser degree in the temporal and frontal lobes. Differential RNA editing levels were observed in 35 target sites within 22 genes. These results may shed light on a possible association between the neurodegenerative processes typical for AD and deficient RNA editing. PMID:26655226

  17. Splicing variants of ADAR2 and ADAR2-mediated RNA editing in glioma

    PubMed Central

    Fu, Yao; Zhao, Xingli; Li, Zhaohui; Wei, Jun; Tian, Yu

    2016-01-01

    The roles of alternative splicing and RNA editing in gene regulation and transcriptome diversity are well documented. Adenosine deaminases acting on RNA (ADARs) are responsible for adenosine-to-inosine (A-to-I) editing and exemplify the complex association between RNA editing and alternative splicing. The self-editing activity of ADAR2, which acts on its own pre-mRNA, leads to its alternative splicing. Alternative splicing occurs independently at nine splicing sites on ADAR2 pre-mRNA, generating numerous alternative splicing variants with various catalytic activities. A-to-I RNA editing is important in a range of physiological processes in humans and is associated with several diseases, including amyotrophic lateral sclerosis, mood disorders, epilepsy and glioma. Reduced editing at the glutamine/arginine site of the AMPA receptor subunit GluA2 in glioma, without any alteration in ADAR2 expression, is a notable phenomenon. Several studies have tried to explain this alteration in the catalytic activity of ADAR2; however, the underlying mechanism remains unclear. The present review summarizes the relevant literature and shares experimental results concerning ADAR2 alternative splicing. In particular, the present review demonstrates that shifts in the relative abundance of the active and inactive splicing variants of ADAR2 may reduce the ADAR2 editing activity in glioma. Dominant expression of ADAR2 splicing variant with low enzyme activity causes reduced RNA editing of GluA2 subunit at the glutamine/arginine site in glioma. PMID:27446352

  18. Evidence for large diversity in the human transcriptome created by Alu RNA editing.

    PubMed

    Barak, Michal; Levanon, Erez Y; Eisenberg, Eli; Paz, Nurit; Rechavi, Gideon; Church, George M; Mehr, Ramit

    2009-11-01

    Adenosine-to-inosine (A-to-I) RNA editing alters the original genomic content of the human transcriptome and is essential for maintenance of normal life in mammals. A-to-I editing in Alu repeats is abundant in the human genome, with many thousands of expressed Alu sequences undergoing editing. Little is known so far about the contribution of Alu editing to transcriptome complexity. Transcripts derived from a single edited Alu sequence can be edited in multiple sites, and thus could theoretically generate a large number of different transcripts. Here we explored whether the combinatorial potential nature of edited Alu sequences is actually fulfilled in the human transcriptome. We analyzed datasets of editing sites and performed an analysis of a detailed transcript set of one edited Alu sequence. We found that editing appears at many more sites than detected by earlier genomic screens. To a large extent, editing of different sites within the same transcript is only weakly correlated. Thus, rather than finding a few versions of each transcript, a large number of edited variants arise, resulting in immense transcript diversity that eclipses alternative splicing as mechanism of transcriptome diversity, although with less impact on the proteome. PMID:19740767

  19. Frequency and fate of microRNA editing in human brain

    PubMed Central

    Kawahara, Yukio; Megraw, Molly; Kreider, Edward; Iizasa, Hisashi; Valente, Louis; Hatzigeorgiou, Artemis G.; Nishikura, Kazuko

    2008-01-01

    Primary transcripts of certain microRNA (miRNA) genes (pri-miRNAs) are subject to RNA editing that converts adenosine to inosine (A→I RNA editing). However, the frequency of the pri-miRNA editing and the fate of edited pri-miRNAs remain largely to be determined. Examination of already known pri-miRNA editing sites indicated that adenosine residues of the UAG triplet sequence might be edited more frequently. In the present study, therefore, we conducted a large-scale survey of human pri-miRNAs containing the UAG triplet sequence. By direct sequencing of RT–PCR products corresponding to pri-miRNAs, we examined 209 pri-miRNAs and identified 43 UAG and also 43 non-UAG editing sites in 47 pri-miRNAs, which were highly edited in human brain. In vitro miRNA processing assay using recombinant Drosha-DGCR8 and Dicer-TRBP (the human immuno deficiency virus transactivating response RNA-binding protein) complexes revealed that a majority of pri-miRNA editing is likely to interfere with the miRNA processing steps. In addition, four new edited miRNAs with altered seed sequences were identified by targeted cloning and sequencing of the miRNAs that would be processed from edited pri-miRNAs. Our studies predict that ∼16% of human pri-miRNAs are subject to A→I editing and, thus, miRNA editing could have a large impact on the miRNA-mediated gene silencing. PMID:18684997

  20. A genome-wide map of hyper-edited RNA reveals numerous new sites.

    PubMed

    Porath, Hagit T; Carmi, Shai; Levanon, Erez Y

    2014-01-01

    Adenosine-to-inosine editing is one of the most frequent post-transcriptional modifications, manifested as A-to-G mismatches when comparing RNA sequences with their source DNA. Recently, a number of RNA-seq data sets have been screened for the presence of A-to-G editing, and hundreds of thousands of editing sites identified. Here we show that existing screens missed the majority of sites by ignoring reads with excessive ('hyper') editing that do not easily align to the genome. We show that careful alignment and examination of the unmapped reads in RNA-seq studies reveal numerous new sites, usually many more than originally discovered, and in precisely those regions that are most heavily edited. Specifically, we discover 327,096 new editing sites in the heavily studied Illumina Human BodyMap data and more than double the number of detected sites in several published screens. We also identify thousands of new sites in mouse, rat, opossum and fly. Our results establish that hyper-editing events account for the majority of editing sites. PMID:25158696

  1. A distant cis acting intronic element induces site-selective RNA editing.

    PubMed

    Daniel, Chammiran; Venø, Morten T; Ekdahl, Ylva; Kjems, Jørgen; Öhman, Marie

    2012-10-01

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABA(A) receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome. PMID:22848101

  2. A distant cis acting intronic element induces site-selective RNA editing

    PubMed Central

    Daniel, Chammiran; Venø, Morten T.; Ekdahl, Ylva; Kjems, Jørgen; Öhman, Marie

    2012-01-01

    Transcripts have been found to be site selectively edited from adenosine-to-inosine (A-to-I) in the mammalian brain, mostly in genes involved in neurotransmission. While A-to-I editing occurs at double-stranded structures, other structural requirements are largely unknown. We have investigated the requirements for editing at the I/M site in the Gabra-3 transcript of the GABAA receptor. We identify an evolutionarily conserved intronic duplex, 150 nt downstream of the exonic hairpin where the I/M site resides, which is required for its editing. This is the first time a distant RNA structure has been shown to be important for A-to-I editing. We demonstrate that the element also can induce editing in related but normally not edited RNA sequences. In human, thousands of genes are edited in duplexes formed by inverted repeats in non-coding regions. It is likely that numerous such duplexes can induce editing of coding regions throughout the transcriptome. PMID:22848101

  3. Involvement of serotonin 2C receptor RNA editing in accumbal neuropeptide Y expression and behavioural despair.

    PubMed

    Aoki, Miku; Watanabe, Yoshihisa; Yoshimoto, Kanji; Tsujimura, Atsushi; Yamamoto, Toshiro; Kanamura, Narisato; Tanaka, Masaki

    2016-05-01

    Serotonin 2C receptors (5-HT2 C Rs) are widely expressed in the central nervous system, and are associated with various neurological disorders. 5-HT2 C R mRNA undergoes adenosine-to-inosine RNA editing at five sites within its coding sequence, resulting in expression of 24 different isoforms. Several edited isoforms show reduced activity, suggesting that RNA editing modulates serotonergic systems in the brain with causative relevance to neuropsychiatric disorders. Transgenic mice solely expressing the non-edited 5-HT2 C R INI-isoform (INI) or the fully edited VGV-isoform exhibit various phenotypes including metabolic abnormalities, aggressive behaviour, anxiety-like behaviour, and depression-like behaviour. Here, we examined the behavioural phenotype and molecular changes of INI mice on a C57BL/6J background. INI mice showed an enhanced behavioural despair in the forced swimming test, elevated sensitivity to the tricyclic antidepressant desipramine, and significantly decreased serotonin in the nucleus accumbens (NAc), amygdala, and striatum. They also showed reduced expression of neuropeptide Y (NPY) mRNA in the NAc. In addition, by stereotactic injection of adeno-associated virus encoding NPY into the NAc, we demonstrated that accumbal NPY overexpression relieved behavioural despair. Our results suggest that accumbal NPY expression may be regulated by 5-HT2 C R RNA editing, and its impairment may be linked to mood disorders. PMID:26950265

  4. Altered A-to-I RNA editing in human embryogenesis.

    PubMed

    Shtrichman, Ronit; Germanguz, Igal; Mandel, Rachel; Ziskind, Anna; Nahor, Irit; Safran, Michal; Osenberg, Sivan; Sherf, Ofra; Rechavi, Gideon; Itskovitz-Eldor, Joseph

    2012-01-01

    Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1-p110 isoform. We were unable to achieve overexpression of ADAR1-p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24-48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development. PMID:22859999

  5. Altered A-to-I RNA Editing in Human Embryogenesis

    PubMed Central

    Mandel, Rachel; Ziskind, Anna; Nahor, Irit; Safran, Michal; Osenberg, Sivan; Sherf, Ofra; Rechavi, Gideon; Itskovitz-Eldor, Joseph

    2012-01-01

    Post-transcriptional events play an important role in human development. The question arises as to whether Adenosine to Inosine RNA editing, catalyzed by the ADAR (Adenosine Deaminase acting on RNA) enzymes, differs in human embryogenesis and in adulthood. We tested the editing of various target genes in coding (FLNA, BLCAP, CYFIP2) and non-coding sequences at their Alu elements (BRCA1, CARD11, RBBP9, MDM4, FNACC), as well as the transcriptional levels of the ADAR1 enzymes. This analysis was performed on five fetal and adult human tissues: brain, heart, liver, kidney, and spleen, as well as on human embryonic stem cells (hESCs), which represent the blastocyst stage in early human development. Our results show substantially greater editing activity for most adult tissue samples relative to fetal ones, in six of the eight genes tested. To test the effect of reduced A-to-I RNA editing activity in early human development we used human embryonic stem cells (hESCs) as a model and tried to generate hESC clones that overexpress the ADAR1–p110 isoform. We were unable to achieve overexpression of ADAR1–p110 by either transfection or lentiviral infection, though we easily generated hESC clones that expressed the GFP transgene and overexpressed ADAR1-p110 in 293T cells and in primary human foreskin fibroblast (HFF) cells. Moreover, in contrast to the expected overexpression of ADAR1-p110 protein following its introduction into hESCs, the expression levels of this protein decreased dramatically 24–48 hr post infection. Similar results were obtained when we tried to overexpress ADAR1-p110 in pluripotent embryonal carcinoma cells. This suggests that ADAR1 protein is substantially regulated in undifferentiated pluripotent hESCs. Overall, our data suggest that A-to-I RNA editing plays a critical role during early human development. PMID:22859999

  6. Alu elements shape the primate transcriptome by cis-regulation of RNA editing

    PubMed Central

    2014-01-01

    Background RNA editing by adenosine to inosine deamination is a widespread phenomenon, particularly frequent in the human transcriptome, largely due to the presence of inverted Alu repeats and their ability to form double-stranded structures – a requisite for ADAR editing. While several hundred thousand editing sites have been identified within these primate-specific repeats, the function of Alu-editing has yet to be elucidated. Results We show that inverted Alu repeats, expressed in the primate brain, can induce site-selective editing in cis on sites located several hundred nucleotides from the Alu elements. Furthermore, a computational analysis, based on available RNA-seq data, finds that site-selective editing occurs significantly closer to edited Alu elements than expected. These targets are poorly edited upon deletion of the editing inducers, as well as in homologous transcripts from organisms lacking Alus. Sequences surrounding sites near edited Alus in UTRs, have been subjected to a lesser extent of evolutionary selection than those far from edited Alus, indicating that their editing generally depends on cis-acting Alus. Interestingly, we find an enrichment of primate-specific editing within encoded sequence or the UTRs of zinc finger-containing transcription factors. Conclusions We propose a model whereby primate-specific editing is induced by adjacent Alu elements that function as recruitment elements for the ADAR editing enzymes. The enrichment of site-selective editing with potentially functional consequences on the expression of transcription factors indicates that editing contributes more profoundly to the transcriptomic regulation and repertoire in primates than previously thought. PMID:24485196

  7. ADAR Proteins: Double-stranded RNA and Z-DNA Binding Domains

    PubMed Central

    Barraud, Pierre; Allain, Frédéric H.-T

    2012-01-01

    Adenosine deaminases acting on RNA (ADARs) catalyze adenosine to inosine editing within double-stranded RNA (dsRNA) substrates. Inosine is read as a guanine by most cellular processes and therefore these changes create codons for a different amino acid, stop codons or even a new splice-site allowing protein diversity generated from a single gene. We are reviewing here the current structural and molecular knowledge on RNA editing by the ADAR family of protein. We focus especially on two types of nucleic acid binding domains present in ADARs, namely the double-stranded RNA and Z-DNA binding domains. PMID:21728134

  8. Reciprocal regulation of A-to-I RNA editing and the vertebrate nervous system

    PubMed Central

    Penn, Andrew C.; Balik, Ales; Greger, Ingo H.

    2013-01-01

    The fine control of molecules mediating communication in the nervous system is key to adjusting neuronal signaling during development and in maintaining the stability of established networks in the face of altered sensory input. To prevent the culmination of pathological recurrent network excitation or debilitating periods of quiescence, adaptive alterations occur in the signaling molecules and ion channels that control membrane excitability and synaptic transmission. However, rather than encoding (and thus “hardwiring”) modified gene copies, the nervous systems of metazoa have opted for expanding on post-transcriptional pre-mRNA splicing by altering key encoded amino acids using a conserved mechanism of A-to-I RNA editing: the enzymatic deamination of adenosine to inosine. Inosine exhibits similar base-pairing properties to guanosine with respect to tRNA codon recognition, replication by polymerases, and RNA secondary structure (i.e.,: forming-capacity). In addition to recoding within the open reading frame, adenosine deamination also occurs with high frequency throughout the non-coding transcriptome, where it affects multiple aspects of RNA metabolism and gene expression. Here, we describe the recoding function of key RNA editing targets in the mammalian central nervous system and their potential to be regulated. We will then discuss how interactions of A-to-I editing with gene expression and alternative splicing could play a wider role in regulating the neuronal transcriptome. Finally, we will highlight the increasing complexity of this multifaceted control hub by summarizing new findings from high-throughput studies. PMID:23616744

  9. Gene Amplification-Associated Overexpression of the RNA Editing Enzyme ADAR1 Enhances Human Lung Tumorigenesis

    PubMed Central

    Anadón, Carmen; Guil, Sonia; Simó-Riudalbas, Laia; Moutinho, Catia; Setien, Fernando; Martínez-Cardús, Anna; Moran, Sebastian; Villanueva, Alberto; Calaf, Monica; Vidal, August; Lazo, Pedro A.; Zondervan, Ilse; Savola, Suvi; Kohno, Takashi; Yokota, Jun; Ribas de Pouplana, Lluís; Esteller, Manel

    2015-01-01

    The introduction of new therapies against particular genetic mutations in non-small cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis. PMID:26640150

  10. Gene amplification-associated overexpression of the RNA editing enzyme ADAR1 enhances human lung tumorigenesis.

    PubMed

    Anadón, C; Guil, S; Simó-Riudalbas, L; Moutinho, C; Setien, F; Martínez-Cardús, A; Moran, S; Villanueva, A; Calaf, M; Vidal, A; Lazo, P A; Zondervan, I; Savola, S; Kohno, T; Yokota, J; de Pouplana, L R; Esteller, M

    2016-08-18

    The introduction of new therapies against particular genetic mutations in non-small-cell lung cancer is a promising avenue for improving patient survival, but the target population is small. There is a need to discover new potential actionable genetic lesions, to which end, non-conventional cancer pathways, such as RNA editing, are worth exploring. Herein we show that the adenosine-to-inosine editing enzyme ADAR1 undergoes gene amplification in non-small cancer cell lines and primary tumors in association with higher levels of the corresponding mRNA and protein. From a growth and invasion standpoint, the depletion of ADAR1 expression in amplified cells reduces their tumorigenic potential in cell culture and mouse models, whereas its overexpression has the opposite effects. From a functional perspective, ADAR1 overexpression enhances the editing frequencies of target transcripts such as NEIL1 and miR-381. In the clinical setting, patients with early-stage lung cancer, but harboring ADAR1 gene amplification, have poor outcomes. Overall, our results indicate a role for ADAR1 as a lung cancer oncogene undergoing gene amplification-associated activation that affects downstream RNA editing patterns and patient prognosis. PMID:26640150

  11. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates

    PubMed Central

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome. PMID:26024316

  12. Genome-Wide Characterization of RNA Editing in Chicken Embryos Reveals Common Features among Vertebrates.

    PubMed

    Frésard, Laure; Leroux, Sophie; Roux, Pierre-François; Klopp, Christophe; Fabre, Stéphane; Esquerré, Diane; Dehais, Patrice; Djari, Anis; Gourichon, David; Lagarrigue, Sandrine; Pitel, Frédérique

    2015-01-01

    RNA editing results in a post-transcriptional nucleotide change in the RNA sequence that creates an alternative nucleotide not present in the DNA sequence. This leads to a diversification of transcription products with potential functional consequences. Two nucleotide substitutions are mainly described in animals, from adenosine to inosine (A-to-I) and from cytidine to uridine (C-to-U). This phenomenon is described in more details in mammals, notably since the availability of next generation sequencing technologies allowing whole genome screening of RNA-DNA differences. The number of studies recording RNA editing in other vertebrates like chicken is still limited. We chose to use high throughput sequencing technologies to search for RNA editing in chicken, and to extend the knowledge of its conservation among vertebrates. We performed sequencing of RNA and DNA from 8 embryos. Being aware of common pitfalls inherent to sequence analyses that lead to false positive discovery, we stringently filtered our datasets and found fewer than 40 reliable candidates. Conservation of particular sites of RNA editing was attested by the presence of 3 edited sites previously detected in mammals. We then characterized editing levels for selected candidates in several tissues and at different time points, from 4.5 days of embryonic development to adults, and observed a clear tissue-specificity and a gradual increase of editing level with time. By characterizing the RNA editing landscape in chicken, our results highlight the extent of evolutionary conservation of this phenomenon within vertebrates, attest to its tissue and stage specificity and provide support of the absence of non A-to-I events from the chicken transcriptome. PMID:26024316

  13. How stress and fluoxetine modulate serotonin 2C receptor pre-mRNA editing.

    PubMed

    Englander, Michael T; Dulawa, Stephanie C; Bhansali, Punita; Schmauss, Claudia

    2005-01-19

    In two inbred strains of mice, C57BL/6 and 129Sv, the majority of forebrain neocortical pre-mRNA encoding the serotonin 2C (5-HT2C) receptor is altered by adenosine-to-inosine editing. As a result, >60% of all mRNAs encode receptors with reduced constitutive and agonist-stimulated activity. However, in the BALB/c strain, a genetically distinct inbred strain with lower forebrain serotonin levels, spontaneously elevated anxiety, and increased stress reactivity, the majority of 5-HT2C mRNA is nonedited and encodes receptors with the highest constitutive activity and the highest agonist affinity and potency. Neither acute stress (the forced swim test) nor chronic treatment with the serotonin-selective reuptake inhibitor fluoxetine elicit significant changes in 5-HT2C pre-mRNA editing in C57BL/6 mice. In contrast, exposure of BALB/c mice to acute stress and chronic treatment of nonstressed BALB/c mice with fluoxetine elicit significant, site-specific increases in 5-HT2C pre-mRNA editing that increase the pool of mRNA encoding receptors with reduced function. These changes in 5-HT2C pre-mRNA editing resemble those detected previously in the prefrontal cortex of subjects with major depression. However, when chronic fluoxetine treatment is combined with stress exposure of BALB/c mice, these changes in 5-HT2C pre-mRNA editing are no longer detected. These findings illustrate that 5-HT2C pre-mRNA editing responses to stress and chronic fluoxetine are modulated by the genetic background, as well as the behavioral state of the animal. They suggest further that the changes in 5-HT2C pre-mRNA editing found in major depression reflect a previously unrecognized molecular response to stress that can be prevented by chronic antidepressant treatment. PMID:15659601

  14. Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions

    PubMed Central

    Hood, Jennifer L.; Morabito, Michael V.; Martinez, Charles R.; Gilbert, James A.; Ferrick, Elizabeth A.; Ayers, Gregory D.; Chappell, James D.; Dermody, Terence S.; Emeson, Ronald B.

    2014-01-01

    Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neuro-tropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo. PMID:24906008

  15. A-to-I pre-mRNA editing of the serotonin 2C receptor: comparisons among inbred mouse strains.

    PubMed

    Du, Yunzhi; Davisson, Muriel T; Kafadar, Karen; Gardiner, Katheleen

    2006-11-01

    The serotonin receptor 5HT2CR pre-mRNA is subject to adenosine deamination (RNA editing) at five residues located within a 15 nucleotide stretch of the coding region. Such changes of adenosine to inosine (A-to-I) can produce 32 mRNA variants, encoding 24 different protein isoforms, some of which vary in biochemical and pharmacological properties. Because serotonin mediates diverse neurological processes relevant to behavior and because inbred mouse strains vary in their responses to tests of learning and behavior, we have examined the A-to-I editing patterns of the 5HT2CR mRNA in whole brains from eight mouse strains. By sequencing approximately 100 clones from individual mice, we generated detailed information on levels of editing at each site and patterns of editing that identify a total of 28 mRNA and 20 protein isoforms. Significant differences between individuals from different strains were found in total editing frequency, in the proportion of transcripts with 1 and 4 edited sites, in editing frequency at the A, B, E and D sites, in amino acid frequencies at positions 157 and 161, and in subsets of major protein isoforms. Primer extension assays were used to show that individuals within strains (six C3H.B-+rd1 and four 129SvImrJ) displayed no significant differences in any feature. These findings suggest that genetic background contributes to subtle variation in 5HT2CR mRNA editing patterns which may have consequences for pharmacological treatments and behavioral testing. PMID:16904273

  16. A-to-I editing of coding and non-coding RNAs by ADARs

    PubMed Central

    Nishikura, Kazuko

    2016-01-01

    Adenosine deaminases acting on RNA (ADARs) convert adenosine to inosine in double-stranded RNA. This A-to-I editing occurs not only in protein-coding regions of mRNAs, but also frequently in non-coding regions that contain inverted Alu repeats. Editing of coding sequences can result in the expression of functionally altered proteins that are not encoded in the genome, whereas the significance of Alu editing remains largely unknown. Certain microRNA (miRNA) precursors are also edited, leading to reduced expression or altered function of mature miRNAs. Conversely, recent studies indicate that ADAR1 forms a complex with Dicer to promote miRNA processing, revealing a new function of ADAR1 in the regulation of RNA interference. PMID:26648264

  17. RNA editing of the human serotonin 5-HT(2C) receptor delays agonist-stimulated calcium release.

    PubMed

    Price, R D; Sanders-Bush, E

    2000-10-01

    RNA encoding the human 5-HT(2C) receptor undergoes adenosine-to-inosine RNA editing events at five positions in the putative second intracellular loop, with a corresponding reduction in receptor/G-protein coupling. Agonist-stimulated calcium release was examined in NIH-3T3 fibroblasts stably expressing the nonedited human INI (hINI) or the edited hVSV or hVGV variants. We hypothesized that different receptor isoforms would show altered dynamics of agonist-induced calcium release. The three isoforms showed a rightward shift in agonist concentration-response curves for eliciting calcium release (EC(50) values: hINI, 2.2 nM; hVSV, 15 nM; hVGV, 49 nM). Additionally, the hVGV receptor showed a blunted and delayed [Ca(2+)](i) peak compared with the hINI or hVSV receptor isoforms. These distinctions in agonist-induced [Ca(2+)](i) release imply that edited 5-HT(2C) receptors may produce distinct physiological responses within the central nervous system. PMID:10999958

  18. ADAR1 and ADAR2 Expression and Editing Activity during Forebrain Development

    PubMed Central

    Jacobs, Michelle M.; Fogg, Rachel L.; Emeson, Ronald B.; Stanwood, Gregg D.

    2009-01-01

    The conversion of adenosine-to-inosine within RNA transcripts is regulated by the ADAR family of enzymes. Little is known regarding the developmental expression of ADAR family members or the mechanisms responsible for the specific patterns of editing observed for ADAR substrates. We have examined the spatiotemporal expression patterns for ADAR1 and ADAR2 in mouse forebrain. ADAR1 and ADAR2 are broadly distributed in most regions of the mouse forebrain by P0, including the cerebral cortex, hippocampus, and diencephalon. High expression levels were maintained into adulthood. Co-localization studies demonstrated ADAR1 and ADAR2 expression in neurons but not astrocytes. Editing for specific ADAR mRNA targets precedes high expression of ADAR proteins, suggesting that region-specific differences in editing patterns may not be mediated solely by ADAR expression levels. PMID:19325227

  19. Small RNA and A-to-I Editing in Autism Spectrum Disorders

    NASA Astrophysics Data System (ADS)

    Eran, Alal

    One in every 88 children is diagnosed with Autism Spectrum Disorders (ASDs), a set of neurodevelopmental conditions characterized by social impairments, communication deficits, and repetitive behavior. ASDs have a substantial genetic component, but the specific cause of most cases remains unknown. Understanding gene-environment interactions underlying ASD is essential for improving early diagnosis and identifying critical targets for intervention and prevention. Towards this goal, we surveyed adenosine-to-inosine (A-to-I) RNA editing in autistic brains. A-to-I editing is an epigenetic mechanism that fine-tunes synaptic function in response to environmental stimuli, shown to modulate complex behavior in animals. We used ultradeep sequencing to quantify A-to-I receding of candidate synaptic genes in postmortem cerebella from individuals with ASD and neurotypical controls. We found unexpectedly wide distributions of human A-to-I editing levels, whose extremes were consistently populated by individuals with ASD. We correlated A-to-I editing with isoform usage, identified clusters of correlated sites, and examined differential editing patterns. Importantly, we found that individuals with ASD commonly use a dysfunctional form of the editing enzyme ADARB1. We next profiled small RNAs thought to regulate A-to-I editing, which originate from one of the most commonly altered loci in ASD, 15q11. Deep targeted sequencing of SNORD115 and SNORD116 transcripts enabled their high-resolution detection in human brains, and revealed a strong gender bias underlying their expression. The consistent 2-fold upregulation of 15q11 small RNAs in male vs. female cerebella could be important in delineating the role of this locus in ASD, a male dominant disorder. Overall, these studies provide an accurate population-level view of small RNA and A-to-I editing in human cerebella, and suggest that A-to-I editing of synaptic genes may be informative for assessing the epigenetic risk for autism

  20. Endonuclease V cleaves at inosines in RNA.

    PubMed

    Vik, Erik Sebastian; Nawaz, Meh Sameen; Strøm Andersen, Pernille; Fladeby, Cathrine; Bjørås, Magnar; Dalhus, Bjørn; Alseth, Ingrun

    2013-01-01

    Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABA(A) neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism. PMID:23912683

  1. Endonuclease V cleaves at inosines in RNA

    PubMed Central

    Sebastian Vik, Erik; Sameen Nawaz, Meh; Strøm Andersen, Pernille; Fladeby, Cathrine; Bjørås, Magnar; Dalhus, Bjørn; Alseth, Ingrun

    2013-01-01

    Endonuclease V orthologues are highly conserved proteins found in all kingdoms of life. While the prokaryotic enzymes are DNA repair proteins for removal of deaminated adenosine (inosine) from the genome, no clear role for the eukaryotic counterparts has hitherto been described. Here we report that human endonuclease V (ENDOV) and also Escherichia coli endonuclease V are highly active ribonucleases specific for inosine in RNA. Inosines are normal residues in certain RNAs introduced by specific deaminases. Adenosine-to-inosine editing is essential for proper function of these transcripts and defects are linked to various human disease. Here we show that human ENDOV cleaves an RNA substrate containing inosine in a position corresponding to a biologically important site for deamination in the Gabra-3 transcript of the GABAA neurotransmitter. Further, human ENDOV specifically incises transfer RNAs with inosine in the wobble position. This previously unknown RNA incision activity may suggest a role for endonuclease V in normal RNA metabolism. PMID:23912683

  2. ADAR2 editing enzyme is a novel human immunodeficiency virus-1 proviral factor.

    PubMed

    Doria, Margherita; Tomaselli, Sara; Neri, Francesca; Ciafrè, Silvia Anna; Farace, Maria Giulia; Michienzi, Alessandro; Gallo, Angela

    2011-05-01

    The adenosine deaminases acting on RNA (ADAR) enzymes catalyse conversion of adenosine to inosine in dsRNA. A positive effect of ADAR1 on human immunodeficiency virus type 1 (HIV-1) replication has recently been reported. Here, we show that another ADAR enzyme, ADAR2, positively affects the replication process of HIV-1. We found that, analogously to ADAR1, ADAR2 enhances the release of progeny virions by an editing-dependent mechanism. However, differently from the ADAR1 enzyme, ADAR2 does not increase the infectious potential of the virus. Importantly, downregulation of ADAR2 in Jurkat cells significantly impairs viral replication. Therefore, ADAR2 shares some but not all proviral functions of ADAR1. These results suggest a novel role of ADAR2 as a viral regulator. PMID:21289159

  3. APOBEC3A is implicated in a novel class of G-to-A mRNA editing in WT1 transcripts.

    PubMed

    Niavarani, Ahmadreza; Currie, Erin; Reyal, Yasmin; Anjos-Afonso, Fernando; Horswell, Stuart; Griessinger, Emmanuel; Luis Sardina, Jose; Bonnet, Dominique

    2015-01-01

    Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease. PMID:25807502

  4. APOBEC3A Is Implicated in a Novel Class of G-to-A mRNA Editing in WT1 Transcripts

    PubMed Central

    Niavarani, Ahmadreza; Anjos-Afonso, Fernando; Horswell, Stuart; Griessinger, Emmanuel; Luis Sardina, Jose; Bonnet, Dominique

    2015-01-01

    Classic deamination mRNA changes, including cytidine to uridine (C-to-U) and adenosine to inosine (A-to-I), are important exceptions to the central dogma and lead to significant alterations in gene transcripts and products. Although there are a few reports of non-classic mRNA alterations, as yet there is no molecular explanation for these alternative changes. Wilms Tumor 1 (WT1) mutations and variants are implicated in several diseases, including Wilms tumor and acute myeloid leukemia (AML). We observed two alternative G-to-A changes, namely c.1303G>A and c.1586G>A in cDNA clones and found them to be recurrent in a series of 21 umbilical cord blood mononuclear cell (CBMC) samples studied. Two less conserved U-to-C changes were also observed. These alternative changes were found to be significantly higher in non-progenitor as compared to progenitor CBMCs, while they were found to be absent in a series of AML samples studied, indicating they are targeted, cell type-specific mRNA editing modifications. Since APOBEC/ADAR family members are implicated in RNA/DNA editing, we screened them by RNA-interference (RNAi) for WT1-mRNA changes and observed near complete reversal of WT1 c.1303G>A alteration upon APOBEC3A (A3A) knockdown. The role of A3A in mediating this change was confirmed by A3A overexpression in Fujioka cells, which led to a significant increase in WT1 c.1303G>A mRNA editing. Non-progenitor CBMCs showed correspondingly higher levels of A3A-mRNA and protein as compared to the progenitor ones. To our knowledge, this is the first report of mRNA modifying activity for an APOBEC3 protein and implicates A3A in a novel G-to-A form of editing. These findings open the way to further investigations into the mechanisms of other potential mRNA changes, which will help to redefine the RNA editing paradigm in both health and disease. PMID:25807502

  5. FRET analysis of in vivo dimerization by RNA-editing enzymes.

    PubMed

    Chilibeck, Kaari A; Wu, Tao; Liang, Chao; Schellenberg, Matthew J; Gesner, Emily M; Lynch, Jeffrey M; MacMillan, Andrew M

    2006-06-16

    Members of the ADAR (adenosine deaminase that acts on RNA) enzyme family catalyze the hydrolytic deamination of adenosine to inosine within double-stranded RNAs, a poorly understood process that is critical to mammalian development. We have performed fluorescence resonance energy transfer experiments in mammalian cells transfected with fluorophore-bearing ADAR1 and ADAR2 fusion proteins to investigate the relationship between these proteins. These studies conclusively demonstrate the homodimerization of ADAR1 and ADAR2 and also show that ADAR1 and ADAR2 form heterodimers in human cells. RNase treatment of cells expressing these fusion proteins changes their localization but does not affect dimerization. Taken together these results suggest that homo- and heterodimerization are important for the activity of ADAR family members in vivo and that these associations are RNA independent. PMID:16618704

  6. REDIdb: the RNA editing database

    PubMed Central

    Picardi, Ernesto; Regina, Teresa Maria Rosaria; Brennicke, Axel; Quagliariello, Carla

    2007-01-01

    The RNA Editing Database (REDIdb) is an interactive, web-based database created and designed with the aim to allocate RNA editing events such as substitutions, insertions and deletions occurring in a wide range of organisms. The database contains both fully and partially sequenced DNA molecules for which editing information is available either by experimental inspection (in vitro) or by computational detection (in silico). Each record of REDIdb is organized in a specific flat-file containing a description of the main characteristics of the entry, a feature table with the editing events and related details and a sequence zone with both the genomic sequence and the corresponding edited transcript. REDIdb is a relational database in which the browsing and identification of editing sites has been simplified by means of two facilities to either graphically display genomic or cDNA sequences or to show the corresponding alignment. In both cases, all editing sites are highlighted in colour and their relative positions are detailed by mousing over. New editing positions can be directly submitted to REDIdb after a user-specific registration to obtain authorized secure access. This first version of REDIdb database stores 9964 editing events and can be freely queried at . PMID:17175530

  7. Profiling the RNA editomes of wild-type C. elegans and ADAR mutants.

    PubMed

    Zhao, Han-Qing; Zhang, Pan; Gao, Hua; He, Xiandong; Dou, Yanmei; Huang, August Y; Liu, Xi-Ming; Ye, Adam Y; Dong, Meng-Qiu; Wei, Liping

    2015-01-01

    RNA editing increases transcriptome diversity through post-transcriptional modifications of RNA. Adenosine deaminases that act on RNA (ADARs) catalyze the adenosine-to-inosine (A-to-I) conversion, the most common type of RNA editing in higher eukaryotes. Caenorhabditis elegans has two ADARs, ADR-1 and ADR-2, but their functions remain unclear. Here, we profiled the RNA editomes of C. elegans at different developmental stages of wild-type and ADAR mutants. We developed a new computational pipeline with a "bisulfite-seq-mapping-like" step and achieved a threefold increase in identification sensitivity. A total of 99.5% of the 47,660 A-to-I editing sites were found in clusters. Of the 3080 editing clusters, 65.7% overlapped with DNA transposons in noncoding regions and 73.7% could form hairpin structures. The numbers of editing sites and clusters were highest at the L1 and embryonic stages. The editing frequency of a cluster positively correlated with the number of editing sites within it. Intriguingly, for 80% of the clusters with 10 or more editing sites, almost all expressed transcripts were edited. Deletion of adr-1 reduced the editing frequency but not the number of editing clusters, whereas deletion of adr-2 nearly abolished RNA editing, indicating a modulating role of ADR-1 and an essential role of ADR-2 in A-to-I editing. Quantitative proteomics analysis showed that adr-2 mutant worms altered the abundance of proteins involved in aging and lifespan regulation. Consistent with this finding, we observed that worms lacking RNA editing were short-lived. Taken together, our results reveal a sophisticated landscape of RNA editing and distinct modes of action of different ADARs. PMID:25373143

  8. RNA Binding-independent Dimerization of Adenosine Deaminases Acting on RNA and Dominant Negative Effects of Nonfunctional Subunits on Dimer Functions*

    PubMed Central

    Valente, Louis; Nishikura, Kazuko

    2010-01-01

    RNA editing that converts adenosine to inosine in double-stranded RNA (dsRNA) is mediated by adenosine deaminases acting on RNA (ADAR). ADAR1 and ADAR2 form respective homodimers, and this association is essential for their enzymatic activities. In this investigation, we set out experiments aiming to determine whether formation of the homodimer complex is mediated by an amino acid interface made through protein-protein interactions of two monomers or via binding of the two subunits to a dsRNA substrate. Point mutations were created in the dsRNA binding domains (dsRBDs) that abolished all RNA binding, as tested for two classes of ADAR ligands, long and short dsRNA. The mutant ADAR dimer complexes were intact, as demonstrated by their ability to co-purify in a sequential affinity-tagged purification and also by their elution at the dimeric fraction position on a size fractionation column. Our results demonstrated ADAR dimerization independent of their binding to dsRNA, establishing the importance of protein-protein interactions for dimer formation. As expected, these mutant ADARs could no longer perform their catalytic function due to the loss in substrate binding. Surprisingly, a chimeric dimer consisting of one RNA binding mutant monomer and a wild type partner still abolished its ability to bind and edit its substrate, indicating that ADAR dimers require two subunits with functional dsRBDs for binding to a dsRNA substrate and then for editing activity to occur. PMID:17428802

  9. A structural determinant required for RNA editing

    PubMed Central

    Tian, Nan; Yang, Yun; Sachsenmaier, Nora; Muggenhumer, Dominik; Bi, Jingpei; Waldsich, Christina; Jantsch, Michael F.; Jin, Yongfeng

    2011-01-01

    RNA editing by adenosine deaminases acting on RNAs (ADARs) can be both specific and non-specific, depending on the substrate. Specific editing of particular adenosines may depend on the overall sequence and structural context. However, the detailed mechanisms underlying these preferences are not fully understood. Here, we show that duplex structures mimicking an editing site in the Gabra3 pre-mRNA unexpectedly fail to support RNA editing at the Gabra3 I/M site, although phylogenetic analysis suggest an evolutionarily conserved duplex structure essential for efficient RNA editing. These unusual results led us to revisit the structural requirement for this editing by mutagenesis analysis. In vivo nuclear injection experiments of mutated editing substrates demonstrate that a non-conserved structure is a determinant for editing. This structure contains bulges either on the same or the strand opposing the edited adenosine. The position of these bulges and the distance to the edited base regulate editing. Moreover, elevated folding temperature can lead to a switch in RNA editing suggesting an RNA structural change. Our results indicate the importance of RNA tertiary structure in determining RNA editing. PMID:21427087

  10. Functional characterizations and expression profiles of ADAR2 gene, responsible for RNA editing, in response to GCRV challenge in grass carp (Ctenopharyngodon idella).

    PubMed

    Su, Juanjuan; Han, Baoquan; Rao, Youliang; Feng, Xiaoli; Su, Jianguo

    2016-09-01

    ADAR (adenosine deaminases acting on RNA)-mediated adenosine-to-inosine (A-to-I) editing to double-stranded RNA (dsRNA) is a critical arm of the antiviral response. The present study focused on the structural and functional characterizations of grass carp (Ctenopharyngodon idella) ADAR2 (CiADAR2) gene. The complete genomic sequence of CiADAR2 is 150,458 bp in length, containing 12 exons and 11 introns. The open reading frame (ORF) of 2100 bp encodes a polypeptide of 699 amino acids (aa) which contains three highly conservative domains - two N-terminal dsRNA binding domains (dsRBDs) and one C-terminal deaminase domain. The predicted crystal structure of CiADAR2 deaminase domain suggested a catalytic center form in the enzyme active site. CiADAR2 mRNA was ubiquitously expressed in the fifteen tested tissues, and was induced post GCRV challenge in spleen and head kidney and C. idella kidney (CIK) cells. The ex vivo expression of CiADAR2 protein was verified by the Flag (tag)-based western blot assay. Antiviral activity assay of CiADAR2 was manifested by the delayed appearance of cytopathic effect (CPE) and inhibition of GCRV yield at 48 h post infection. Furthermore, in CiADAR2 overexpression cells, mRNA expression levels of CiIFN1, CiTLR7 and CiTLR8 were facilitated at different time points after GCRV infection, comparing to those in control group. Taken together, it was indicated that ADAR2 was an antiviral cytokine against GCRV and anti-GCRV function mechanism might involve in the TLR7/8-regulated IFN-signaling. These findings suggested that CiADAR2 was a novel member engaging in antiviral innate immune defense in C. idella, which laid a foundation for the further mechanism research of ADAR2 in fishes. PMID:27514783

  11. The evolution of chloroplast RNA editing.

    PubMed

    Tillich, Michael; Lehwark, Pascal; Morton, Brian R; Maier, Uwe G

    2006-10-01

    RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria. PMID:16835291

  12. The Landscape of A-to-I RNA Editome Is Shaped by Both Positive and Purifying Selection

    PubMed Central

    Kong, Yimeng; Pan, Bohu; Chen, Longxian; Wang, Hongbing; Hao, Pei; Li, Xuan

    2016-01-01

    The hydrolytic deamination of adenosine to inosine (A-to-I editing) in precursor mRNA induces variable gene products at the post-transcription level. How and to what extent A-to-I RNA editing diversifies transcriptome is not fully characterized in the evolution, and very little is known about the selective constraints that drive the evolution of RNA editing events. Here we present a study on A-to-I RNA editing, by generating a global profile of A-to-I editing for a phylogeny of seven Drosophila species, a model system spanning an evolutionary timeframe of approximately 45 million years. Of totally 9281 editing events identified, 5150 (55.5%) are located in the coding sequences (CDS) of 2734 genes. Phylogenetic analysis places these genes into 1,526 homologous families, about 5% of total gene families in the fly lineages. Based on conservation of the editing sites, the editing events in CDS are categorized into three distinct types, representing events on singleton genes (type I), and events not conserved (type II) or conserved (type III) within multi-gene families. While both type I and II events are subject to purifying selection, notably type III events are positively selected, and highly enriched in the components and functions of the nervous system. The tissue profiles are documented for three editing types, and their critical roles are further implicated by their shifting patterns during holometabolous development and in post-mating response. In conclusion, three A-to-I RNA editing types are found to have distinct evolutionary dynamics. It appears that nervous system functions are mainly tested to determine if an A-to-I editing is beneficial for an organism. The coding plasticity enabled by A-to-I editing creates a new class of binary variations, which is a superior alternative to maintain heterozygosity of expressed genes in a diploid mating system. PMID:27467689

  13. Exploring the RNA editing potential of RNA-Seq data by ExpEdit.

    PubMed

    D'Antonio, Mattia; Picardi, Ernesto; Castrignanò, Tiziana; D'Erchia, Anna Maria; Pesole, Graziano

    2015-01-01

    Revealing the impact of A-to-I RNA editing in RNA-Seq experiments is relevant in humans because RNA editing can influence gene expression. In addition, its deregulation has been linked to a variety of human diseases. Exploiting the RNA editing potential in complete RNA-Seq datasets, however, is a challenging task. Indeed, no dedicated software is available, and sometimes deep computational skills and appropriate hardware resources are required. To explore the impact of known RNA editing events in massive transcriptome sequencing experiments, we developed the ExpEdit web service application. In the present work, we provide an overview of ExpEdit as well as methodologies to investigate known RNA editing in human RNA-Seq datasets. PMID:25577388

  14. Statistical Physics Approaches to RNA Editing

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf

    2012-02-01

    The central dogma of molecular Biology states that DNA is transcribed base by base into RNA which is in turn translated into proteins. However, some organisms edit their RNA before translation by inserting, deleting, or substituting individual or short stretches of bases. In many instances the mechanisms by which an organism recognizes the positions at which to edit or by which it performs the actual editing are unknown. One model system that stands out by its very high rate of on average one out of 25 bases being edited are the Myxomycetes, a class of slime molds. In this talk we will show how the computational methods and concepts from statistical Physics can be used to analyze DNA and protein sequence data to predict editing sites in these slime molds and to guide experiments that identified previously unknown types of editing as well as the complete set of editing events in the slime mold Physarum polycephalum.

  15. Changing genetic information through RNA editing

    NASA Technical Reports Server (NTRS)

    Maas, S.; Rich, A.

    2000-01-01

    RNA editing, the post-transcriptional alteration of a gene-encoded sequence, is a widespread phenomenon in eukaryotes. As a consequence of RNA editing, functionally distinct proteins can be produced from a single gene. The molecular mechanisms involved include single or multiple base insertions or deletions as well as base substitutions. In mammals, one type of substitutional RNA editing, characterized by site-specific base-modification, was shown to modulate important physiological processes. The underlying reaction mechanism of substitutional RNA editing involves hydrolytic deamination of cytosine or adenosine bases to uracil or inosine, respectively. Protein factors have been characterized that are able to induce RNA editing in vitro. A supergene family of RNA-dependent deaminases has emerged with the recent addition of adenosine deaminases specific for tRNA. Here we review the developments that have substantially increased our understanding of base-modification RNA editing over the past few years, with an emphasis on mechanistic differences, evolutionary aspects and the first insights into the regulation of editing activity.

  16. Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

    PubMed Central

    Sarvestani, Soroush T.; Tate, Michelle D.; Moffat, Jessica M.; Jacobi, Ashley M.; Behlke, Mark A.; Miller, Alistair R.; Beckham, Simone A.; McCoy, Claire E.; Chen, Weisan; Mintern, Justine D.; O'Keeffe, Meredith; John, Matthias

    2014-01-01

    RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5′-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA. PMID:24227841

  17. Are substitution rates and RNA editing correlated?

    PubMed Central

    2010-01-01

    Background RNA editing is a post-transcriptional process that, in seed plants, involves a cytosine to uracil change in messenger RNA, causing the translated protein to differ from that predicted by the DNA sequence. RNA editing occurs extensively in plant mitochondria, but large differences in editing frequencies are found in some groups. The underlying processes responsible for the distribution of edited sites are largely unknown, but gene function, substitution rate, and gene conversion have been proposed to influence editing frequencies. Results We studied five mitochondrial genes in the monocot order Alismatales, all showing marked differences in editing frequencies among taxa. A general tendency to lose edited sites was observed in all taxa, but this tendency was particularly strong in two clades, with most of the edited sites lost in parallel in two different areas of the phylogeny. This pattern is observed in at least four of the five genes analyzed. Except in the groups that show an unusually low editing frequency, the rate of C-to-T changes in edited sites was not significantly higher that in non-edited 3rd codon positions. This may indicate that selection is not actively removing edited sites in nine of the 12 families of the core Alismatales. In all genes but ccmB, a significant correlation was found between frequency of change in edited sites and synonymous substitution rate. In general, taxa with higher substitution rates tend to have fewer edited sites, as indicated by the phylogenetically independent correlation analyses. The elimination of edited sites in groups that lack or have reduced levels of editing could be a result of gene conversion involving a cDNA copy (retroprocessing). If so, this phenomenon could be relatively common in the Alismatales, and may have affected some groups recurrently. Indirect evidence of retroprocessing without a necessary correlation with substitution rate was found mostly in families Alismataceae and Hydrocharitaceae

  18. PRUNE2 is a human prostate cancer suppressor regulated by the intronic long noncoding RNA PCA3.

    PubMed

    Salameh, Ahmad; Lee, Alessandro K; Cardó-Vila, Marina; Nunes, Diana N; Efstathiou, Eleni; Staquicini, Fernanda I; Dobroff, Andrey S; Marchiò, Serena; Navone, Nora M; Hosoya, Hitomi; Lauer, Richard C; Wen, Sijin; Salmeron, Carolina C; Hoang, Anh; Newsham, Irene; Lima, Leandro A; Carraro, Dirce M; Oliviero, Salvatore; Kolonin, Mikhail G; Sidman, Richard L; Do, Kim-Anh; Troncoso, Patricia; Logothetis, Christopher J; Brentani, Ricardo R; Calin, George A; Cavenee, Webster K; Dias-Neto, Emmanuel; Pasqualini, Renata; Arap, Wadih

    2015-07-01

    Prostate cancer antigen 3 (PCA3) is the most specific prostate cancer biomarker but its function remains unknown. Here we identify PRUNE2, a target protein-coding gene variant, which harbors the PCA3 locus, thereby classifying PCA3 as an antisense intronic long noncoding (lnc)RNA. We show that PCA3 controls PRUNE2 levels via a unique regulatory mechanism involving formation of a PRUNE2/PCA3 double-stranded RNA that undergoes adenosine deaminase acting on RNA (ADAR)-dependent adenosine-to-inosine RNA editing. PRUNE2 expression or silencing in prostate cancer cells decreased and increased cell proliferation, respectively. Moreover, PRUNE2 and PCA3 elicited opposite effects on tumor growth in immunodeficient tumor-bearing mice. Coregulation and RNA editing of PRUNE2 and PCA3 were confirmed in human prostate cancer specimens, supporting the medical relevance of our findings. These results establish PCA3 as a dominant-negative oncogene and PRUNE2 as an unrecognized tumor suppressor gene in human prostate cancer, and their regulatory axis represents a unique molecular target for diagnostic and therapeutic intervention. PMID:26080435

  19. RCARE: RNA Sequence Comparison and Annotation for RNA Editing

    PubMed Central

    2015-01-01

    The post-transcriptional sequence modification of transcripts through RNA editing is an important mechanism for regulating protein function and is associated with human disease phenotypes. The identification of RNA editing or RNA-DNA difference (RDD) sites is a fundamental step in the study of RNA editing. However, a substantial number of false-positive RDD sites have been identified recently. A major challenge in identifying RDD sites is to distinguish between the true RNA editing sites and the false positives. Furthermore, determining the location of condition-specific RDD sites and elucidating their functional roles will help toward understanding various biological phenomena that are mediated by RNA editing. The present study developed RNA-sequence comparison and annotation for RNA editing (RCARE) for searching, annotating, and visualizing RDD sites using thousands of previously known editing sites, which can be used for comparative analyses between multiple samples. RCARE also provides evidence for improving the reliability of identified RDD sites. RCARE is a web-based comparison, annotation, and visualization tool, which provides rich biological annotations and useful summary plots. The developers of previous tools that identify or annotate RNA-editing sites seldom mention the reliability of their respective tools. In order to address the issue, RCARE utilizes a number of scientific publications and databases to find specific documentations respective to a particular RNA-editing site, which generates evidence levels to convey the reliability of RCARE. Sequence-based alignment files can be converted into VCF files using a Python script and uploaded to the RCARE server for further analysis. RCARE is available for free at http://www.snubi.org/software/rcare/. PMID:26043858

  20. Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA

    PubMed Central

    Jayachandran, Uma; Grey, Heather; Cook, Atlanta G.

    2016-01-01

    Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3′ untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs. PMID:26712564

  1. Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA.

    PubMed

    Jayachandran, Uma; Grey, Heather; Cook, Atlanta G

    2016-02-29

    Nuclear factors 90 and 45 (NF90 and NF45) form a protein complex involved in the post-transcriptional control of many genes in vertebrates. NF90 is a member of the dsRNA binding domain (dsRBD) family of proteins. RNA binding partners identified so far include elements in 3' untranslated regions of specific mRNAs and several non-coding RNAs. In NF90, a tandem pair of dsRBDs separated by a natively unstructured segment confers dsRNA binding activity. We determined a crystal structure of the tandem dsRBDs of NF90 in complex with a synthetic dsRNA. This complex shows surprising similarity to the tandem dsRBDs from an adenosine-to-inosine editing enzyme, ADAR2 in complex with a substrate RNA. Residues involved in unusual base-specific recognition in the minor groove of dsRNA are conserved between NF90 and ADAR2. These data suggest that, like ADAR2, underlying sequences in dsRNA may influence how NF90 recognizes its target RNAs. PMID:26712564

  2. Evolution of RNA editing in trypanosome mitochondria

    PubMed Central

    Simpson, Larry; Thiemann, Otavio H.; Savill, Nicholas J.; Alfonzo, Juan D.; Maslov, D. A.

    2000-01-01

    Two different RNA editing systems have been described in the kinetoplast-mitochondrion of trypanosomatid protists. The first involves the precise insertion and deletion of U residues mostly within the coding regions of maxicircle-encoded mRNAs to produce open reading frames. This editing is mediated by short overlapping complementary guide RNAs encoded in both the maxicircle and the minicircle molecules and involves a series of enzymatic cleavage-ligation steps. The second editing system is a C34 to U34 modification in the anticodon of the imported tRNATrp, thereby permitting the decoding of the UGA stop codon as tryptophan. U-insertion editing probably originated in an ancestor of the kinetoplastid lineage and appears to have evolved in some cases by the replacement of the original pan-edited cryptogene with a partially edited cDNA. The driving force for the evolutionary fixation of these retroposition events was postulated to be the stochastic loss of entire minicircle sequence classes and their encoded guide RNAs upon segregation of the single kinetoplast DNA network into daughter cells at cell division. A large plasticity in the relative abundance of minicircle sequence classes has been observed during cell culture in the laboratory. Computer simulations provide theoretical evidence for this plasticity if a random distribution and segregation model of minicircles is assumed. The possible evolutionary relationship of the C to U and U-insertion editing systems is discussed. PMID:10860961

  3. RNA editing generates cellular subsets with diverse sequence within populations.

    PubMed

    Harjanto, Dewi; Papamarkou, Theodore; Oates, Chris J; Rayon-Estrada, Violeta; Papavasiliou, F Nina; Papavasiliou, Anastasia

    2016-01-01

    RNA editing is a mutational mechanism that specifically alters the nucleotide content in transcribed RNA. However, editing rates vary widely, and could result from equivalent editing amongst individual cells, or represent an average of variable editing within a population. Here we present a hierarchical Bayesian model that quantifies the variance of editing rates at specific sites using RNA-seq data from both single cells, and a cognate bulk sample to distinguish between these two possibilities. The model predicts high variance for specific edited sites in murine macrophages and dendritic cells, findings that we validated experimentally by using targeted amplification of specific editable transcripts from single cells. The model also predicts changes in variance in editing rates for specific sites in dendritic cells during the course of LPS stimulation. Our data demonstrate substantial variance in editing signatures amongst single cells, supporting the notion that RNA editing generates diversity within cellular populations. PMID:27418407

  4. RNA editing generates cellular subsets with diverse sequence within populations

    PubMed Central

    Harjanto, Dewi; Papamarkou, Theodore; Oates, Chris J.; Rayon-Estrada, Violeta; Papavasiliou, F. Nina; Papavasiliou, Anastasia

    2016-01-01

    RNA editing is a mutational mechanism that specifically alters the nucleotide content in transcribed RNA. However, editing rates vary widely, and could result from equivalent editing amongst individual cells, or represent an average of variable editing within a population. Here we present a hierarchical Bayesian model that quantifies the variance of editing rates at specific sites using RNA-seq data from both single cells, and a cognate bulk sample to distinguish between these two possibilities. The model predicts high variance for specific edited sites in murine macrophages and dendritic cells, findings that we validated experimentally by using targeted amplification of specific editable transcripts from single cells. The model also predicts changes in variance in editing rates for specific sites in dendritic cells during the course of LPS stimulation. Our data demonstrate substantial variance in editing signatures amongst single cells, supporting the notion that RNA editing generates diversity within cellular populations. PMID:27418407

  5. Evidence for auto-inhibition by the N terminus of hADAR2 and activation by dsRNA binding

    PubMed Central

    MACBETH, MARK R.; LINGAM, ARUNTH T.; BASS, BRENDA L.

    2004-01-01

    Adenosine deaminases that act on RNA (ADARs) catalyze adenosine to inosine conversion in RNA that is largely double stranded. Human ADAR2 (hADAR2) contains two double-stranded RNA binding motifs (dsRBMs), separated by a 90-amino acid linker, and these are followed by the C-terminal catalytic domain. We assayed enzymatic activity of N-terminal deletion constructs of hADAR2 to determine the role of the dsRBMs and the intervening linker peptide. We found that a truncated protein consisting of one dsRBM and the deaminase domain was capable of deaminating a short 15-bp substrate. In contrast, full-length hADAR2 was inactive on this short substrate. In addition, we observed that the N terminus, which was deleted from the truncated protein, inhibits editing activity when added in trans. We propose that the N-terminal domain of hADAR2 contains sequences that cause auto-inhibition of the enzyme. Our results suggest activation requires binding to an RNA substrate long enough to accommodate interactions with both dsRBMs. PMID:15383678

  6. The Impact of mRNA Structure on Guide RNA Targeting in Kinetoplastid RNA Editing

    PubMed Central

    Reifur, Larissa; Yu, Laura E.; Cruz-Reyes, Jorge; vanHartesvelt, Michelle; Koslowsky, Donna J.

    2010-01-01

    Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5′ end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different “sets” of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a “bank” of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins. PMID:20808932

  7. The role of RNA editing in dynamic environments

    SciTech Connect

    Rocha, L. M.; Huang, C. F.

    2004-01-01

    This paper presents a computational methodology based on Genetic Algorithms with Genotype Editing (GAE) for investigating the role of RNA editing in dynamic environments. This model is constructed based on several genetic editing characteristics that are gleaned from the RNA editing system as observed in several organisms. We have previously expanded the traditional Genetic Algorithm (GA) with artificial editing mechanisms (Rocha, 1995, 1997), and studied the benefits of including straightforward Genotype Editing in GA for several machine learning problems (Huang and Rocha, 2003, 2004). We show that the incorporation of genotype editing provides a means for artificial agents with genetic descriptions to gain greater phenotypic plasticity. Artificial agents use genotype edition to their advantage by linking it to environmental context. The ability to link changes in the environment with editing parameters gives organisms an adaptive advantage as genotype expression can become contextually regulated. The study of this RNA editing model in changing environments has shed some light into the evolutionary implications of RNA editing. We expect that our methodology will both facilitate determining the evolutionary role of RNA editing in biology, and advance the current state of research in Evolutionary Computation and Artificial Life.

  8. Ebola virus RNA editing depends on the primary editing site sequence and an upstream secondary structure.

    PubMed

    Mehedi, Masfique; Hoenen, Thomas; Robertson, Shelly; Ricklefs, Stacy; Dolan, Michael A; Taylor, Travis; Falzarano, Darryl; Ebihara, Hideki; Porcella, Stephen F; Feldmann, Heinz

    2013-01-01

    Ebolavirus (EBOV), the causative agent of a severe hemorrhagic fever and a biosafety level 4 pathogen, increases its genome coding capacity by producing multiple transcripts encoding for structural and nonstructural glycoproteins from a single gene. This is achieved through RNA editing, during which non-template adenosine residues are incorporated into the EBOV mRNAs at an editing site encoding for 7 adenosine residues. However, the mechanism of EBOV RNA editing is currently not understood. In this study, we report for the first time that minigenomes containing the glycoprotein gene editing site can undergo RNA editing, thereby eliminating the requirement for a biosafety level 4 laboratory to study EBOV RNA editing. Using a newly developed dual-reporter minigenome, we have characterized the mechanism of EBOV RNA editing, and have identified cis-acting sequences that are required for editing, located between 9 nt upstream and 9 nt downstream of the editing site. Moreover, we show that a secondary structure in the upstream cis-acting sequence plays an important role in RNA editing. EBOV RNA editing is glycoprotein gene-specific, as a stretch encoding for 7 adenosine residues located in the viral polymerase gene did not serve as an editing site, most likely due to an absence of the necessary cis-acting sequences. Finally, the EBOV protein VP30 was identified as a trans-acting factor for RNA editing, constituting a novel function for this protein. Overall, our results provide novel insights into the RNA editing mechanism of EBOV, further understanding of which might result in novel intervention strategies against this viral pathogen. PMID:24146620

  9. RNA editing of microRNA prevents RNA-induced silencing complex recognition of target mRNA

    PubMed Central

    Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

    2015-01-01

    MicroRNAs (miRNAs) integrate with Argonaut (Ago) to create the RNA-induced silencing complex, and regulate gene expression by silencing target mRNAs. RNA editing of miRNA may affect miRNA processing, assembly of the Ago complex and target mRNA binding. However, the function of edited miRNA, assembled within the Ago complex, has not been extensively investigated. In this study, sequence analysis of the Ago complex of Marsupenaeus japonicus shrimp infected with white spot syndrome virus (WSSV) revealed that host ADAR (adenosine deaminase acting on RNA) catalysed A-to-I RNA editing of a viral miRNA (WSSV-miR-N12) at the +16 site. This editing of the non-seed sequence did not affect association of the edited miRNA with the Ago protein, but inhibited interaction between the miRNA and its target gene (wsv399). The WSSV early gene wsv399 inhibited WSSV infection. As a result, the RNA editing of miRNA caused virus latency. Our results highlight a novel example of miRNA editing in the miRNA-induced silencing complex. PMID:26674414

  10. Re-editing the paradigm of Cytidine (C) to Uridine (U) RNA editing

    PubMed Central

    Fossat, Nicolas; Tam, Patrick P L

    2014-01-01

    Cytidine (C) to Uridine (U) RNA editing is a post-trancriptional modification that until recently was known to only affect Apolipoprotein b (Apob) RNA and minimally require 2 components of the C to U editosome, the deaminase APOBEC1 and the RNA-binding protein A1CF. Our latest work has identified a novel RNA-binding protein, RBM47, as a core component of the editosome, which can substitute A1CF for the editing of ApoB mRNA. In addition, new RNA species that are subjected to C to U editing have been identified. Here, we highlight these recent discoveries and discuss how they change our view of the composition of the C to U editing machinery and expand our knowledge of the functional attributes of C to U RNA editing. PMID:25585043

  11. The emerging role of RNA editing in plasticity

    PubMed Central

    Rosenthal, Joshua J. C.

    2015-01-01

    ABSTRACT All true metazoans modify their RNAs by converting specific adenosine residues to inosine. Because inosine binds to cytosine, it is a biological mimic for guanosine. This subtle change, termed RNA editing, can have diverse effects on various RNA-mediated cellular pathways, including RNA interference, innate immunity, retrotransposon defense and messenger RNA recoding. Because RNA editing can be regulated, it is an ideal tool for increasing genetic diversity, adaptation and environmental acclimation. This review will cover the following themes related to RNA editing: (1) how it is used to modify different cellular RNAs, (2) how frequently it is used by different organisms to recode mRNA, (3) how specific recoding events regulate protein function, (4) how it is used in adaptation and (5) emerging evidence that it can be used for acclimation. Organismal biologists with an interest in adaptation and acclimation, but with little knowledge of RNA editing, are the intended audience. PMID:26085659

  12. Genome-wide identification of RNA editing in hepatocellular carcinoma.

    PubMed

    Kang, Lin; Liu, Xiaoqiao; Gong, Zhoulin; Zheng, Hancheng; Wang, Jun; Li, Yingrui; Yang, Huanming; Hardwick, James; Dai, Hongyue; Poon, Ronnie T P; Lee, Nikki P; Mao, Mao; Peng, Zhiyu; Chen, Ronghua

    2015-02-01

    We did whole-transcriptome sequencing and whole-genome sequencing on nine pairs of Hepatocellular carcinoma (HCC) tumors and matched adjacent tissues to identify RNA editing events. We identified mean 26,982 editing sites with mean 89.5% canonical A→G edits in each sample using an improved bioinformatics pipeline. The editing rate was significantly higher in tumors than adjacent normal tissues. Comparing the difference between tumor and normal tissues of each patient, we found 7 non-synonymous tissue specific editing events including 4 tumor-specific edits and 3 normal-specific edits in the coding region, as well as 292 edits varying in editing degree. The significant expression changes of 150 genes associated with RNA editing were found in tumors, with 3 of the 4 most significant genes being cancer related. Our results show that editing might be related to higher gene expression. These findings indicate that RNA editing modification may play an important role in the development of HCC. PMID:25462863

  13. Identification of candidate mitochondrial RNA editing ligases from Trypanosoma brucei.

    PubMed Central

    McManus, M T; Shimamura, M; Grams, J; Hajduk, S L

    2001-01-01

    Most mitochondrial genes of Trypanosoma brucei do not contain the necessary information to make translatable mRNAs. These transcripts must undergo RNA editing, a posttranscriptional process by which uridine residues are added and deleted from mitochondrial mRNAs. RNA editing is believed to be catalyzed by a ribonucleoprotein complex containing endonucleolytic, terminal uridylyl transferase (TUTase), 3' uridine-specific exonucleolytic (U-exo), and ligase activities. None of the catalytic enzymes for RNA editing have been identified. Here we describe the identification of two candidate RNA ligases (48 and 52 kDa) that are core catalytic components of the T. brucei ribonucleoprotein editing complex. Both enzymes share homology to the covalent nucleotidyl transferase superfamily and contain five key signature motifs, including the active site KXXG. In this report, we present data on the proposed 48 kDa RNA editing ligase. We have prepared polyclonal antibodies against recombinant 48 kDa ligase that specifically recognize the trypanosome enzyme. When expressed in trypanosomes as an epitope-tagged fusion protein, the recombinant ligase localizes to the mitochondrion, associates with RNA editing complexes, and adenylates with ATP. These findings provide strong support for the enzymatic cascade model for kinetoplastid RNA editing. PMID:11233974

  14. Uncovering RNA Editing Sites in Long Non-Coding RNAs

    PubMed Central

    Picardi, Ernesto; D’Erchia, Anna Maria; Gallo, Angela; Montalvo, Antonio; Pesole, Graziano

    2014-01-01

    RNA editing is an important co/post-transcriptional molecular process able to modify RNAs by nucleotide insertions/deletions or substitutions. In human, the most common RNA editing event involves the deamination of adenosine (A) into inosine (I) through the adenosine deaminase acting on RNA proteins. Although A-to-I editing can occur in both coding and non-coding RNAs, recent findings, based on RNA-seq experiments, have clearly demonstrated that a large fraction of RNA editing events alter non-coding RNAs sequences including untranslated regions of mRNAs, introns, long non-coding RNAs (lncRNAs), and low molecular weight RNAs (tRNA, miRNAs, and others). An accurate detection of A-to-I events occurring in non-coding RNAs is of utmost importance to clarify yet unknown functional roles of RNA editing in the context of gene expression regulation and maintenance of cell homeostasis. In the last few years, massive transcriptome sequencing has been employed to identify putative RNA editing changes at genome scale. Despite several efforts, the computational prediction of A-to-I sites in complete eukaryotic genomes is yet a challenging task. We have recently developed a software package, called REDItools, in order to simplify the detection of RNA editing events from deep sequencing data. In the present work, we show the potential of our tools in recovering A-to-I candidates from RNA-Seq experiments as well as guidelines to improve the RNA editing detection in non-coding RNAs, with specific attention to the lncRNAs. PMID:25538940

  15. A-to-I RNA editing is induced upon hypoxia.

    PubMed

    Nevo-Caspi, Yael; Amariglio, Ninette; Rechavi, Gideon; Paret, Gideon

    2011-06-01

    Regulating gene expression is part of a cell's response to hypoxia. A-to-I RNA editing is an epigenetic phenomenon that can contribute to RNA and protein levels and to isoform diversity. In this study, we identified alterations in the levels of RNA editing following hypoxic stress in three genes: MED13, STAT3, and F11R. Changes in editing levels were associated with changes in RNA levels. These results suggest that A-to-I RNA editing may be one of the mechanisms used by cells to regulate changes in gene expression after hypoxia. These findings could lead to a novel therapeutic approach and better health care for children with hypoxemia. PMID:21330951

  16. RNA editing, epitranscriptomics, and processing in cancer progression

    PubMed Central

    Witkin, Keren L; Hanlon, Sean E; Strasburger, Jennifer A; Coffin, John M; Jaffrey, Samie R; Howcroft, T Kevin; Dedon, Peter C; Steitz, Joan A; Daschner, Phil J; Read-Connole, Elizabeth

    2015-01-01

    The transcriptome is extensively and dynamically regulated by a network of RNA modifying factors. RNA editing enzymes APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) and ADAR (adenosine deaminase, RNA-specific) irreversibly recode primary RNA sequences, whereas newly described methylases (writers) and de-methylases (erasers) dynamically alter RNA molecules in response to environmental conditions. RNA modifications can affect RNA splicing, nuclear-cytoplasmic transport, translation, and regulation of gene expression by RNA interference. In addition, tRNA base modifications, processing, and regulated cleavage have been shown to alter global patterns of mRNA translation in response to cellular stress pathways. Recent studies, some of which were discussed at this workshop, have rekindled interest in the emerging roles of RNA modifications in health and disease. On September 10th, 2014, the Division of Cancer Biology, NCI sponsored a workshop to explore the role of epitranscriptomic RNA modifications and tRNA processing in cancer progression. The workshop attendees spanned a scientific range including chemists, virologists, and RNA and cancer biologists. The goal of the workshop was to explore the interrelationships between RNA editing, epitranscriptomics, and RNA processing and the enzymatic pathways that regulate these activities in cancer initiation and progression. At the conclusion of the workshop, a general discussion focused on defining the major challenges and opportunities in this field, as well as identifying the tools, technologies, resources and community efforts required to accelerate research in this emerging area. PMID:25455629

  17. Profiling RNA editing in human tissues: towards the inosinome Atlas

    PubMed Central

    Picardi, Ernesto; Manzari, Caterina; Mastropasqua, Francesca; Aiello, Italia; D’Erchia, Anna Maria; Pesole, Graziano

    2015-01-01

    Adenine to Inosine RNA editing is a widespread co- and post-transcriptional mechanism mediated by ADAR enzymes acting on double stranded RNA. It has a plethora of biological effects, appears to be particularly pervasive in humans with respect to other mammals, and is implicated in a number of diverse human pathologies. Here we present the first human inosinome atlas comprising 3,041,422 A-to-I events identified in six tissues from three healthy individuals. Matched directional total-RNA-Seq and whole genome sequence datasets were generated and analysed within a dedicated computational framework, also capable of detecting hyper-edited reads. Inosinome profiles are tissue specific and edited gene sets consistently show enrichment of genes involved in neurological disorders and cancer. Overall frequency of editing also varies, but is strongly correlated with ADAR expression levels. The inosinome database is available at: http://srv00.ibbe.cnr.it/editing/. PMID:26449202

  18. Nuclease Tudor-SN Is Involved in Tick dsRNA-Mediated RNA Interference and Feeding but Not in Defense against Flaviviral or Anaplasma phagocytophilum Rickettsial Infection

    PubMed Central

    Ayllón, Nieves; Naranjo, Victoria; Hajdušek, Ondrej; Villar, Margarita; Galindo, Ruth C.; Kocan, Katherine M.; Alberdi, Pilar; Šíma, Radek; Cabezas-Cruz, Alejandro; Rückert, Claudia; Bell-Sakyi, Lesley; Kazimírová, Mária; Havlíková, Sabína; Klempa, Boris; Kopáček, Petr; de la Fuente, José

    2015-01-01

    Tudor staphylococcal nuclease (Tudor-SN) and Argonaute (Ago) are conserved components of the basic RNA interference (RNAi) machinery with a variety of functions including immune response and gene regulation. The RNAi machinery has been characterized in tick vectors of human and animal diseases but information is not available on the role of Tudor-SN in tick RNAi and other cellular processes. Our hypothesis is that tick Tudor-SN is part of the RNAi machinery and may be involved in innate immune response and other cellular processes. To address this hypothesis, Ixodes scapularis and I. ricinus ticks and/or cell lines were used to annotate and characterize the role of Tudor-SN in dsRNA-mediated RNAi, immune response to infection with the rickettsia Anaplasma phagocytophilum and the flaviviruses TBEV or LGTV and tick feeding. The results showed that Tudor-SN is conserved in ticks and involved in dsRNA-mediated RNAi and tick feeding but not in defense against infection with the examined viral and rickettsial pathogens. The effect of Tudor-SN gene knockdown on tick feeding could be due to down-regulation of genes that are required for protein processing and blood digestion through a mechanism that may involve selective degradation of dsRNAs enriched in G:U pairs that form as a result of adenosine-to-inosine RNA editing. These results demonstrated that Tudor-SN plays a role in tick RNAi pathway and feeding but no strong evidence for a role in innate immune responses to pathogen infection was found. PMID:26186700

  19. Model for Codon Position Bias in RNA Editing

    NASA Astrophysics Data System (ADS)

    Liu, Tsunglin; Bundschuh, Ralf

    2005-08-01

    RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.

  20. A model for codon position bias in RNA editing

    NASA Astrophysics Data System (ADS)

    Bundschuh, Ralf; Liu, Tsunglin

    2006-03-01

    RNA editing can be crucial for the expression of genetic information via inserting, deleting, or substituting a few nucleotides at specific positions in an RNA sequence. Within coding regions in an RNA sequence, editing usually occurs with a certain bias in choosing the positions of the editing sites. In the mitochondrial genes of Physarum polycephalum, many more editing events have been observed at the third codon position than at the first and second, while in some plant mitochondria the second codon position dominates. Here we propose an evolutionary model that explains this bias as the basis of selection at the protein level. The model predicts a distribution of the three positions rather close to the experimental observation in Physarum. This suggests that the codon position bias in Physarum is mainly a consequence of selection at the protein level.

  1. The Chloroplast Genome of Pellia endiviifolia: Gene Content, RNA-Editing Pattern, and the Origin of Chloroplast Editing

    PubMed Central

    Grosche, Christopher; Funk, Helena T.; Maier, Uwe G.; Zauner, Stefan

    2012-01-01

    RNA editing is a post-transcriptional process that can act upon transcripts from mitochondrial, nuclear, and chloroplast genomes. In chloroplasts, single-nucleotide conversions in mRNAs via RNA editing occur at different frequencies across the plant kingdom. These range from several hundred edited sites in some mosses and ferns to lower frequencies in seed plants and the complete lack of RNA editing in the liverwort Marchantia polymorpha. Here, we report the sequence and edited sites of the chloroplast genome from the liverwort Pellia endiviifolia. The type and frequency of chloroplast RNA editing display a pattern highly similar to that in seed plants. Analyses of the C to U conversions and the genomic context in which the editing sites are embedded provide evidence in favor of the hypothesis that chloroplast RNA editing evolved to compensate mutations in the first land plants. PMID:23221608

  2. Editing of plastid RNA in Arabidopsis thaliana ecotypes.

    PubMed

    Tillich, Michael; Funk, Helena T; Schmitz-Linneweber, Christian; Poltnigg, Peter; Sabater, Bartolomé; Martin, Mercedes; Maier, Rainer M

    2005-09-01

    Post-transcriptional maturation of plastid-encoded mRNAs from land plants includes editing by making cytidine to uridine alterations at highly specific positions; this usually restores codon identities for conserved amino acids that are important for the proper function of the affected proteins. In contrast to the rather constant number of editing sites their location varies greatly, even between closely related taxa. Here, we experimentally determined the specific pattern of editing sites (the editotype) of the plastid genome of Arabidopsis thaliana ecotype Columbia (Col-0). Based on phylogenetic analyses of plastid open reading frames, we identified 28 editing sites. Two editing events in the genes matK and ndhB seem to have evolved late during the evolution of flowering plants. Strikingly, they are embedded in almost identical sequence elements and seem to be phylogenetically co-processed. This suggests that the two sites are recognized by the same trans-factor, which could help to explain the hitherto enigmatic gain of editing sites in evolution. In order to trace variations in editotype at the subspecies level we examined two other A. thaliana accessions, Cape Verde Islands (Cvi-0) and Wassilewskija (Ws-2), for the Col-0 editing sites. Both Cvi-0 and Ws-2 possess and process the whole set of editing sites as determined in Col-0, but the consequences of RNA editing differ at one position between the ecotypes. PMID:16115067

  3. The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

    PubMed Central

    Linnstaedt, Sarah D.; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Casey, John L.

    2006-01-01

    RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification. PMID:16790843

  4. Divergent RNA editing frequencies in hornwort mitochondrial nad5 sequences.

    PubMed

    Duff, R Joel

    2006-02-01

    Hornwort mitochondrial genomes have some of the highest rates of RNA editing among plants. Comparison of eleven partial mitochondrial nad5 genomic and cDNA sequences from diverse taxa of hornworts reveal 125 edited sites in only 1107 nt. No single sample exhibits more than half of these sites. Ten of the 11 hornwort taxa have between 35 and 54 edited sties each; whereas, the eleventh taxon, Leiosporoceros, which represents a potential sister taxa to all other hornworts, has only eight sites. Comparison of multiple cDNA sequences from several individuals reveals the presence of many immature transcripts showing the heterogonous nature of the progression of editing. Phylogenetic analyses of hornwort genomic and cDNAs sequences reveal that 65 of the 94 phylogenetically informative sites within the hornwort clade are edited positions. PMID:16376027

  5. RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species.

    PubMed

    Wang, Wenqin; Zhang, Wei; Wu, Yongrui; Maliga, Pal; Messing, Joachim

    2015-01-01

    RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor. PMID:26517707

  6. RNA Editing in Chloroplasts of Spirodela polyrhiza, an Aquatic Monocotelydonous Species

    PubMed Central

    Wang, Wenqin; Zhang, Wei; Wu, Yongrui; Maliga, Pal; Messing, Joachim

    2015-01-01

    RNA editing is the post-transcriptional conversion from C to U before translation, providing a unique feature in the regulation of gene expression. Here, we used a robust and efficient method based on RNA-seq from non-ribosomal total RNA to simultaneously measure chloroplast-gene expression and RNA editing efficiency in the Greater Duckweed, Spirodela polyrhiza, a species that provides a new reference for the phylogenetic studies of monocotyledonous plants. We identified 66 editing sites at the genome-wide level, with an average editing efficiency of 76%. We found that the expression levels of chloroplast genes were relatively constant, but 11 RNA editing sites show significant changes in editing efficiency, when fronds turn into turions. Thus, RNA editing efficiency contributes more to the yield of translatable transcripts than steady state mRNA levels. Comparison of RNA editing sites in coconut, Spirodela, maize, and rice suggests that RNA editing originated from a common ancestor. PMID:26517707

  7. RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

    PubMed Central

    Salavati, Reza

    2014-01-01

    Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based “mix and measure” hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function. PMID:25079143

  8. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis

    PubMed Central

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R.

    2016-01-01

    ABSTRACT Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  9. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis.

    PubMed

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R

    2016-05-01

    Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  10. The boundaries of partially edited transcripts are not conserved in kinetoplastids: implications for the guide RNA model of editing.

    PubMed Central

    Landweber, L F; Fiks, A G; Gilbert, W

    1993-01-01

    We have studied partially edited molecules for the cytochrome-c oxidase subunit III (COIII) transcript from two species of the insect trypanosome Herpetomonas. We found unexpected patterns of editing, in which editing does not proceed strictly 3' to 5', in 24 of 61 partially edited clones. A comparison of the partially edited molecules between the two kinetoplastid species revealed an 8- to 10-nt shift in precisely defined editing boundaries, sites at which editing pauses before binding of the next guide RNA after formation of a stable duplex between a guide RNA and mRNA. This suggests that the region of base pairing between individual guide RNAs and the COIII transcript is not strictly conserved in kinetoplastids, implying gradual evolution of the editing process. Images Fig. 2 Fig. 3 PMID:8415685

  11. A Transition State Analogue for an RNA-Editing Reaction

    PubMed Central

    Haudenschild, Brittany L.; Maydanovych, Olena; Véliz, Eduardo A.; Macbeth, Mark R.; Bass, Brenda L.; Beal, Peter A.

    2007-01-01

    Deamination at C6 of adenosine in RNA catalyzed by the ADAR enzymes generates inosine at the corresponding position. Because inosine is decoded as guanosine during translation, this modification can lead to codon changes in messenger RNA. Hydration of 8-azanebularine across the C6–N1 double bond generates an excellent mimic of the transition state proposed for the hydrolytic deamination reaction catalyzed by ADARs. Here, we report the synthesis of a phosphoramidite of 8-azanebularine and its use in the preparation of RNAs mimicking the secondary structure found at a known editing site in the glutamate receptor B subunit pre-mRNA. The binding properties of analogue-containing RNAs indicate that a tight binding ligand for an ADAR can be generated by incorporation of 8-azanebularine. The observed high-affinity binding is dependent on a functional active site, the presence of one, but not the other, of ADAR2’s two double-stranded RNA-binding motifs (dsRBMs), and the correct placement of the nucleoside analogue into the sequence/structural context of a known editing site. These results advance our understanding of substrate recognition during ADAR-catalyzed RNA editing and are important for structural studies of ADAR· RNA complexes. PMID:15355102

  12. Occurrence of plastid RNA editing in all major lineages of land plants

    PubMed Central

    Freyer, Regina; Kiefer-Meyer, Marie-Christine; Kössel, Hans

    1997-01-01

    RNA editing changes posttranscriptionally single nucleotides in chloroplast-encoded transcripts. Although much work has been done on mechanistic and functional aspects of plastid editing, little is known about evolutionary aspects of this RNA processing step. To gain a better understanding of the evolution of RNA editing in plastids, we have investigated the editing patterns in ndhB and rbcL transcripts from various species comprising all major groups of land plants. Our results indicate that RNA editing occurs in plastids of bryophytes, fern allies, true ferns, gymnosperms, and angiosperms. Both editing frequencies and editing patterns show a remarkable degree of interspecies variation. Furthermore, we have found that neither plastid editing frequencies nor the editing pattern of a specific transcript correlate with the phylogenetic tree of the plant kingdom. The poor evolutionary conservation of editing sites among closely related species as well as the occurrence of single species-specific editing sites suggest that the differences in the editing patterns and editing frequencies are probably due both to independent loss and to gain of editing sites. In addition, our results indicate that RNA editing is a relatively ancient process that probably predates the evolution of land plants. This supposition is in good agreement with the phylogenetic data obtained for plant mitochondrial RNA editing, thus providing additional evidence for common evolutionary roots of the two plant organellar editing systems. PMID:9177209

  13. Variable Frequency of Plastid RNA Editing among Ferns and Repeated Loss of Uridine-to-Cytidine Editing from Vascular Plants

    PubMed Central

    Guo, Wenhu; Grewe, Felix; Mower, Jeffrey P.

    2015-01-01

    The distinct distribution and abundance of C-to-U and U-to-C RNA editing among land plants suggest that these two processes originated and evolve independently, but the paucity of information from several key lineages limits our understanding of their evolution. To examine the evolutionary diversity of RNA editing among ferns, we sequenced the plastid transcriptomes from two early diverging species, Ophioglossum californicum and Psilotum nudum. Using a relaxed automated approach to minimize false negatives combined with manual inspection to eliminate false positives, we identified 297 C-to-U and three U-to-C edit sites in the O. californicum plastid transcriptome but only 27 C-to-U and no U-to-C edit sites in the P. nudum plastid transcriptome. A broader comparison of editing content with the leptosporangiate fern Adiantum capillus-veneris and the hornwort Anthoceros formosae uncovered large variance in the abundance of plastid editing, indicating that the frequency and type of RNA editing is highly labile in ferns. Edit sites that increase protein conservation among species are more abundant and more efficiently edited than silent and non-conservative sites, suggesting that selection maintains functionally important editing. The absence of U-to-C editing from P. nudum plastid transcripts and other vascular plants demonstrates that U-to-C editing loss is a recurrent phenomenon in vascular plant evolution. PMID:25568947

  14. RNA editing of the Drosophila para Na(+) channel transcript. Evolutionary conservation and developmental regulation.

    PubMed Central

    Hanrahan, C J; Palladino, M J; Ganetzky, B; Reenan, R A

    2000-01-01

    Post-transcriptional editing of pre-mRNAs through the action of dsRNA adenosine deaminases results in the modification of particular adenosine (A) residues to inosine (I), which can alter the coding potential of the modified transcripts. We describe here three sites in the para transcript, which encodes the major voltage-activated Na(+) channel polypeptide in Drosophila, where RNA editing occurs. The occurrence of RNA editing at the three sites was found to be developmentally regulated. Editing at two of these sites was also conserved across species between the D. melanogaster and D. virilis. In each case, a highly conserved region was found in the intron downstream of the editing site and this region was shown to be complementary to the region of the exonic editing site. Thus, editing at these sites would appear to involve a mechanism whereby the edited exon forms a base-paired secondary structure with the distant conserved noncoding sequences located in adjacent downstream introns, similar to the mechanism shown for A-to-I RNA editing of mammalian glutamate receptor subunits (GluRs). For the third site, neither RNA editing nor the predicted RNA secondary structures were evolutionarily conserved. Transcripts from transgenic Drosophila expressing a minimal editing site construct for this site were shown to faithfully undergo RNA editing. These results demonstrate that Na(+) channel diversity in Drosophila is increased by RNA editing via a mechanism analogous to that described for transcripts encoding mammalian GluRs. PMID:10880477

  15. Altered RNA editing in 3′ UTR perturbs microRNA-mediated regulation of oncogenes and tumor-suppressors

    PubMed Central

    Zhang, Liye; Yang, Chih-Sheng; Varelas, Xaralabos; Monti, Stefano

    2016-01-01

    RNA editing is a molecular event that alters specific nucleotides in RNA post-transcriptionally. RNA editing has the potential to impact a variety of cellular processes and is implicated in diseases such as cancer. Yet, the precise mechanisms by which RNA editing controls cellular processes are poorly understood. Here, we characterize sequences altered by RNA editing in patient samples from lymphoma, neuroblastoma and head and neck cancers. We show that A-to-I RNA editing sites are highly conserved across samples of the same tissue type and that most editing sites identified in tumors are also detectable in normal tissues. Next, we identify the significant changes in editing levels of known sites between tumor and paired “normal” tissues across 14 cancer types (627 pairs) from The Cancer Genome Atlas project and show that the complexity of RNA editing regulation cannot be captured by the activity of ADAR family genes alone. Our pan-cancer analysis confirms previous results on individual tumor types and suggests that changes of RNA editing levels in coding and 3′UTR regions could be a general mechanism to promote tumor growth. We also propose a model explaining how altered RNA editing levels affect microRNA-mediated post-transcriptional regulation of oncogenes and tumor-suppressors. PMID:26980570

  16. Crystal Structure of Staphylococcus aureus tRNA Adenosine Deaminase TadA in Complex with RNA

    SciTech Connect

    Losey,H.; Ruthenburg, A.; Verdine, G.

    2006-01-01

    Bacterial tRNA adenosine deaminases (TadAs) catalyze the hydrolytic deamination of adenosine to inosine at the wobble position of tRNA(Arg2), a process that enables this single tRNA to recognize three different arginine codons in mRNA. In addition, inosine is also introduced at the wobble position of multiple eukaryotic tRNAs. The genes encoding these deaminases are essential in bacteria and yeast, demonstrating the importance of their biological activity. Here we report the crystallization and structure determination to 2.0 A of Staphylococcus aureus TadA bound to the anticodon stem-loop of tRNA(Arg2) bearing nebularine, a non-hydrolyzable adenosine analog, at the wobble position. The cocrystal structure reveals the basis for both sequence and structure specificity in the interactions of TadA with RNA, and it additionally provides insight into the active site architecture that promotes efficient hydrolytic deamination.

  17. A hammerhead ribozyme substrate and reporter for in vitro kinetoplastid RNA editing.

    PubMed Central

    Wang, Bingbing; Salavati, Reza; Heidmann, Stefan; Stuart, Kenneth

    2002-01-01

    Current in vitro assays for RNA editing in kinetoplastids directly examine the products generated by incubation of pre-mRNA substrate with guide RNA (gRNA) and mitochondrial (mt) extract. RNA editing substrates that are modeled on hammerhead ribozymes were designed with catalytic cores that contained or lacked additional uridylates (Us). They proved to be sensitive reporters of editing activity when used for in vitro assays. A deletion editing substrate that is based on A6 pre-mRNA had no ribozyme activity, but its incubation with gRNA and mt extract resulted in its deletion editing and production of a catalytically active ribozyme. Hammerhead ribozymes are thus sensitive tools to assay in vitro RNA editing. PMID:11991648

  18. Dynamic regulation of RNA editing in human brain development and disease.

    PubMed

    Hwang, Taeyoung; Park, Chul-Kee; Leung, Anthony K L; Gao, Yuan; Hyde, Thomas M; Kleinman, Joel E; Rajpurohit, Anandita; Tao, Ran; Shin, Joo Heon; Weinberger, Daniel R

    2016-08-01

    RNA editing is increasingly recognized as a molecular mechanism regulating RNA activity and recoding proteins. Here we surveyed the global landscape of RNA editing in human brain tissues and identified three unique patterns of A-to-I RNA editing rates during cortical development: stable high, stable low and increasing. RNA secondary structure and the temporal expression of adenosine deaminase acting on RNA (ADAR) contribute to cis- and trans-regulatory mechanisms of these RNA editing patterns, respectively. Interestingly, the increasing pattern was associated with neuronal maturation, correlated with mRNA abundance and potentially influenced miRNA binding energy. Gene ontology analyses implicated the increasing pattern in vesicle or organelle membrane-related genes and glutamate signaling pathways. We also found that the increasing pattern was selectively perturbed in spinal cord injury and glioblastoma. Our findings reveal global and dynamic aspects of RNA editing in brain, providing new insight into epitranscriptional regulation of sequence diversity. PMID:27348216

  19. Naturally Occurring Isoleucyl-tRNA Synthetase without tRNA-dependent Pre-transfer Editing*

    PubMed Central

    Cvetesic, Nevena; Dulic, Morana; Bilus, Mirna; Sostaric, Nikolina; Lenhard, Boris; Gruic-Sovulj, Ita

    2016-01-01

    Isoleucyl-tRNA synthetase (IleRS) is unusual among aminoacyl-tRNA synthetases in having a tRNA-dependent pre-transfer editing activity. Alongside the typical bacterial IleRS (such as Escherichia coli IleRS), some bacteria also have the enzymes (eukaryote-like) that cluster with eukaryotic IleRSs and exhibit low sensitivity to the antibiotic mupirocin. Our phylogenetic analysis suggests that the ileS1 and ileS2 genes of contemporary bacteria are the descendants of genes that might have arisen by an ancient duplication event before the separation of bacteria and archaea. We present the analysis of evolutionary constraints of the synthetic and editing reactions in eukaryotic/eukaryote-like IleRSs, which share a common origin but diverged through adaptation to different cell environments. The enzyme from the yeast cytosol exhibits tRNA-dependent pre-transfer editing analogous to E. coli IleRS. This argues for the presence of this proofreading in the common ancestor of both IleRS types and an ancient origin of the synthetic site-based quality control step. Yet surprisingly, the eukaryote-like enzyme from Streptomyces griseus IleRS lacks this capacity; at the same time, its synthetic site displays the 103-fold drop in sensitivity to antibiotic mupirocin relative to the yeast enzyme. The discovery that pre-transfer editing is optional in IleRSs lends support to the notion that the conserved post-transfer editing domain is the main checkpoint in these enzymes. We substantiated this by showing that under error-prone conditions S. griseus IleRS is able to rescue the growth of an E. coli lacking functional IleRS, providing the first evidence that tRNA-dependent pre-transfer editing in IleRS is not essential for cell viability. PMID:26921320

  20. Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing.

    PubMed

    Huang, Zhenqiu; Faktorová, Drahomíra; Křížová, Adéla; Kafková, Lucie; Read, Laurie K; Lukeš, Julius; Hashimi, Hassan

    2015-12-01

    Trypanosoma brucei is the causative agent of the human and veterinarian diseases African sleeping sickness and nagana. A majority of its mitochondrial-encoded transcripts undergo RNA editing, an essential process of post-transcriptional uridine insertion and deletion to produce translatable mRNA. Besides the well-characterized RNA editing core complex, the mitochondrial RNA-binding 1 (MRB1) complex is one of the key players. It comprises a core complex of about six proteins, guide RNA-associated proteins (GAPs) 1/2, which form a heterotetramer that binds and stabilizes gRNAs, plus MRB5390, MRB3010, and MRB11870, which play roles in initial stages of RNA editing, presumably guided by the first gRNA:mRNA duplex in the case of the latter two proteins. To better understand all functions of the MRB1 complex, we performed a functional analysis of the MRB8620 core subunit, the only one not characterized so far. Here we show that MRB8620 plays a role in RNA editing in both procyclic and bloodstream stages of T. brucei, which reside in the tsetse fly vector and mammalian circulatory system, respectively. While RNAi silencing of MRB8620 does not affect procyclic T. brucei fitness when grown in glucose-containing media, it is somewhat compromised in cells grown in the absence of this carbon source. MRB8620 is crucial for integrity of the MRB1 core, such as its association with GAP1/2, which presumably acts to deliver gRNAs to this complex. In contrast, GAP1/2 is not required for the fabrication of the MRB1 core. Disruption of the MRB1 core assembly is followed by the accumulation of mRNAs associated with GAP1/2. PMID:26447184

  1. APOBEC3A cytidine deaminase induces RNA editing in monocytes and macrophages

    PubMed Central

    Sharma, Shraddha; Patnaik, Santosh K.; Thomas Taggart, R.; Kannisto, Eric D.; Enriquez, Sally M.; Gollnick, Paul; Baysal, Bora E.

    2015-01-01

    The extent, regulation and enzymatic basis of RNA editing by cytidine deamination are incompletely understood. Here we show that transcripts of hundreds of genes undergo site-specific C>U RNA editing in macrophages during M1 polarization and in monocytes in response to hypoxia and interferons. This editing alters the amino acid sequences for scores of proteins, including many that are involved in pathogenesis of viral diseases. APOBEC3A, which is known to deaminate cytidines of single-stranded DNA and to inhibit viruses and retrotransposons, mediates this RNA editing. Amino acid residues of APOBEC3A that are known to be required for its DNA deamination and anti-retrotransposition activities were also found to affect its RNA deamination activity. Our study demonstrates the cellular RNA editing activity of a member of the APOBEC3 family of innate restriction factors and expands the understanding of C>U RNA editing in mammals. PMID:25898173

  2. Trans-splicing and RNA editing of LSU rRNA in Diplonema mitochondria

    PubMed Central

    Valach, Matus; Moreira, Sandrine; Kiethega, Georgette N.; Burger, Gertraud

    2014-01-01

    Mitochondrial ribosomal RNAs (rRNAs) often display reduced size and deviant secondary structure, and sometimes are fragmented, as are their corresponding genes. Here we report a mitochondrial large subunit rRNA (mt-LSU rRNA) with unprecedented features. In the protist Diplonema, the rnl gene is split into two pieces (modules 1 and 2, 534- and 352-nt long) that are encoded by distinct mitochondrial chromosomes, yet the rRNA is continuous. To reconstruct the post-transcriptional maturation pathway of this rRNA, we have catalogued transcript intermediates by deep RNA sequencing and RT-PCR. Gene modules are transcribed separately. Subsequently, transcripts are end-processed, the module-1 transcript is polyuridylated and the module-2 transcript is polyadenylated. The two modules are joined via trans-splicing that retains at the junction ∼26 uridines, resulting in an extent of insertion RNA editing not observed before in any system. The A-tail of trans-spliced molecules is shorter than that of mono-module 2, and completely absent from mitoribosome-associated mt-LSU rRNA. We also characterize putative antisense transcripts. Antisense-mono-modules corroborate bi-directional transcription of chromosomes. Antisense-mt-LSU rRNA, if functional, has the potential of guiding concomitantly trans-splicing and editing of this rRNA. Together, these findings open a window on the investigation of complex regulatory networks that orchestrate multiple and biochemically diverse post-transcriptional events. PMID:24259427

  3. PREPACT 2.0: Predicting C-to-U and U-to-C RNA Editing in Organelle Genome Sequences with Multiple References and Curated RNA Editing Annotation

    PubMed Central

    Lenz, Henning; Knoop, Volker

    2013-01-01

    RNA editing is vast in some genetic systems, with up to thousands of targeted C-to-U and U-to-C substitutions in mitochondria and chloroplasts of certain plants. Efficient prognoses of RNA editing in organelle genomes will help to reveal overlooked cases of editing. We present PREPACT 2.0 (http://www.prepact.de) with numerous enhancements of our previously developed Plant RNA Editing Prediction & Analysis Computer Tool. Reference organelle transcriptomes for editing prediction have been extended and reorganized to include 19 curated mitochondrial and 13 chloroplast genomes, now allowing to distinguish RNA editing sites from “pre-edited” sites. Queries may be run against multiple references and a new “commons” function identifies and highlights orthologous candidate editing sites congruently predicted by multiple references. Enhancements to the BLASTX mode in PREPACT 2.0 allow querying of complete novel organelle genomes within a few minutes, identifying protein genes and candidate RNA editing sites simultaneously without prior user analyses. PMID:23362369

  4. Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes

    PubMed Central

    Liu, Huiquan; Wang, Qinhu; He, Yi; Chen, Lingfeng; Hao, Chaofeng; Jiang, Cong; Li, Yang; Dai, Yafeng; Kang, Zhensheng; Xu, Jin-Rong

    2016-01-01

    Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with the PUK1 (FGRRES_01058) pseudokinase gene important for sexual reproduction in Fusarium graminearum, we found that two tandem stop codons, UA1831GUA1834G, in its kinase domain were changed to UG1831GUG1834G by RNA editing in perithecia. To confirm A-to-I editing of PUK1 transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia. PUK1 was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG1831GUG1834G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites in F. graminearum occur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69 PUK1-like pseudogenes with stop codons in ORFs. PUK1 orthologs and other pseudogenes also displayed stage-specific expression and editing in Neurospora crassa and F. verticillioides. Furthermore, F. graminearum differs from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing in F. graminearum preferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs. PMID:26934920

  5. Genome-wide A-to-I RNA editing in fungi independent of ADAR enzymes.

    PubMed

    Liu, Huiquan; Wang, Qinhu; He, Yi; Chen, Lingfeng; Hao, Chaofeng; Jiang, Cong; Li, Yang; Dai, Yafeng; Kang, Zhensheng; Xu, Jin-Rong

    2016-04-01

    Yeasts and filamentous fungi do not have adenosine deaminase acting on RNA (ADAR) orthologs and are believed to lack A-to-I RNA editing, which is the most prevalent editing of mRNA in animals. However, during this study with thePUK1(FGRRES_01058) pseudokinase gene important for sexual reproduction inFusarium graminearum, we found that two tandem stop codons, UA(1831)GUA(1834)G, in its kinase domain were changed to UG(1831)GUG(1834)G by RNA editing in perithecia. To confirm A-to-I editing ofPUK1transcripts, strand-specific RNA-seq data were generated with RNA isolated from conidia, hyphae, and perithecia.PUK1was almost specifically expressed in perithecia, and 90% of transcripts were edited to UG(1831)GUG(1834)G. Genome-wide analysis identified 26,056 perithecium-specific A-to-I editing sites. Unlike those in animals, 70.5% of A-to-I editing sites inF. graminearumoccur in coding regions, and more than two-thirds of them result in amino acid changes, including editing of 69PUK1-like pseudogenes with stop codons in ORFs.PUK1orthologs and other pseudogenes also displayed stage-specific expression and editing inNeurospora crassaandF. verticillioides Furthermore,F. graminearumdiffers from animals in the sequence preference and structure selectivity of A-to-I editing sites. Whereas A's embedded in RNA stems are targeted by ADARs, RNA editing inF. graminearumpreferentially targets A's in hairpin loops, which is similar to the anticodon loop of tRNA targeted by adenosine deaminases acting on tRNA (ADATs). Overall, our results showed that A-to-I RNA editing occurs specifically during sexual reproduction and mainly in the coding regions in filamentous ascomycetes, involving adenosine deamination mechanisms distinct from metazoan ADARs. PMID:26934920

  6. Mitochondrial tRNA 5'-editing in Dictyostelium discoideum and Polysphondylium pallidum.

    PubMed

    Abad, Maria G; Long, Yicheng; Kinchen, R Dimitri; Schindel, Elinor T; Gray, Michael W; Jackman, Jane E

    2014-05-30

    Mitochondrial tRNA (mt-tRNA) 5'-editing was first described more than 20 years ago; however, the first candidates for 5'-editing enzymes were only recently identified in a eukaryotic microbe (protist), the slime mold Dictyostelium discoideum. In this organism, eight of 18 mt-tRNAs are predicted to be edited based on the presence of genomically encoded mismatched nucleotides in their aminoacyl-acceptor stem sequences. Here, we demonstrate that mt-tRNA 5'-editing occurs at all predicted sites in D. discoideum as evidenced by changes in the sequences of isolated mt-tRNAs compared with the expected sequences encoded by the mitochondrial genome. We also identify two previously unpredicted editing events in which G-U base pairs are edited in the absence of any other genomically encoded mismatches. A comparison of 5'-editing in D. discoideum with 5'-editing in another slime mold, Polysphondylium pallidum, suggests organism-specific idiosyncrasies in the treatment of U-G/G-U pairs. In vitro activities of putative D. discoideum editing enzymes are consistent with the observed editing reactions and suggest an overall lack of tRNA substrate specificity exhibited by the repair component of the editing enzyme. Although the presence of terminal mismatches in mt-tRNA sequences is highly predictive of the occurrence of mt-tRNA 5'-editing, the variability in treatment of U-G/G-U base pairs observed here indicates that direct experimental evidence of 5'-editing must be obtained to understand the complete spectrum of mt-tRNA editing events in any species. PMID:24737330

  7. microRNA editing in seed region aligns with cellular changes in hypoxic conditions.

    PubMed

    Nigita, Giovanni; Acunzo, Mario; Romano, Giulia; Veneziano, Dario; Laganà, Alessandro; Vitiello, Marika; Wernicke, Dorothee; Ferro, Alfredo; Croce, Carlo M

    2016-07-27

    RNA editing is a finely tuned, dynamic mechanism for post-transcriptional gene regulation that has been thoroughly investigated in the last decade. Nevertheless, RNA editing in non-coding RNA, such as microRNA (miRNA), have caused great debate and have called for deeper investigation. Until recently, in fact, inadequate methodologies and experimental contexts have been unable to provide detailed insights for further elucidation of RNA editing affecting miRNAs, especially in cancer.In this work, we leverage on recent innovative bioinformatics approaches applied to a more informative experimental context in order to analyze the variations in miRNA seed region editing activity during a time course of a hypoxia-exposed breast cancer cell line. By investigating its behavior in a dynamic context, we found that miRNA editing events in the seed region are not depended on miRNA expression, unprecedentedly providing insights on the targetome shifts derived from these modifications. This reveals that miRNA editing acts under the influence of environmentally induced stimuli.Our results show a miRNA editing activity trend aligning with cellular pathways closely associated to hypoxia, such as the VEGF and PI3K/Akt pathways, providing important novel insights on this poorly elucidated phenomenon. PMID:27298257

  8. microRNA editing in seed region aligns with cellular changes in hypoxic conditions

    PubMed Central

    Nigita, Giovanni; Acunzo, Mario; Romano, Giulia; Veneziano, Dario; Laganà, Alessandro; Vitiello, Marika; Wernicke, Dorothee; Ferro, Alfredo; Croce, Carlo M.

    2016-01-01

    RNA editing is a finely tuned, dynamic mechanism for post-transcriptional gene regulation that has been thoroughly investigated in the last decade. Nevertheless, RNA editing in non-coding RNA, such as microRNA (miRNA), have caused great debate and have called for deeper investigation. Until recently, in fact, inadequate methodologies and experimental contexts have been unable to provide detailed insights for further elucidation of RNA editing affecting miRNAs, especially in cancer. In this work, we leverage on recent innovative bioinformatics approaches applied to a more informative experimental context in order to analyze the variations in miRNA seed region editing activity during a time course of a hypoxia-exposed breast cancer cell line. By investigating its behavior in a dynamic context, we found that miRNA editing events in the seed region are not depended on miRNA expression, unprecedentedly providing insights on the targetome shifts derived from these modifications. This reveals that miRNA editing acts under the influence of environmentally induced stimuli. Our results show a miRNA editing activity trend aligning with cellular pathways closely associated to hypoxia, such as the VEGF and PI3K/Akt pathways, providing important novel insights on this poorly elucidated phenomenon. PMID:27298257

  9. Maxicircle DNA and edited mRNA sequences of closely related trypanosome species: implications of kRNA editing for evolution of maxicircle genomes.

    PubMed Central

    Read, L K; Fish, W R; Muthiani, A M; Stuart, K

    1993-01-01

    kRNA editing produces functional mRNAs by uridine insertion and deletion. We analyzed portions of the apocytochrome b and NADH dehydrogenase subunits 7 and 8 (ND7 and 8) genes and their edited mRNAs in Trypanosoma congolense and compared these to the corresponding sequences in T.brucei. We find that these genes are highly diverged between the two species, especially in the positions of thymidines and in nucleotide transitions. Editing eliminates differences in encoded uridines producing edited mRNAs that are identical except for the nucleotide substitutions. The resulting predicted proteins are identical since all nucleotide substitutions are silent. A T.congolense minicircle-encoded gRNA which can specify editing of ND8 mRNA was identified. This gRNA can basepair with both T.congolense and T.brucei ND8 mRNA despite nucleotide transitions due to the flexibility of G:U base-pairing. These results illustrate how editing affects the characteristics of maxicircle sequence divergence and allows protein sequence conservation despite a level of DNA sequence divergence which would be predicted to be intolerable in the absence of editing. PMID:8396763

  10. Parallel Evolution and Lineage-Specific Expansion of RNA Editing in Ctenophores.

    PubMed

    Kohn, Andrea B; Sanford, Rachel S; Yoshida, Masa-aki; Moroz, Leonid L

    2015-12-01

    RNA editing is a process of targeted alterations of nucleotides in all types of RNA molecules (e.g., rRNA, tRNA, mRNA, and miRNA). As a result, the transcriptional output differs from its genomic DNA template. RNA editing can be defined both by biochemical mechanisms and by enzymes that perform these reactions. There are high levels of RNA editing detected in the mammalian nervous system, suggesting that nervous systems use this mechanism to increase protein diversity, because the post-transcription modifications lead to new gene products with novel functions. By re-annotating the ctenophore genomes, we found that the number of predicted RNA-editing enzymes is comparable to the numbers in mammals, but much greater than in other non-bilaterian basal metazoans. However, the overall molecular diversity of RNA-editing enzymes in ctenophores is lower, suggesting a possible "compensation" by an expansion of the ADAT1-like subfamily in this lineage. In two genera of ctenophores, Pleurobrachia and Mnemiopsis, there are high levels of expression for RNA-editing enzymes in their aboral organs, the integrative center involved in control of locomotion and geotaxis. This finding supports the hypothesis that RNA editing is correlated with the complexity of tissues and behaviors. Smaller numbers of RNA-editing enzymes in Porifera and Placozoa also correlates with the primary absence of neural and muscular systems in these lineages. In ctenophores, the expansion of the RNA-editing machinery can also provide mechanisms that support the remarkable capacity for regeneration in these animals. In summary, despite their compact genomes, a wide variety of epigenomic mechanisms employed by ctenophores and other non-bilaterian basal metazoans can provide novel insights into the evolutionary origins of biological novelties. PMID:26089435

  11. Pervasive RNA editing among hornwort rbcL transcripts except Leiosporoceros.

    PubMed

    Duff, R Joel; Moore, Francisco B-G

    2005-11-01

    RNA editing affecting chloroplast and mitochondrial genomes has been identified in all major clades of land plants. The frequency of edited sites varies greatly between lineages but hornworts represent an extreme in propensity for editing in both their chloroplast and mitochondrial genomes. cDNA sequences from seven taxonomically diverse hornwort rbcL sequences combined with a survey of 13 additional DNA sequences for potential edited sites demonstrate the presence of 62 edited sites and predict a minimum of 10 additional sites. These 72 total edited sites represent 43 C-to-U and 28 U-to-C nucleotide conversions, with 1 site exhibiting editing in both directions. With one exception, all taxa are heavily edited, with each having from 20 to 34 edited sites. However, a single sample, Leiosporoceros, is shown to lack edited sites. Phylogenetic reconstruction of hornworts results in ambiguous resolution of Leiosporoceros depending on whether edited sites are maintained or eliminated from the analyses. Depending on the inferred relationship of Leiosporoceros to the hornworts, at least two explanations for the origin and maintenance of pervasive editing in hornworts are possible. The absence of edited sites in Leiosporoceros could represent either the absence or a low level of editing ability in the common ancestor of hornworts, as represented by Leiosporoceros, or the loss of editing sites in this lineage after the primary diversification events in the group. PMID:16177870

  12. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites.

    PubMed

    Ammerman, Michelle L; Presnyak, Vladimir; Fisk, John C; Foda, Bardees M; Read, Laurie K

    2010-11-01

    TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5' ends of pan-edited RNAs than at their 3' ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3' to 5' progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3' ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA-RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3' to 5' progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs. PMID:20855539

  13. RNA editing in bryophytes and a molecular phylogeny of land plants.

    PubMed Central

    Malek, O; Lättig, K; Hiesel, R; Brennicke, A; Knoop, V

    1996-01-01

    RNA editing has been observed to date in all groups of vascular plants, but not in bryophytes. Its occurrence was therefore assumed to correlate with the evolution of tracheophytes. To gain more insight into both the phylogeny of early land plants and the evolution of mitochondrial RNA editing we have investigated a number of vascular and non-vascular plant species. Contrary to the belief that editing is absent from bryophytes, here we report mitochondrial RNA editing in cox3 mRNA of the liverwort Pellia epiphylla, the mosses Tetraphis pellucida and Ceratodon purpureus and the hornwort Anthroceros crispulus. RNA editing in plants consequently predates the evolution of tracheophytes. Editing is also found in the eusporangiate ferns Ophioglossum petiolatum and Angiopteris palmiformis, the whisk fern Tmesipteris elongata and the gnetopsid Ephedra gerardiana, but was not detected in Gnetum gnemon.cox3 mRNA of the lycopsid Isoetes lacustris shows the highest frequency of RNA editing ever observed in a plant, with 39% of all cytidine residues converted to uridines. The frequency of RNA editing correlates with the genomic GC content rather than with the phylogenetic position of a species. Phylogenetic trees derived from the slowly evolving mitochondrial sequences find external support from the assessments of classical systematics. Images PMID:8635473

  14. TbRGG2 facilitates kinetoplastid RNA editing initiation and progression past intrinsic pause sites

    PubMed Central

    Ammerman, Michelle L.; Presnyak, Vladimir; Fisk, John C.; Foda, Bardees M.; Read, Laurie K.

    2010-01-01

    TbRGG2 is an essential kinetoplastid RNA editing accessory factor that acts specifically on pan-edited RNAs. To understand the mechanism of TbRGG2 action, we undertook an in-depth analysis of edited RNA populations in TbRGG2 knockdown cells and an in vitro examination of the biochemical activities of the protein. We demonstrate that TbRGG2 down-regulation more severely impacts editing at the 5′ ends of pan-edited RNAs than at their 3′ ends. The initiation of editing is reduced to some extent in TbRGG2 knockdown cells. In addition, TbRGG2 plays a post-initiation role as editing becomes stalled in TbRGG2-depleted cells, resulting in an overall decrease in the 3′ to 5′ progression of editing. Detailed analyses of edited RNAs from wild-type and TbRGG2-depleted cells reveal that TbRGG2 facilitates progression of editing past intrinsic pause sites that often correspond to the 3′ ends of cognate guide RNAs (gRNAs). In addition, noncanonically edited junction regions are either absent or significantly shortened in TbRGG2-depleted cells, consistent with impaired gRNA transitions. Sequence analysis further suggests that TbRGG2 facilitates complete utilization of certain gRNAs. In vitro RNA annealing and in vivo RNA unwinding assays demonstrate that TbRGG2 can modulate RNA–RNA interactions. Collectively, these data are consistent with a model in which TbRGG2 facilitates initiation and 3′ to 5′ progression of editing through its ability to affect gRNA utilization, both during the transition between specific gRNAs and during usage of certain gRNAs. PMID:20855539

  15. Reprogramming, Circular Reasoning and Self versus Non-self: One-Stop Shopping with RNA Editing.

    PubMed

    Savva, Yiannis A; Rezaei, Ali; St Laurent, Georges; Reenan, Robert A

    2016-01-01

    Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self. PMID:27458478

  16. Reprogramming, Circular Reasoning and Self versus Non-self: One-Stop Shopping with RNA Editing

    PubMed Central

    Savva, Yiannis A.; Rezaei, Ali; St. Laurent, Georges; Reenan, Robert A.

    2016-01-01

    Transcription of genetic information from archival DNA into RNA molecule working copies is vital for proper cellular function and is highly accurate. In turn, RNAs serve structural, enzymatic, and regulatory roles, as well as being informational templates for the ribosomal translation of proteins. Following RNA synthesis, maturing of RNA molecules occurs through various RNA processing events. One component of the collection of processes involving RNA species, broadly defined as RNA metabolism, is the RNA-editing pathway and is found in all animals. Acting specifically on RNA substrates with double-stranded character, RNA editing has been shown to regulate a plethora of genomic outputs, including gene recoding, RNA splicing, biogenesis and targeting actions of microRNAs and small interfering RNAs, and global gene expression. Recent evidence suggests that RNA modifications mediated via RNA editing influence the biogenesis of circular RNAs and safeguard against aberrant innate immune responses generated to endogenous RNA sources. These novel roles have the potential to contribute new insights into molecular mechanisms underlying pathogenesis mediated by mishandling of double-stranded RNA. Here, we discuss recent advances in the field, which highlight novel roles associated with the RNA-editing process and emphasize their importance during cellular RNA metabolism. In addition, we highlight the relevance of these newly discovered roles in the context of neurological disorders and the more general concept of innate recognition of self versus non-self. PMID:27458478

  17. F11R Expression upon Hypoxia Is Regulated by RNA Editing

    PubMed Central

    Ben-Zvi, Michal; Amariglio, Ninette

    2013-01-01

    F11R is a cell adhesion molecule found on the surface of human platelets. It plays a role in platelet aggregation, cell migration and cell proliferation. F11R is subjected to RNA editing, a post-transcriptional modification which affects RNA structure, stability, localization, translation and splicing. RNA editing in the 3'UTR of F11R and RNA levels are increased upon hypoxia. We therefore set to examine if RNA editing plays a role in the increase of F11R RNA seen upon hypoxic conditions. We show that ADAR1, but not ADAR2, takes part in the editing of F11R however editing alone is not sufficient for obtaining an elevation in RNA levels. In addition we show that hyper-edited mature mRNAs are retained in the nucleus and are associated with p54nrb. We therefore conclude that hypoxia-induced edited RNAs of F11R are preferentially stabilized and accumulate in the nucleus preventing their export to the cytoplasm for translation. This mechanism may be used by additional proteins in the cell as part of the cell's effort to reduce metabolism upon hypoxic stress. PMID:24147060

  18. F11R expression upon hypoxia is regulated by RNA editing.

    PubMed

    Ben-Zvi, Michal; Amariglio, Ninette; Paret, Gideon; Nevo-Caspi, Yael

    2013-01-01

    F11R is a cell adhesion molecule found on the surface of human platelets. It plays a role in platelet aggregation, cell migration and cell proliferation. F11R is subjected to RNA editing, a post-transcriptional modification which affects RNA structure, stability, localization, translation and splicing. RNA editing in the 3'UTR of F11R and RNA levels are increased upon hypoxia. We therefore set to examine if RNA editing plays a role in the increase of F11R RNA seen upon hypoxic conditions. We show that ADAR1, but not ADAR2, takes part in the editing of F11R however editing alone is not sufficient for obtaining an elevation in RNA levels. In addition we show that hyper-edited mature mRNAs are retained in the nucleus and are associated with p54(nrb). We therefore conclude that hypoxia-induced edited RNAs of F11R are preferentially stabilized and accumulate in the nucleus preventing their export to the cytoplasm for translation. This mechanism may be used by additional proteins in the cell as part of the cell's effort to reduce metabolism upon hypoxic stress. PMID:24147060

  19. Charge Reduction and Thermodynamic Stabilization of Substrate RNAs Inhibit RNA Editing

    PubMed Central

    Leeder, W.-Matthias; Reuss, Andreas J.; Brecht, Michael; Kratz, Katja; Wachtveitl, Josef; Göringer, H. Ulrich

    2015-01-01

    African trypanosomes cause a parasitic disease known as sleeping sickness. Mitochondrial transcript maturation in these organisms requires a RNA editing reaction that is characterized by the insertion and deletion of U-nucleotides into otherwise non-functional mRNAs. Editing represents an ideal target for a parasite-specific therapeutic intervention since the reaction cycle is absent in the infected host. In addition, editing relies on a macromolecular protein complex, the editosome, that only exists in the parasite. Therefore, all attempts to search for editing interfering compounds have been focused on molecules that bind to proteins of the editing machinery. However, in analogy to other RNA-driven biochemical pathways it should be possible to stall the reaction by targeting its substrate RNAs. Here we demonstrate inhibition of editing by specific aminoglycosides. The molecules bind into the major groove of the gRNA/pre-mRNA editing substrates thereby causing a stabilization of the RNA molecules through charge compensation and an increase in stacking. The data shed light on mechanistic details of the editing process and identify critical parameters for the development of new trypanocidal compounds. PMID:25742417

  20. Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation.

    PubMed

    Das, Mom; Vargas-Rodriguez, Oscar; Goto, Yuki; Suga, Hiroaki; Musier-Forsyth, Karin

    2014-04-01

    Errors in protein synthesis due to mispairing of amino acids with tRNAs jeopardize cell viability. Several checkpoints to prevent formation of Ala- and Cys-tRNA(Pro) have been described, including the Ala-specific editing domain (INS) of most bacterial prolyl-tRNA synthetases (ProRSs) and an autonomous single-domain INS homolog, YbaK, which clears Cys-tRNA(Pro) in trans. In many species where ProRS lacks an INS domain, ProXp-ala, another single-domain INS-like protein, is responsible for editing Ala-tRNA(Pro). Although the amino acid specificity of these editing domains has been established, the role of tRNA sequence elements in substrate selection has not been investigated in detail. Critical recognition elements for aminoacylation by bacterial ProRS include acceptor stem elements G72/A73 and anticodon bases G35/G36. Here, we show that ProXp-ala and INS require these same acceptor stem and anticodon elements, respectively, whereas YbaK lacks inherent tRNA specificity. Thus, these three related domains use divergent approaches to recognize tRNAs and prevent mistranslation. Whereas some editing domains have borrowed aspects of tRNA recognition from the parent aminoacyl-tRNA synthetase, relaxed tRNA specificity leading to semi-promiscuous editing may offer advantages to cells. PMID:24371276

  1. Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata

    PubMed Central

    Oldenkott, Bastian; Yamaguchi, Kazuo; Tsuji-Tsukinoki, Sumika; Knie, Nils

    2014-01-01

    RNA editing in chloroplasts and mitochondria of land plants differs significantly in abundance. For example, some 200–500 sites of cytidine-to-uridine RNA editing exist in flowering plant mitochondria as opposed to only 30–50 such C-to-U editing events in their chloroplasts. In contrast, we predicted significantly more chloroplast RNA editing for the protein-coding genes in the available complete plastome sequences of two species of the spike moss genus Selaginella (Lycopodiophyta). To evaluate these predictions we investigated the Selaginella uncinata chloroplast transcriptome. Our exhaustive cDNA studies identified the extraordinary number of 3415 RNA-editing events, exclusively of the C-to-U type, in the 74 mRNAs encoding intact reading frames in the S. uncinata chloroplast. We find the overwhelming majority (61%) of the 428 silent editing events leaving codon meanings unaltered directly neighboring other editing events, possibly suggesting a sterically more flexible RNA-editing deaminase activity in Selaginella. No evidence of RNA editing was found for tRNAs or rRNAs but we identified a total of 74 editing sites in cDNA sequences of four group II introns (petBi6g2, petDi8g2, ycf3i124g2, and ycf3i354g2) retained in partially matured transcripts, which strongly contribute to improved base-pairing in the intron secondary structures as a likely prerequisite for their splicing. PMID:25142065

  2. RESOPS: A Database for Analyzing the Correspondence of RNA Editing Sites to Protein Three-Dimensional Structures

    PubMed Central

    Yura, Kei; Sulaiman, Sintawee; Hatta, Yosuke; Shionyu, Masafumi; Go, Mitiko

    2009-01-01

    Transcripts from mitochondrial and chloroplast DNA of land plants often undergo cytidine to uridine conversion-type RNA editing events. RESOPS is a newly built database that specializes in displaying RNA editing sites of land plant organelles on protein three-dimensional (3D) structures to help elucidate the mechanisms of RNA editing for gene expression regulation. RESOPS contains the following information: unedited and edited cDNA sequences with notes for the target nucleotides of RNA editing, conceptual translation from the edited cDNA sequence in pseudo-UniProt format, a list of proteins under the influence of RNA editing, multiple amino acid sequence alignments of edited proteins, the location of amino acid residues coded by codons under the influence of RNA editing in protein 3D structures and the statistics of biased distributions of the edited residues with respect to protein structures. Most of the data processing procedures are automated; hence, it is easy to keep abreast of updated genome and protein 3D structural data. In the RESOPS database, we clarified that the locations of residues switched by RNA editing are significantly biased to protein structural cores. The integration of different types of data in the database also help advance the understanding of RNA editing mechanisms. RESOPS is accessible at http://cib.cf.ocha.ac.jp/RNAEDITING/. PMID:19808808

  3. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    PubMed Central

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  4. Comprehensive analysis of human small RNA sequencing data provides insights into expression profiles and miRNA editing

    PubMed Central

    Gong, Jing; Wu, Yuliang; Zhang, Xiantong; Liao, Yifang; Sibanda, Vusumuzi Leroy; Liu, Wei; Guo, An-Yuan

    2014-01-01

    MicroRNAs (miRNAs) play key regulatory roles in various biological processes and diseases. A comprehensive analysis of large scale small RNA sequencing data (smRNA-seq) will be very helpful to explore tissue or disease specific miRNA markers and uncover miRNA variants. Here, we systematically analyzed 410 human smRNA-seq datasets, which samples are from 24 tissue/disease/cell lines. We tested the mapping strategies and found that it was necessary to make multiple-round mappings with different mismatch parameters. miRNA expression profiles revealed that on average ∼70% of known miRNAs were expressed at low level or not expressed (RPM < 1) in a sample and only ∼9% of known miRNAs were relatively highly expressed (RPM > 100). About 30% known miRNAs were not expressed in all of our used samples. The miRNA expression profiles were compiled into an online database (HMED, http://bioinfo.life.hust.edu.cn/smallRNA/). Dozens of tissue/disease specific miRNAs, disease/control dysregulated miRNAs and miRNAs with arm switching events were discovered. Further, we identified some highly confident editing sites including 24 A-to-I sites and 23 C-to-U sites. About half of them were widespread miRNA editing sites in different tissues. We characterized that the 2 types of editing sites have different features with regard to location, editing level and frequency. Our analyses for expression profiles, specific miRNA markers, arm switching, and editing sites, may provide valuable information for further studies of miRNA function and biomarker finding. PMID:25692236

  5. Altered expression and editing of miRNA-100 regulates iTreg differentiation

    PubMed Central

    Negi, Vinny; Paul, Deepanjan; Das, Sudipta; Bajpai, Prashant; Singh, Suchita; Mukhopadhyay, Arijit; Agrawal, Anurag; Ghosh, Balaram

    2015-01-01

    RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4+ T cell differentiation. CD4+ T cells are an integral component of the adaptive immune system. Naïve CD4+ T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end. PMID:26209130

  6. Altered expression and editing of miRNA-100 regulates iTreg differentiation.

    PubMed

    Negi, Vinny; Paul, Deepanjan; Das, Sudipta; Bajpai, Prashant; Singh, Suchita; Mukhopadhyay, Arijit; Agrawal, Anurag; Ghosh, Balaram

    2015-09-18

    RNA editing of miRNAs, especially in the seed region, adds another layer to miRNA mediated gene regulation which can modify its targets, altering cellular signaling involved in important processes such as differentiation. In this study, we have explored the role of miRNA editing in CD4(+) T cell differentiation. CD4(+) T cells are an integral component of the adaptive immune system. Naïve CD4(+) T cells, on encountering an antigen, get differentiated either into inflammatory subtypes like Th1, Th2 or Th17, or into immunosuppressive subtype Treg, depending on the cytokine milieu. We found C-to-U editing at fifth position of mature miR-100, specifically in Treg. The C-to-U editing of miR-100 is functionally associated with at least one biologically relevant target change, from MTOR to SMAD2. Treg cell polarization by TGFβ1 was reduced by both edited and unedited miR-100 mimics, but percentage of Treg in PBMCs was only reduced by edited miR-100 mimics, suggesting a model in which de-repression of MTOR due to loss of unedited mir-100, promotes tolerogenic signaling, while gain of edited miR-100 represses SMAD2, thereby limiting the Treg. Such delicately counterbalanced systems are a hallmark of immune plasticity and we propose that miR-100 editing is a novel mechanism toward this end. PMID:26209130

  7. The crystal structure of an oligo(U):pre-mRNA duplex from a trypanosome RNA editing substrate.

    PubMed

    Mooers, Blaine H M; Singh, Amritanshu

    2011-10-01

    Guide RNAs bind antiparallel to their target pre-mRNAs to form editing substrates in reaction cycles that insert or delete uridylates (Us) in most mitochondrial transcripts of trypanosomes. The 5' end of each guide RNA has an anchor sequence that binds to the pre-mRNA by base-pair complementarity. The template sequence in the middle of the guide RNA directs the editing reactions. The 3' ends of most guide RNAs have ∼15 contiguous Us that bind to the purine-rich unedited pre-mRNA upstream of the editing site. The resulting U-helix is rich in G·U wobble base pairs. To gain insights into the structure of the U-helix, we crystallized 8 bp of the U-helix in one editing substrate for the A6 mRNA of Trypanosoma brucei. The fragment provides three samples of the 5'-AGA-3'/5'-UUU-3' base-pair triple. The fusion of two identical U-helices head-to-head promoted crystallization. We obtained X-ray diffraction data with a resolution limit of 1.37 Å. The U-helix had low and high twist angles before and after each G·U wobble base pair; this variation was partly due to shearing of the wobble base pairs as revealed in comparisons with a crystal structure of a 16-nt RNA with all Watson-Crick base pairs. Both crystal structures had wider major grooves at the junction between the poly(U) and polypurine tracts. This junction mimics the junction between the template helix and the U-helix in RNA-editing substrates and may be a site of major groove invasion by RNA editing proteins. PMID:21878548

  8. Did RNA editing in plant organellar genomes originate under natural selection or through genetic drift?

    PubMed Central

    Jobson, Richard W; Qiu, Yin-Long

    2008-01-01

    Background The C↔U substitution types of RNA editing have been observed frequently in organellar genomes of land plants. Although various attempts have been made to explain why such a seemingly inefficient genetic mechanism would have evolved, no satisfactory explanation exists in our view. In this study, we examined editing patterns in chloroplast genomes of the hornwort Anthoceros formosae and the fern Adiantum capillus-veneris and in mitochondrial genomes of the angiosperms Arabidopsis thaliana, Beta vulgaris and Oryza sativa, to gain an understanding of the question of how RNA editing originated. Results We found that 1) most editing sites were distributed at the 2nd and 1st codon positions, 2) editing affected codons that resulted in larger hydrophobicity and molecular size changes much more frequently than those with little change involved, 3) editing uniformly increased protein hydrophobicity, 4) editing occurred more frequently in ancestrally T-rich sequences, which were more abundant in genes encoding membrane-bound proteins with many hydrophobic amino acids than in genes encoding soluble proteins, and 5) editing occurred most often in genes found to be under strong selective constraint. Conclusion These analyses show that editing mostly affects functionally important and evolutionarily conserved codon positions, codons and genes encoding membrane-bound proteins. In particular, abundance of RNA editing in plant organellar genomes may be associated with disproportionately large percentages of genes in these two genomes that encode membrane-bound proteins, which are rich in hydrophobic amino acids and selectively constrained. These data support a hypothesis that natural selection imposed by protein functional constraints has contributed to selective fixation of certain editing sites and maintenance of the editing activity in plant organelles over a period of more than four hundred millions years. The retention of genes encoding RNA editing activity may be

  9. Uridine insertion/deletion editing in trypanosomes: a playground for RNA-guided information transfer.

    PubMed

    Aphasizhev, Ruslan; Aphasizheva, Inna

    2011-01-01

    RNA editing is a collective term referring to enzymatic processes that change RNA sequence apart from splicing, 5' capping or 3' extension. In this article, we focus on uridine insertion/deletion mRNA editing found exclusively in mitochondria of kinetoplastid protists. This type of editing corrects frameshifts, introduces start and stops codons, and often adds much of the coding sequence to create an open reading frame. The mitochondrial genome of trypanosomatids, the most extensively studied clade within the order Kinetoplastida, is composed of ∼50 maxicircles with limited coding capacity and thousands of minicircles. To produce functional mRNAs, a multitude of nuclear-encoded factors mediate interactions of maxicircle-encoded pre-mRNAs with a vast repertoire of minicircle-encoded guide RNAs. Editing reactions of mRNA cleavage, U-insertions or U-deletions, and ligation are catalyzed by the RNA editing core complex (RECC, the 20S editosome) while each step of this enzymatic cascade is directed by guide RNAs. These 50-60 nucleotide (nt) molecules are 3' uridylated by RET1 TUTase and stabilized via association with the gRNA binding complex (GRBC). Remarkably, the information transfer between maxicircle and minicircle transcriptomes does not rely on template-dependent polymerization of nucleic acids. Instead, intrinsic substrate specificities of key enzymes are largely responsible for the fidelity of editing. Conversely, the efficiency of editing is enhanced by assembling enzymes and RNA binding proteins into stable multiprotein complexes. WIREs RNA 2011 2 669-685 DOI: 10.1002/wrna.82 For further resources related to this article, please visit the WIREs website. PMID:21823228

  10. Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing

    PubMed Central

    Leu, Kuan-Chieh; Hsieh, Ming-Hsiun; Wang, Huei-Jing; Hsieh, Hsu-Liang

    2016-01-01

    ABSTRACT The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg300-to-Trp300 and Pro313-to-Ser313 amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles. PMID:27149614

  11. Distinct role of Arabidopsis mitochondrial P-type pentatricopeptide repeat protein-modulating editing protein, PPME, in nad1 RNA editing.

    PubMed

    Leu, Kuan-Chieh; Hsieh, Ming-Hsiun; Wang, Huei-Jing; Hsieh, Hsu-Liang; Jauh, Guang-Yuh

    2016-06-01

    The mitochondrion is an important power generator in most eukaryotic cells. To preserve its function, many essential nuclear-encoded factors play specific roles in mitochondrial RNA metabolic processes, including RNA editing. RNA editing consists of post-transcriptional deamination, which alters specific nucleotides in transcripts to mediate gene expression. In plant cells, many pentatricopeptide repeat proteins (PPRs) participate in diverse organellar RNA metabolic processes, but only PLS-type PPRs are involved in RNA editing. Here, we report a P-type PPR protein from Arabidopsis thaliana, P-type PPR-Modulating Editing (PPME), which has a distinct role in mitochondrial nad1 RNA editing via RNA binding activity. In the homozygous ppme mutant, cytosine (C)-to-uracil (U) conversions at both the nad1-898 and 937 sites were abolished, disrupting Arg(300)-to-Trp(300) and Pro(313)-to-Ser(313) amino acid changes in the mitochondrial NAD1 protein. NAD1 is a critical component of mitochondrial respiration complex I; its activity is severely reduced in the homozygous ppme mutant, resulting in significantly altered growth and development. Both abolished RNA editing and defective complex I activity were completely rescued by CaMV 35S promoter- and PPME native promoter-driven PPME genomic fragments tagged with GFP in a homozygous ppme background. Our experimental results demonstrate a distinct role of a P-type PPR protein, PPME, in RNA editing in plant organelles. PMID:27149614

  12. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.

    PubMed

    Xie, Kabin; Minkenberg, Bastian; Yang, Yinong

    2015-03-17

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  13. RNA editing by G-nucleotide insertion in mumps virus P-gene mRNA transcripts.

    PubMed Central

    Paterson, R G; Lamb, R A

    1990-01-01

    A guanine nucleotide insertion event has been shown to occur at a specific site within mumps virus P-gene mRNA transcripts. The region of the mRNA containing the site expected to be used for RNA editing and the complementary portion of the genomic RNA were cloned, and their nucleotide sequences were obtained. The genomic RNA was found to possess six C residues at the insertion site, whereas 63% of the P-gene-specific mRNA transcripts were found to have from two to five G residues inserted at this position in the RNA. An unedited mRNA was shown to encode the mumps virus cysteine-rich protein V, and mRNA transcripts containing two and four inserted G residues were translated to yield the mumps virus P and I proteins, respectively. Images PMID:2166809

  14. Altering the Enantioselectivity of Tyrosyl-tRNA Synthetase by Insertion of a Stereospecific Editing Domain.

    PubMed

    Richardson, Charles J; First, Eric A

    2016-03-15

    Translation of mRNAs by the ribosome is stereospecific, with only l-amino acids being incorporated into the nascent polypeptide chain. This stereospecificity results from the exclusion of d-amino acids at three steps during protein synthesis: (1) the aminoacylation of tRNA by aminoacyl-tRNA synthetases, (2) binding of aminoacyl-tRNAs to EF-Tu, and (3) recognition of aminoacyl-tRNAs by the ribosome. As a first step toward incorporating d-amino acids during protein synthesis, we have altered the enantioselectivity of tyrosyl-tRNA synthetase. This enzyme is unusual among aminoacyl-tRNA synthetases, as it can aminoacylate tRNA with d-tyrosine (albeit at a reduced rate compared to l-tyrosine). To change the enantioselectivity of tyrosyl-tRNA synthetase, we introduced the post-transfer editing domain from Pyrococcus horikoshii phenylalanyl-tRNA synthetase into the connective polypeptide 1 (CP1) domain of Geobacillus stearothermophilus tyrosyl-tRNA synthetase (henceforth designated TyrRS-FRSed). We show that the phenylalanyl-tRNA synthetase editing domain is stereospecific, hydrolyzing l-Tyr-tRNA(Tyr), but not d-Tyr-tRNA(Tyr). We further show that inserting the phenylalanyl-tRNA synthetase editing domain into the CP1 domain of tyrosyl-tRNA synthetase decreases the activity of the synthetic site in tyrosyl-tRNA synthetase. This decrease in activity is critical, as it prevents the rate of synthesis from overwhelming the ability of the editing domain to hydrolyze the l-Tyr-tRNA(Tyr) product. Overall, inserting the phenylalanyl-tRNA synthetase editing domain results in a 2-fold shift in the enantioselectivity of tyrosyl-tRNA synthetase toward the d-Tyr-tRNA(Tyr) product. When a 4-fold excess of d-tyrosine is used, approximately 40% of the tRNA(Tyr) is aminoacylated with d-tyrosine. PMID:26890980

  15. Massive Gene Transfer and Extensive RNA Editing of a Symbiotic Dinoflagellate Plastid Genome

    PubMed Central

    Mungpakdee, Sutada; Shinzato, Chuya; Takeuchi, Takeshi; Kawashima, Takeshi; Koyanagi, Ryo; Hisata, Kanako; Tanaka, Makiko; Goto, Hiroki; Fujie, Manabu; Lin, Senjie; Satoh, Nori; Shoguchi, Eiichi

    2014-01-01

    Genome sequencing of Symbiodinium minutum revealed that 95 of 109 plastid-associated genes have been transferred to the nuclear genome and subsequently expanded by gene duplication. Only 14 genes remain in plastids and occur as DNA minicircles. Each minicircle (1.8–3.3 kb) contains one gene and a conserved noncoding region containing putative promoters and RNA-binding sites. Nine types of RNA editing, including a novel G/U type, were discovered in minicircle transcripts but not in genes transferred to the nucleus. In contrast to DNA editing sites in dinoflagellate mitochondria, which tend to be highly conserved across all taxa, editing sites employed in DNA minicircles are highly variable from species to species. Editing is crucial for core photosystem protein function. It restores evolutionarily conserved amino acids and increases peptidyl hydropathy. It also increases protein plasticity necessary to initiate photosystem complex assembly. PMID:24881086

  16. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system

    PubMed Central

    Xie, Kabin; Minkenberg, Bastian

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA–gRNA architecture were efficiently and precisely processed into gRNAs with desired 5′ targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits. PMID:25733849

  17. A Zinc Finger Motif-Containing Protein Is Essential for Chloroplast RNA Editing

    PubMed Central

    Sun, Tao; Shi, Xiaowen; Friso, Giulia; Van Wijk, Klaas; Bentolila, Stephane; Hanson, Maureen R.

    2015-01-01

    C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing. PMID:25768119

  18. In vitro RNA editing-like activity in a mitochondrial extract from Leishmania tarentolae.

    PubMed Central

    Frech, G C; Bakalara, N; Simpson, L; Simpson, A M

    1995-01-01

    A mitochondrial extract from Leishmania tarentolae directs the incorporation of uridylate (U) residues within the pre-edited domain of synthetic cytochrome b (CYb) and NADH dehydrogenase subunit 7 mRNA. This has several characteristics of an in vitro RNA editing activity, but no direct evidence for involvement of guide RNAs was obtained. Inhibition by micrococcal nuclease suggests a requirement for some type of endogenous RNA. The limitation of internal U-incorporation to the pre-edited region in the CYb mRNA and the inhibition by deletion or substitution of both mRNA anchor sequences for CYb gRNA-I and -II could be consistent either with a gRNA-mediated process or a secondary structure-mediated process. A low level of incorporation of [alpha-32P]CTP occurs at the same sites as UTP. Internal U-incorporation activity is selectively inhibited by heterologous RNAs, suggesting an involvement of low affinity RNA-binding proteins which can be competed by the added RNA. Images PMID:7828590

  19. Disruption of RNA editing in Leishmania tarentolae by the loss of minicircle-encoded guide RNA genes.

    PubMed Central

    Thiemann, O H; Maslov, D A; Simpson, L

    1994-01-01

    RNA editing in kinetoplastids appears to be a labile genetic trait that is affected by prolonged cell culture. The transcripts of the G1-G5 cryptogenes are pan-edited in the recently isolated LEM125 strain of Leishmania tarentolae, but not in the UC strain which has been in culture for 55 years. At least 32 minicircle-encoded guide RNAs (gRNAs) for the editing of G1-G5 transcripts are present in LEM125 and absent in UC. We hypothesize that specific minicircle sequence classes encoding gRNAs for the editing of these transcripts were lost during the long culture history of the UC strain. The protein products, which include components of complex I of the respiratory chain, are probably not required during the culture stage of the Leishmania life cycle. Images PMID:7988566

  20. Gene Loss and Error-Prone RNA Editing in the Mitochondrion of Perkinsela, an Endosymbiotic Kinetoplastid

    PubMed Central

    David, Vojtěch; Flegontov, Pavel; Gerasimov, Evgeny; Tanifuji, Goro; Hashimi, Hassan; Logacheva, Maria D.; Maruyama, Shinichiro; Onodera, Naoko T.; Gray, Michael W.; Archibald, John M.

    2015-01-01

    ABSTRACT Perkinsela is an enigmatic early-branching kinetoplastid protist that lives as an obligate endosymbiont inside Paramoeba (Amoebozoa). We have sequenced the highly reduced mitochondrial genome of Perkinsela, which possesses only six protein-coding genes (cox1, cox2, cox3, cob, atp6, and rps12), despite the fact that the organelle itself contains more DNA than is present in either the host or endosymbiont nuclear genomes. An in silico analysis of two Perkinsela strains showed that mitochondrial RNA editing and processing machineries typical of kinetoplastid flagellates are generally conserved, and all mitochondrial transcripts undergo U-insertion/deletion editing. Canonical kinetoplastid mitochondrial ribosomes are also present. We have developed software tools for accurate and exhaustive mapping of transcriptome sequencing (RNA-seq) reads with extensive U-insertions/deletions, which allows detailed investigation of RNA editing via deep sequencing. With these methods, we show that up to 50% of reads for a given edited region contain errors of the editing system or, less likely, correspond to alternatively edited transcripts. PMID:26628723

  1. RNA-guided genome editing in plants using a CRISPR-Cas system.

    PubMed

    Xie, Kabin; Yang, Yinong

    2013-11-01

    Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants. PMID:23956122

  2. Synthetic CRISPR RNA-Cas9–guided genome editing in human cells

    PubMed Central

    Rahdar, Meghdad; McMahon, Moira A.; Prakash, Thazha P.; Swayze, Eric E.; Bennett, C. Frank; Cleveland, Don W.

    2015-01-01

    Genome editing with the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 nuclease system is a powerful technology for manipulating genomes, including introduction of gene disruptions or corrections. Here we develop a chemically modified, 29-nucleotide synthetic CRISPR RNA (scrRNA), which in combination with unmodified transactivating crRNA (tracrRNA) is shown to functionally replace the natural guide RNA in the CRISPR-Cas9 nuclease system and to mediate efficient genome editing in human cells. Incorporation of rational chemical modifications known to protect against nuclease digestion and stabilize RNA–RNA interactions in the tracrRNA hybridization region of CRISPR RNA (crRNA) yields a scrRNA with enhanced activity compared with the unmodified crRNA and comparable gene disruption activity to the previously published single guide RNA. Taken together, these findings provide a platform for therapeutic applications, especially for nervous system disease, using successive application of cell-permeable, synthetic CRISPR RNAs to activate and then silence Cas9 nuclease activity. PMID:26589814

  3. RNA Editing of Androgen Receptor Gene Transcripts in Prostate Cancer Cells*S⃞

    PubMed Central

    Martinez, Harryl D.; Jasavala, Rohini J.; Hinkson, Izumi; Fitzgerald, Latricia D.; Trimmer, James S.; Kung, Hsing-Jien; Wright, Michael E.

    2008-01-01

    Reactivation of the androgen receptor (AR) signaling pathway represents a critical step in the growth and survival of androgen-independent (AI) prostate cancer (CaP). In this study we show the DU145 and PC3 AI human CaP cell lines respond to androgens and require AR expression for optimal proliferation in vitro. Interestingly, AR gene transcripts in DU145 and PC3 cells harbored a large number of single base pair nucleotide transitions that resulted in missense mutations in selected AR codons. The most notable lesion detected in AR gene transcripts included the oncogenic codon 877T→A gain-of-function mutation. Surprisingly, AR gene transcript nucleotide transitions were not genome-encoded substitutions, but instead the mutations co-localized to putative A-to-I, U-to-C, C-to-U, and G-to-A RNA editing sites, suggesting the lesions were mediated through RNA editing mechanisms. Higher levels of mRNA encoding the A-to-I RNA editing enzymes ADAR1 and ADARB1 were observed in DU145 and PC3 cells relative to the androgen-responsive LNCaP and 22Rv1 human CaP cell lines, which correlated with higher levels of AR gene transcript A-to-I editing detected in DU145 and PC3 cells. Our results suggest that AR gene transcripts are targeted by different RNA editing enzymes in DU145 and PC3 cells. Thus RNA editing of AR gene transcripts may contribute to the etiology of hormone-refractory phenotypes in advanced stage AI CaP. PMID:18708348

  4. Inositol Hexakisphosphate Is Bound in the ADAR2 Core and Required for RNA Editing

    PubMed Central

    Macbeth, Mark R.; Schubert, Heidi L.; VanDemark, Andrew P.; Lingam, Arunth T.; Hill, Christopher P.; Bass, Brenda L.

    2007-01-01

    We report the crystal structure of the catalytic domain of human ADAR2, an RNA editing enzyme, at 1.7 angstrom resolution. The structure reveals a zinc ion in the active site and suggests how the substrate adenosine is recognized. Unexpectedly, inositol hexakisphosphate (IP6) is buried within the enzyme core, contributing to the protein fold. Although there are no reports that adenosine deaminases that act on RNA (ADARs) require a cofactor, we show that IP6 is required for activity. Amino acids that coordinate IP6 in the crystal structure are conserved in some adenosine deaminases that act on transfer RNA (tRNA) (ADATs), related enzymes that edit tRNA. Indeed, IP6 is also essential for in vivo and in vitro deamination of adenosine 37 of tRNAala by ADAT1. PMID:16141067

  5. A Novel Computational Strategy to Identify A-to-I RNA Editing Sites by RNA-Seq Data: De Novo Detection in Human Spinal Cord Tissue

    PubMed Central

    Picardi, Ernesto; Gallo, Angela; Galeano, Federica; Tomaselli, Sara; Pesole, Graziano

    2012-01-01

    RNA editing is a post-transcriptional process occurring in a wide range of organisms. In human brain, the A-to-I RNA editing, in which individual adenosine (A) bases in pre-mRNA are modified to yield inosine (I), is the most frequent event. Modulating gene expression, RNA editing is essential for cellular homeostasis. Indeed, its deregulation has been linked to several neurological and neurodegenerative diseases. To date, many RNA editing sites have been identified by next generation sequencing technologies employing massive transcriptome sequencing together with whole genome or exome sequencing. While genome and transcriptome reads are not always available for single individuals, RNA-Seq data are widespread through public databases and represent a relevant source of yet unexplored RNA editing sites. In this context, we propose a simple computational strategy to identify genomic positions enriched in novel hypothetical RNA editing events by means of a new two-steps mapping procedure requiring only RNA-Seq data and no a priori knowledge of RNA editing characteristics and genomic reads. We assessed the suitability of our procedure by confirming A-to-I candidates using conventional Sanger sequencing and performing RNA-Seq as well as whole exome sequencing of human spinal cord tissue from a single individual. PMID:22957051

  6. The expression of apoB mRNA editing factors is not the sole determinant for the induction of editing in differentiating Caco-2 cells

    SciTech Connect

    Galloway, Chad A.; Smith, Harold C.

    2010-01-01

    Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is {approx}80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However, transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.

  7. The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster.

    PubMed

    Li, Xianghua; Overton, Ian M; Baines, Richard A; Keegan, Liam P; O'Connell, Mary A

    2014-01-01

    RNA editing by deamination of specific adenosine bases to inosines during pre-mRNA processing generates edited isoforms of proteins. Recoding RNA editing is more widespread in Drosophila than in vertebrates. Editing levels rise strongly at metamorphosis, and Adar(5G1) null mutant flies lack editing events in hundreds of CNS transcripts; mutant flies have reduced viability, severely defective locomotion and age-dependent neurodegeneration. On the other hand, overexpressing an adult dADAR isoform with high enzymatic activity ubiquitously during larval and pupal stages is lethal. Advantage was taken of this to screen for genetic modifiers; Adar overexpression lethality is rescued by reduced dosage of the Rdl (Resistant to dieldrin), gene encoding a subunit of inhibitory GABA receptors. Reduced dosage of the Gad1 gene encoding the GABA synthetase also rescues Adar overexpression lethality. Drosophila Adar(5G1) mutant phenotypes are ameliorated by feeding GABA modulators. We demonstrate that neuronal excitability is linked to dADAR expression levels in individual neurons; Adar-overexpressing larval motor neurons show reduced excitability whereas Adar(5G1) null mutant or targeted Adar knockdown motor neurons exhibit increased excitability. GABA inhibitory signalling is impaired in human epileptic and autistic conditions, and vertebrate ADARs may have a relevant evolutionarily conserved control over neuronal excitability. PMID:24137011

  8. Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.

    PubMed Central

    Morrison, J R; Pászty, C; Stevens, M E; Hughes, S D; Forte, T; Scott, J; Rubin, E M

    1996-01-01

    RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport. Images Fig. 1 Fig. 2 Fig. 3 PMID:8692961

  9. Cas9 gRNA engineering for genome editing, activation and repression

    PubMed Central

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-01-01

    We demonstrate that by altering the length of Cas9-associated guide RNA(gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  10. Cas9 gRNA engineering for genome editing, activation and repression.

    PubMed

    Kiani, Samira; Chavez, Alejandro; Tuttle, Marcelle; Hall, Richard N; Chari, Raj; Ter-Ovanesyan, Dmitry; Qian, Jason; Pruitt, Benjamin W; Beal, Jacob; Vora, Suhani; Buchthal, Joanna; Kowal, Emma J K; Ebrahimkhani, Mohammad R; Collins, James J; Weiss, Ron; Church, George

    2015-11-01

    We demonstrate that by altering the length of Cas9-associated guide RNA (gRNA) we were able to control Cas9 nuclease activity and simultaneously perform genome editing and transcriptional regulation with a single Cas9 protein. We exploited these principles to engineer mammalian synthetic circuits with combined transcriptional regulation and kill functions governed by a single multifunctional Cas9 protein. PMID:26344044

  11. Extensive and evolutionarily persistent mitochondrial tRNA editing in Velvet Worms (phylum Onychophora).

    PubMed

    Segovia, Romulo; Pett, Walker; Trewick, Steve; Lavrov, Dennis V

    2011-10-01

    Mitochondrial genomes of onychophorans (velvet worms) present an interesting problem: Some previous studies reported them lacking several transfer RNA (tRNA) genes, whereas others found that all their tRNA genes were present but severely reduced. To resolve this discrepancy, we determined complete mitochondrial DNA (mtDNA) sequences of the onychophorans Oroperipatus sp. and Peripatoides sympatrica as well as cDNA sequences from 14 and 10 of their tRNAs, respectively. We show that tRNA genes in these genomes are indeed highly reduced and encode truncated molecules, which are restored to more conventional structures by extensive tRNA editing. During this editing process, up to 34 nucleotides are added to the tRNA sequences encoded in Oroperipatus sp. mtDNA, rebuilding the aminoacyl acceptor stem, the TΨC arm, and in some extreme cases, the variable arm and even a part of the anticodon stem. The editing is less extreme in P. sympatrica in which at least a part of the TΨC arm is always encoded in mtDNA. When the entire TΨC arm is added de novo in Oroperipatus sp., the sequence of this arm is either identical or similar among different tRNA species, yet the sequences show substantial variation for each tRNA. These observations suggest that the arm is rebuilt, at least in part, by a template-independent mechanism and argue against the alternative possibility that tRNA genes or their parts are imported from the nucleus. By contrast, the 3' end of the aminoacyl acceptor stem is likely restored by a template-dependent mechanism. The extreme tRNA editing reported here has been preserved for >140 My as it was found in both extant families of onychophorans. Furthermore, a similar type of tRNA editing may be present in several other groups of arthropods, which show a high degree of tRNA gene reduction in their mtDNA. PMID:21546355

  12. Epigenome Editing of Potato by Grafting Using Transgenic Tobacco as siRNA Donor.

    PubMed

    Kasai, Atsushi; Bai, Songling; Hojo, Hatsune; Harada, Takeo

    2016-01-01

    In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops. PMID:27564864

  13. Epigenome Editing of Potato by Grafting Using Transgenic Tobacco as siRNA Donor

    PubMed Central

    Hojo, Hatsune; Harada, Takeo

    2016-01-01

    In plants, it is possible to induce heritable transcriptional gene silencing (TGS) via RNA-directed DNA methylation (RdDM) using artificially synthesized small RNA (siRNA) homologous to the 5'-flanking region of the target gene. As the siRNA signal with a specific RNA determinant moves through plasmodesmata and sieve elements, we attempted to induce TGS of a transgene and an endogenous gene of potato (Solanum tuberosum) rootstock by grafting using siRNA produced in a tobacco (Nicotiana benthamiana) scion. Our results provide evidence that this system can induce TGS of target genes in tubers formed on potato rootstock. The TGS is maintained in the progeny tubers lacking the transported siRNAs. Our findings reveal that epigenome editing using mobile RNA has the potential to allow breeding of artificial sport cultivars in vegetative propagation crops. PMID:27564864

  14. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions

    PubMed Central

    Long, Yicheng; Jackman, Jane E.

    2015-01-01

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from A. castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing. PMID:26143376

  15. In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions.

    PubMed

    Long, Yicheng; Jackman, Jane E

    2015-07-22

    Protozoan mitochondrial tRNAs (mt-tRNAs) are repaired by a process known as 5'-editing. Mt-tRNA sequencing revealed organism-specific patterns of editing G-U base pairs, wherein some species remove G-U base pairs during 5'-editing, while others retain G-U pairs in the edited tRNA. We tested whether 3'-5' polymerases that catalyze the repair step of 5'-editing exhibit organism-specific preferences that explain the treatment of G-U base pairs. Biochemical and kinetic approaches revealed that a 3'-5' polymerase from Acanthamoeba castellanii tolerates G-U wobble pairs in editing substrates much more readily than several other enzymes, consistent with its biological pattern of editing. PMID:26143376

  16. Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids.

    PubMed Central

    Chaudhuri, S; Carrer, H; Maliga, P

    1995-01-01

    In tobacco plastids, functional psbL mRNA is created by editing an ACG codon to an AUG translation initiation codon. To determine if editing may occur in a chimeric mRNA, the N-terminal part of psbL containing the editing site was translationally fused with the aadA and kan bacterial genes. The chimeric constructs were introduced into the tobacco plastid genome by targeted gene insertion. Editing of the chimeric mRNAs indicated that the 98 nt fragment spanning the psbL editing site contains all cis information required for editing. Expression of the chimeric gene transcripts led to a significant decrease in the editing efficiency of the endogenous psbL mRNA. However, the efficiency of editing in the transplastomic lines was unchanged for four sites in the rpoB and ndhB mRNAs. Reduced efficiency of psbL editing, but not of the other four sites, in the transplastomic lines indicates depletion of psbL-specific editing factor(s). This finding implicates the involvement of site-specific factors in editing of plastid mRNAs in higher plants. Images PMID:7796820

  17. Commitment of apolipoprotein B RNA to the splicing pathway regulates cytidine-to-uridine editing-site utilization.

    PubMed Central

    Sowden, M P; Smith, H C

    2001-01-01

    A tripartite motif located in the centre of the 7.5 kb exon 26 of apolipoprotein B (apoB) mRNA directs editosome assembly and site-specific cytidine-to-uridine editing at nucleotide 6666. apoB mRNA editing is a post-transcriptional event, occurring primarily at the time exon 26 is spliced or at a time after splicing, but before nuclear export. We show, through reporter RNA constructs, that RNA splice sites suppress editing of precursor RNAs when placed proximal or distal to the editing site. Processed RNAs were edited more efficiently than precursor RNAs. Mutation of both the splice donor and acceptor sites was necessary for RNAs to be edited efficiently. The results suggested that commitment of pre-mRNA to the splicing and/or nuclear-export pathways may play a role in regulating editing-site utilization. The HIV-1 Rev-Rev response element ('RRE') interaction was utilized to uncouple the commitment of precursor RNAs to the spliceosome assembly pathway and associated nuclear-export pathway. Under these conditions, unspliced reporter RNAs were edited efficiently. We propose that pre-mRNA passage through the temporal or spatial restriction point where they become committed to spliceosome assembly contributes regulatory information for subsequent editosome activity. PMID:11672445

  18. Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants.

    PubMed

    Cheng, Shifeng; Gutmann, Bernard; Zhong, Xiao; Ye, Yongtao; Fisher, Mark F; Bai, Fengqi; Castleden, Ian; Song, Yue; Song, Bo; Huang, Jiaying; Liu, Xin; Xu, Xun; Lim, Boon L; Bond, Charles S; Yiu, Siu-Ming; Small, Ian

    2016-02-01

    The pentatricopeptide repeat (PPR) proteins form one of the largest protein families in land plants. They are characterised by tandem 30-40 amino acid motifs that form an extended binding surface capable of sequence-specific recognition of RNA strands. Almost all of them are post-translationally targeted to plastids and mitochondria, where they play important roles in post-transcriptional processes including splicing, RNA editing and the initiation of translation. A code describing how PPR proteins recognise their RNA targets promises to accelerate research on these proteins, but making use of this code requires accurate definition and annotation of all of the various nucleotide-binding motifs in each protein. We have used a structural modelling approach to define 10 different variants of the PPR motif found in plant proteins, in addition to the putative deaminase motif that is found at the C-terminus of many RNA-editing factors. We show that the super-helical RNA-binding surface of RNA-editing factors is potentially longer than previously recognised. We used the redefined motifs to develop accurate and consistent annotations of PPR sequences from 109 genomes. We report a high error rate in PPR gene models in many public plant proteomes, due to gene fusions and insertions of spurious introns. These consistently annotated datasets across a wide range of species are valuable resources for future comparative genomics studies, and an essential pre-requisite for accurate large-scale computational predictions of PPR targets. We have created a web portal (http://www.plantppr.com) that provides open access to these resources for the community. PMID:26764122

  19. A role of ADAR2 and RNA editing of glutamate receptors in mood disorders and schizophrenia

    PubMed Central

    2014-01-01

    Background Pre-mRNAs of 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)-propanoic acid (AMPA)/kainate glutamate receptors undergo post-transcriptional modification known as RNA editing that is mediated by adenosine deaminase acting on RNA type 2 (ADAR2). This modification alters the amino acid sequence and function of the receptor. Glutamatergic signaling has been suggested to have a role in mood disorders and schizophrenia, but it is unknown whether altered RNA editing of AMPA/kainate receptors has pathophysiological significance in these mental disorders. In this study, we found that ADAR2 expression tended to be decreased in the postmortem brains of patients with schizophrenia and bipolar disorder. Results Decreased ADAR2 expression was significantly correlated with decreased editing of the R/G sites of AMPA receptors. In heterozygous Adar2 knockout mice (Adar2+/− mice), editing of the R/G sites of AMPA receptors was decreased. Adar2+/− mice showed a tendency of increased activity in the open-field test and a tendency of resistance to immobility in the forced swimming test. They also showed enhanced amphetamine-induced hyperactivity. There was no significant difference in amphetamine-induced hyperactivity between Adar2+/− and wild type mice after the treatment with an AMPA/kainate receptor antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline. Conclusions These findings collectively suggest that altered RNA editing efficiency of AMPA receptors due to down-regulation of ADAR2 has a possible role in the pathophysiology of mental disorders. PMID:24443933

  20. DEAH-RHA helicase•Znf cofactor systems in kinetoplastid RNA editing and evolutionarily distant RNA processes

    PubMed Central

    Cruz-Reyes, Jorge; Mooers, Blaine H.M.; Abu-Adas, Zakaria; Kumar, Vikas; Gulati, Shelly

    2016-01-01

    Multi-zinc finger proteins are an emerging class of cofactors in DEAH-RHA RNA helicases across highly divergent eukaryotic lineages. DEAH-RHA helicase•zinc finger cofactor partnerships predate the split of kinetoplastid protozoa, which include several human pathogens, from other eukaryotic lineages 100–400 Ma. Despite a long evolutionary history, the prototypical DEAH-RHA domains remain highly conserved. This short review focuses on a recently identified DEAH-RHA helicase•zinc finger cofactor system in kinetoplastid RNA editing, and its potential functional parallels with analogous systems in embryogenesis control in nematodes and antivirus protection in humans. PMID:27540585

  1. Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN

    PubMed Central

    Li, Zhongsen; Liu, Zhan-Bin; Xing, Aiqiu; Moon, Bryan P.; Koellhoffer, Jessica P.; Huang, Lingxia; Ward, R. Timothy; Clifton, Elizabeth; Falco, S. Carl; Cigan, A. Mark

    2015-01-01

    Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing. PMID:26294043

  2. Dramatic Enhancement of Genome Editing by CRISPR/Cas9 Through Improved Guide RNA Design

    PubMed Central

    Farboud, Behnom; Meyer, Barbara J.

    2015-01-01

    Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3′ end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3′ GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome. PMID:25695951

  3. RNA editing differently affects protein-coding genes in D. melanogaster and H. sapiens

    PubMed Central

    Grassi, Luigi; Leoni, Guido; Tramontano, Anna

    2015-01-01

    When an RNA editing event occurs within a coding sequence it can lead to a different encoded amino acid. The biological significance of these events remains an open question: they can modulate protein functionality, increase the complexity of transcriptomes or arise from a loose specificity of the involved enzymes. We analysed the editing events in coding regions that produce or not a change in the encoded amino acid (nonsynonymous and synonymous events, respectively) in D. melanogaster and in H. sapiens and compared them with the appropriate random models. Interestingly, our results show that the phenomenon has rather different characteristics in the two organisms. For example, we confirm the observation that editing events occur more frequently in non-coding than in coding regions, and report that this effect is much more evident in H. sapiens. Additionally, in this latter organism, editing events tend to affect less conserved residues. The less frequently occurring editing events in Drosophila tend to avoid drastic amino acid changes. Interestingly, we find that, in Drosophila, changes from less frequently used codons to more frequently used ones are favoured, while this is not the case in H. sapiens. PMID:26169954

  4. Dramatic enhancement of genome editing by CRISPR/Cas9 through improved guide RNA design.

    PubMed

    Farboud, Behnom; Meyer, Barbara J

    2015-04-01

    Success with genome editing by the RNA-programmed nuclease Cas9 has been limited by the inability to predict effective guide RNAs and DNA target sites. Not all guide RNAs have been successful, and even those that were, varied widely in their efficacy. Here we describe and validate a strategy for Caenorhabditis elegans that reliably achieved a high frequency of genome editing for all targets tested in vivo. The key innovation was to design guide RNAs with a GG motif at the 3' end of their target-specific sequences. All guides designed using this simple principle induced a high frequency of targeted mutagenesis via nonhomologous end joining (NHEJ) and a high frequency of precise DNA integration from exogenous DNA templates via homology-directed repair (HDR). Related guide RNAs having the GG motif shifted by only three nucleotides showed severely reduced or no genome editing. We also combined the 3' GG guide improvement with a co-CRISPR/co-conversion approach. For this co-conversion scheme, animals were only screened for genome editing at designated targets if they exhibited a dominant phenotype caused by Cas9-dependent editing of an unrelated target. Combining the two strategies further enhanced the ease of mutant recovery, thereby providing a powerful means to obtain desired genetic changes in an otherwise unaltered genome. PMID:25695951

  5. Positive correlation between ADAR expression and its targets suggests a complex regulation mediated by RNA editing in the human brain

    PubMed Central

    Liscovitch, Noa; Bazak, Lily; Levanon, Erez Y; Chechik, Gal

    2014-01-01

    A-to-I RNA editing by adenosine deaminases acting on RNA is a post-transcriptional modification that is crucial for normal life and development in vertebrates. RNA editing has been shown to be very abundant in the human transcriptome, specifically at the primate-specific Alu elements. The functional role of this wide-spread effect is still not clear; it is believed that editing of transcripts is a mechanism for their down-regulation via processes such as nuclear retention or RNA degradation. Here we combine 2 neural gene expression datasets with genome-level editing information to examine the relation between the expression of ADAR genes with the expression of their target genes. Specifically, we computed the spatial correlation across structures of post-mortem human brains between ADAR and a large set of targets that were found to be edited in their Alu repeats. Surprisingly, we found that a large fraction of the edited genes are positively correlated with ADAR, opposing the assumption that editing would reduce expression. When considering the correlations between ADAR and its targets over development, 2 gene subsets emerge, positively correlated and negatively correlated with ADAR expression. Specifically, in embryonic time points, ADAR is positively correlated with many genes related to RNA processing and regulation of gene expression. These findings imply that the suggested mechanism of regulation of expression by editing is probably not a global one; ADAR expression does not have a genome wide effect reducing the expression of editing targets. It is possible, however, that RNA editing by ADAR in non-coding regions of the gene might be a part of a more complex expression regulation mechanism. PMID:25692240

  6. Positive correlation between ADAR expression and its targets suggests a complex regulation mediated by RNA editing in the human brain.

    PubMed

    Liscovitch, Noa; Bazak, Lily; Levanon, Erez Y; Chechik, Gal

    2014-01-01

    A-to-I RNA editing by adenosine deaminases acting on RNA is a post-transcriptional modification that is crucial for normal life and development in vertebrates. RNA editing has been shown to be very abundant in the human transcriptome, specifically at the primate-specific Alu elements. The functional role of this wide-spread effect is still not clear; it is believed that editing of transcripts is a mechanism for their down-regulation via processes such as nuclear retention or RNA degradation. Here we combine 2 neural gene expression datasets with genome-level editing information to examine the relation between the expression of ADAR genes with the expression of their target genes. Specifically, we computed the spatial correlation across structures of post-mortem human brains between ADAR and a large set of targets that were found to be edited in their Alu repeats. Surprisingly, we found that a large fraction of the edited genes are positively correlated with ADAR, opposing the assumption that editing would reduce expression. When considering the correlations between ADAR and its targets over development, 2 gene subsets emerge, positively correlated and negatively correlated with ADAR expression. Specifically, in embryonic time points, ADAR is positively correlated with many genes related to RNA processing and regulation of gene expression. These findings imply that the suggested mechanism of regulation of expression by editing is probably not a global one; ADAR expression does not have a genome wide effect reducing the expression of editing targets. It is possible, however, that RNA editing by ADAR in non-coding regions of the gene might be a part of a more complex expression regulation mechanism. PMID:25692240

  7. Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing.

    PubMed

    Tsai, Shengdar Q; Wyvekens, Nicolas; Khayter, Cyd; Foden, Jennifer A; Thapar, Vishal; Reyon, Deepak; Goodwin, Mathew J; Aryee, Martin J; Joung, J Keith

    2014-06-01

    Monomeric CRISPR-Cas9 nucleases are widely used for targeted genome editing but can induce unwanted off-target mutations with high frequencies. Here we describe dimeric RNA-guided FokI nucleases (RFNs) that can recognize extended sequences and edit endogenous genes with high efficiencies in human cells. RFN cleavage activity depends strictly on the binding of two guide RNAs (gRNAs) to DNA with a defined spacing and orientation substantially reducing the likelihood that a suitable target site will occur more than once in the genome and therefore improving specificities relative to wild-type Cas9 monomers. RFNs guided by a single gRNA generally induce lower levels of unwanted mutations than matched monomeric Cas9 nickases. In addition, we describe a simple method for expressing multiple gRNAs bearing any 5' end nucleotide, which gives dimeric RFNs a broad targeting range. RFNs combine the ease of RNA-based targeting with the specificity enhancement inherent to dimerization and are likely to be useful in applications that require highly precise genome editing. PMID:24770325

  8. The structure of the C-terminal domain of the largest editosome interaction protein and its role in promoting RNA binding by RNA-editing ligase L2

    PubMed Central

    Park, Young-Jun; Budiarto, Tanya; Wu, Meiting; Pardon, Els; Steyaert, Jan; Hol, Wim G. J.

    2012-01-01

    Trypanosomatids, such as the sleeping sickness parasite Trypanosoma brucei, contain a ∼20S RNA-editing complex, also called the editosome, which is required for U-insertion/deletion editing of mitochondrial mRNAs. The editosome contains a core of 12 proteins including the large interaction protein A1, the small interaction protein A6, and the editing RNA ligase L2. Using biochemical and structural data, we identified distinct domains of T. brucei A1 which specifically recognize A6 and L2. We provide evidence that an N-terminal domain of A1 interacts with the C-terminal domain of L2. The C-terminal domain of A1 appears to be required for the interaction with A6 and also plays a key role in RNA binding by the RNA-editing ligase L2 in trans. Three crystal structures of the C-terminal domain of A1 have been elucidated, each in complex with a nanobody as a crystallization chaperone. These structures permitted the identification of putative dsRNA recognition sites. Mutational analysis of conserved residues of the C-terminal domain identified Arg703, Arg731 and Arg734 as key requirements for RNA binding. The data show that the editing RNA ligase activity is modulated by a novel mechanism, i.e. by the trans-acting RNA binding C-terminal domain of A1. PMID:22561373

  9. RNA editing and mitochondrial activity in promastigotes and amastigotes of Leishmania donovani✯

    PubMed Central

    Neboháčová, Martina; Kim, Christine E.; Simpson, Larry; Maslov, Dmitri A.

    2009-01-01

    Kinetoplast maxicircle DNA sequence organization was investigated in Leishmania donovani, strain 1S LdBob. Gene arrangement in the coding (conserved) region of the maxicircle is collinear with that of most trypanosomatids, with individual genes showing 80-90% nucleotide identity to Leishmania tarentolae, strain UC. The notable exception was an integration of a full-size minicircle sequence in the ND1 gene coding region found in L. donovani. Editing patterns of the mitochondrial mRNAs investigated also followed L. tarentolae UC patterns, including productive editing of the components of respiratory complexes III, IV and V, and ribosomal protein S12 (RPS12), as well as the lack of productive editing in five out of six pan-edited cryptogenes (ND3, ND8, ND9, G3, G4) found in these species. Several guide RNAs for the editing events were localized in minicircles and maxicircles in the locations that are conserved between the species. Mitochondrial activity, including rates of oxygen consumption, the presence and the levels of respiratory complexes and their individual subunits and the steady-state levels of several mitochondrial-encoded mRNAs were essentially the same in axenically grown amastigotes and in promastigotes of L. donovani. However, some modulation of mitochondrial activity between these developmental stages was suggested by the finding of an amastigote-specific component in Complex IV, a down-regulation of mitochondrial RNA-binding proteins (MRP) [define] and MRP-associated protein (MRP-AP) in amastigotes, and by variations in the levels of RPS12, ND3, ND9, G3 and G4 pre-edited transcripts. PMID:19109964

  10. Organellar RNA editing and plant-specific extensions of pentatricopeptide repeat proteins in jungermanniid but not in marchantiid liverworts.

    PubMed

    Rüdinger, Mareike; Polsakiewicz, Monika; Knoop, Volker

    2008-07-01

    The pyrimidine exchange type of RNA editing in land plant (embryophyte) organelles has largely remained an enigma with respect to its biochemical mechanisms, the underlying specificities, and its raison d'être. Apparently arising with the earliest embryophytes, RNA editing is conspicuously absent in one clade of liverworts, the complex thalloid Marchantiidae. Several lines of evidence suggest that the large gene family of organelle-targeted RNA-binding pentatricopeptide repeat (PPR) proteins plays a fundamental role in the sequence-specific editing of organelle transcripts. We here describe the identification of PPR protein genes with plant-specific carboxyterminal (C-terminal) sequence signatures (E, E+, and DYW domains) in ferns, lycopodiophytes, mosses, hornworts, and jungermanniid liverworts, one subclass of the basal most clade of embryophytes, on DNA and cDNA level. In contrast, we were unable to identify these genes in a wide sampling of marchantiid liverworts (including the phylogenetic basal genus Blasia)--taxa for which no RNA editing is observed in the organelle transcripts. On the other hand, we found significant diversity of this type of PPR proteins also in Haplomitrium, a genus with an extremely high rate of RNA editing and a phylogenetic placement basal to all other liverworts. Although the presence of modularly extended PPR proteins correlates well with organelle RNA editing, the now apparent complete loss of an entire gene family from one clade of embryophytes, the marchantiid liverworts, remains puzzling. PMID:18400790

  11. Multiple G-quartet structures in pre-edited mRNAs suggest evolutionary driving force for RNA editing in trypanosomes.

    PubMed

    Leeder, W-Matthias; Hummel, Niklas F C; Göringer, H Ulrich

    2016-01-01

    Mitochondrial transcript maturation in African trypanosomes requires a U-nucleotide specific RNA editing reaction. In its most extreme form hundreds of U's are inserted into and deleted from primary transcripts to generate functional mRNAs. Unfortunately, both origin and biological role of the process have remained enigmatic. Here we report a so far unrecognized structural feature of pre-edited mRNAs. We demonstrate that the cryptic pre-mRNAs contain numerous clustered G-nt, which fold into G-quadruplex (GQ) structures. We identified 27 GQ's in the different pre-mRNAs and demonstrate a positive correlation between the steady state abundance of guide (g)RNAs and the sequence position of GQ-elements. We postulate that the driving force for selecting G-rich sequences lies in the formation of DNA/RNA hybrid G-quadruplex (HQ) structures between the pre-edited transcripts and the non-template strands of mitochondrial DNA. HQ's are transcription termination/replication initiation sites and thus guarantee an unperturbed replication of the mt-genome. This is of special importance in the insect-stage of the parasite. In the transcription-on state, the identified GQ's require editing as a GQ-resolving activity indicating a link between replication, transcription and RNA editing. We propose that the different processes have coevolved and suggest the parasite life-cycle and the single mitochondrion as evolutionary driving forces. PMID:27436151

  12. Multiple G-quartet structures in pre-edited mRNAs suggest evolutionary driving force for RNA editing in trypanosomes

    PubMed Central

    Leeder, W.-Matthias; Hummel, Niklas F. C.; Göringer, H. Ulrich

    2016-01-01

    Mitochondrial transcript maturation in African trypanosomes requires a U-nucleotide specific RNA editing reaction. In its most extreme form hundreds of U’s are inserted into and deleted from primary transcripts to generate functional mRNAs. Unfortunately, both origin and biological role of the process have remained enigmatic. Here we report a so far unrecognized structural feature of pre-edited mRNAs. We demonstrate that the cryptic pre-mRNAs contain numerous clustered G-nt, which fold into G-quadruplex (GQ) structures. We identified 27 GQ’s in the different pre-mRNAs and demonstrate a positive correlation between the steady state abundance of guide (g)RNAs and the sequence position of GQ-elements. We postulate that the driving force for selecting G-rich sequences lies in the formation of DNA/RNA hybrid G-quadruplex (HQ) structures between the pre-edited transcripts and the non-template strands of mitochondrial DNA. HQ’s are transcription termination/replication initiation sites and thus guarantee an unperturbed replication of the mt-genome. This is of special importance in the insect-stage of the parasite. In the transcription-on state, the identified GQ’s require editing as a GQ-resolving activity indicating a link between replication, transcription and RNA editing. We propose that the different processes have coevolved and suggest the parasite life-cycle and the single mitochondrion as evolutionary driving forces. PMID:27436151

  13. A Paradigm Shift for the Amino Acid Editing Mechanism of Human Cytoplasmic Leucyl-tRNA Synthetase†

    PubMed Central

    Joy Pang, Yan Ling; Martinis, Susan A.

    2010-01-01

    Leucyl-tRNA synthetase (LeuRS) has been identified as a target for a novel class of boron-containing small molecules that bind to its editing active site. When the 3′ end of tRNALeu binds to the editing active site, the boron crosslinks to the cis diols of its terminal ribose. The crosslinked RNA-protein complex blocks the overall aminoacylation activity of the enzyme. Similar to other LeuRSs, the human cytoplasmic enzyme (hscLeuRS) editing active site resides in a discrete domain called the connective polypeptide 1 domain (CP1), where mischarged tRNA binds for hydrolysis of the noncognate amino acid. The editing site of hscLeuRS includes a highly conserved threonine discriminator and universally conserved aspartic acid that were mutationally characterized. Substitution of the threonine residue to alanine uncoupled specificity similar to other LeuRSs. However, the introduction of bulky residues in the amino acid binding pocket failed to block deacylation of tRNA, indicating that the architecture of the amino acid binding pocket is different compared to other characterized LeuRSs. In addition, mutation of the universally conserved aspartic acid abolished tRNALeu deacylation. Surprisingly though, this editing-defective hscLeuRS maintained fidelity. It is possible that an alternate editing mechanism may have been activated upon failure of the post-transfer editing active site. PMID:19702327

  14. RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering1[OPEN

    PubMed Central

    Germain, Arnaud

    2016-01-01

    Plant RNA editosomes modify cytidines (C) to uridines (U) at specific sites in plastid and mitochondrial transcripts. Members of the RNA-editing factor interacting protein (RIP) family and Organelle RNA Recognition Motif-containing (ORRM) family are essential components of the Arabidopsis (Arabidopsis thaliana) editosome. ORRM2 and ORRM3 have been recently identified as minor mitochondrial editing factors whose silencing reduces editing efficiency at ∼6% of the mitochondrial C targets. Here we report the identification of ORRM4 (for organelle RRM protein 4) as a novel, major mitochondrial editing factor that controls ∼44% of the mitochondrial editing sites. C-to-U conversion is reduced, but not eliminated completely, at the affected sites. The orrm4 mutant exhibits slower growth and delayed flowering time. ORRM4 affects editing in a site-specific way, though orrm4 mutation affects editing of the entire transcript of certain genes. ORRM4 contains an RRM domain at the N terminus and a Gly-rich domain at the C terminus. The RRM domain provides the editing activity of ORRM4, whereas the Gly-rich domain is required for its interaction with ORRM3 and with itself. The presence of ORRM4 in the editosome is further supported by its interaction with RIP1 in a bimolecular fluorescence complementation assay. The identification of ORRM4 as a major mitochondrial editing factor further expands our knowledge of the composition of the RNA editosome and reveals that adequate mitochondrial editing is necessary for normal plant development. PMID:26578708

  15. A core MRB1 complex component is indispensable for RNA editing in insect and human infective stages of Trypanosoma brucei.

    PubMed

    Ammerman, Michelle L; Tomasello, Danielle L; Faktorová, Drahomíra; Kafková, Lucie; Hashimi, Hassan; Lukeš, Julius; Read, Laurie K

    2013-01-01

    Uridine insertion/deletion RNA editing is a unique and vital process in kinetoplastids, required for creation of translatable open reading frames in most mitochondrially-encoded RNAs. Emerging as a key player in this process is the mitochondrial RNA binding 1 (MRB1) complex. MRB1 comprises an RNA-independent core complex of at least six proteins, including the GAP1/2 guide RNA (gRNA) binding proteins. The core interacts in an RNA-enhanced or -dependent manner with imprecisely defined TbRGG2 subcomplexes, Armadillo protein MRB10130, and additional factors that comprise the dynamic MRB1 complex. Towards understanding MRB1 complex function in RNA editing, we present here functional characterization of the pentein domain-containing MRB1 core protein, MRB11870. Inducible RNAi studies demonstrate that MRB11870 is essential for proliferation of both insect vector and human infective stage T. brucei. MRB11870 ablation causes a massive defect in RNA editing, affecting both pan-edited and minimally edited mRNAs, but does not substantially affect mitochondrial RNA stability or processing of precursor transcripts. The editing defect in MRB1-depleted cells occurs at the initiation stage of editing, as pre-edited mRNAs accumulate. However, the gRNAs that direct editing remain abundant in the knockdown cells. To examine the contribution of MRB11870 to MRB1 macromolecular interactions, we tagged core complexes and analyzed their composition and associated proteins in the presence and absence of MRB11870. These studies demonstrated that MRB11870 is essential for association of GAP1/2 with the core, as well as for interaction of the core with other proteins and subcomplexes. Together, these data support a model in which the MRB1 core mediates functional interaction of gRNAs with the editing machinery, having GAP1/2 as its gRNA binding constituents. MRB11870 is a critical component of the core, essential for its structure and function. PMID:24250748

  16. Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module.

    PubMed

    Yan, Wei; Ye, Qing; Tan, Min; Chen, Xi; Eriani, Gilbert; Wang, En-Duo

    2015-05-01

    A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS. PMID:25817995

  17. Editing for an AMPA receptor subunit RNA in prefrontal cortex and striatum in Alzheimer's disease, Huntington's disease and schizophrenia

    NASA Technical Reports Server (NTRS)

    Akbarian, S.; Smith, M. A.; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    Animal studies and cell culture experiments demonstrated that posttranscriptional editing of the transcript of the GluR-2 gene, resulting in substitution of an arginine for glutamine in the second transmembrane region (TM II) of the expressed protein, is associated with a reduction in Ca2+ permeability of the receptor channel. Thus, disturbances in GluR-2 RNA editing with alteration of intracellular Ca2+ homeostasis could lead to neuronal dysfunction and even neuronal degeneration. The present study determined the proportions of edited and unedited GluR-2 RNA in the prefrontal cortex of brains from patients with Alzheimer's disease, in the striatum of brains from patients with Huntington's disease, and in the same areas of brains from age-matched schizophrenics and controls, by using reverse transcriptase-polymerase chain reaction, restriction endonuclease digestion, gel electrophoresis and scintillation radiometry. In the prefrontal cortex of controls, < 0.1% of all GluR-2 RNA molecules were unedited and > 99.9% were edited; in the prefrontal cortex both of schizophrenics and of Alzheimer's patients approximately 1.0% of all GluR-2 RNA molecules were unedited and 99% were edited. In the striatum of controls and of schizophrenics, approximately 0.5% of GluR-2 RNA molecules were unedited and 99.5% were edited; in the striatum of Huntington's patients nearly 5.0% of GluR-2 RNA was unedited. In the prefrontal white matter of controls, approximately 7.0% of GluR-2 RNA was unedited. In the normal human prefrontal cortex and striatum, the large majority of GluR-2 RNA molecules contains a CGG codon for arginine in the TMII coding region; this implies that the corresponding AMPA receptors have a low Ca2+ permeability, as previously demonstrated for the rat brain. The process of GluR-2 RNA editing is compromised in a region-specific manner in schizophrenia, in Alzheimer's disease and Huntington's Chorea although in each of these disorders there is still a large excess of

  18. Chemical modification of nucleotide bases and mRNA editing depend on hexamer or nucleoprotein phase in Sendai virus nucleocapsids.

    PubMed Central

    Iseni, Frédéric; Baudin, Florence; Garcin, Dominique; Marq, Jean-Baptiste; Ruigrok, Rob W H; Kolakofsky, Daniel

    2002-01-01

    The minus-strand genome of Sendai virus is an assembly of the nucleocapsid protein (N) and RNA, in which each N subunit is associated with precisely 6 nt. Only genomes that are a multiple of 6 nt long replicate efficiently or are found naturally, and their replication promoters contain sequence elements with hexamer repeats. Paramyxoviruses that are governed by this hexamer rule also edit their P gene mRNA during its synthesis, by G insertions, via a controlled form of viral RNA polymerase "stuttering" (pseudo-templated transcription). This stuttering is directed by a cis-acting sequence (3' UNN UUUUUU CCC), whose hexamer phase is conserved within each virus group. To determine whether the hexamer phase of a given nucleotide sequence within nucleocapsids affected its sensitivity to chemical modification, and whether hexamer phase of the mRNA editing site was important for the editing process, we prepared a matched set of viruses in which a model editing site was displaced 1 nt at a time relative to the genome ends. The relative abilities of these Sendai viruses to edit their mRNAs in cell culture infections were examined, and the ability of DMS to chemically modify the nucleotides of this cis-acting signal within resting viral nucleocapsids was also studied. Cytidines at hexamer phases 1 and 6 were the most accessible to chemical modification, whereas mRNA editing was most extensive when the stutter-site C was in positions 2 to 5. Apparently, the N subunit imprints the nucleotide sequence it is associated with, and affects both the initiation of viral RNA synthesis and mRNA editing. The N-subunit assembly thus appears to superimpose another code upon the genetic code. PMID:12212849

  19. dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing.

    PubMed Central

    Palladino, M J; Keegan, L P; O'Connell, M A; Reenan, R A

    2000-01-01

    We have identified a homolog of the ADAR (adenosine deaminases that act on RNA) class of RNA editases from Drosophila, dADAR. The dADAR locus has been localized to the 2B6-7 region of the X chromosome and the complete genomic sequence organization is reported here. dADAR is most homologous to the mammalian RNA editing enzyme ADAR2, the enzyme that specifically edits the Q/R site in the pre-mRNA encoding the glutamate receptor subunit GluR-B. Partially purified dADAR expressed in Pichia pastoris has robust nonspecific A-to-I deaminase activity on synthetic dsRNA substrates. Transcripts of the dADAR locus originate from two regulated promoters. In addition, alternative splicing generates at least four major dADAR isoforms that differ at their amino-termini as well as altering the spacing between their dsRNA binding motifs. dADAR is expressed in the developing nervous system, making it a candidate for the editase that acts on para voltage-gated Na+ channel transcripts in the central nervous system. Surprisingly, dADAR itself undergoes developmentally regulated RNA editing that changes a conserved residue in the catalytic domain. Taken together, these findings show that both transcription and processing of dADAR transcripts are under strict developmental control and suggest that the process of RNA editing in Drosophila is dynamically regulated. PMID:10917596

  20. Analysis of the Resistance Mechanism of a Benzoxaborole Inhibitor Reveals Insight into the Leucyl-tRNA Synthetase Editing Mechanism.

    PubMed

    Zhao, Hanchao; Palencia, Andres; Seiradake, Elena; Ghaemi, Zhaleh; Cusack, Stephen; Luthey-Schulten, Zaida; Martinis, Susan

    2015-10-16

    A new class of antimicrobial benzoxaborole compounds was identified as a potent inhibitor of leucyl-tRNA synthetase (LeuRS) and therefore of protein synthesis. In a novel mechanism, AN2690 (5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole) blocks fungal cytoplasmic LeuRS by covalently trapping tRNA(Leu) in the editing site of the enzyme's CP1 domain. However, some resistant mutation sites are located outside of the CP1 hydrolytic editing active site. Thus, their mode of action that undermines drug inhibition was not understood. A combination of X-ray crystallography, molecular dynamics, metadynamics, biochemical experiments, and mutational analysis of a distal benzoxaborole-resistant mutant uncovered a eukaryote-specific tyrosine "switch" that is critical to tRNA-dependent post-transfer editing. The tyrosine "switch" has three states that shift between interactions with a lysine and the 3'-hydroxyl of the tRNA terminus, to inhibit or promote post-transfer editing. The oxaborole's mechanism of action capitalizes upon one of these editing active site states. This tunable editing mechanism in eukaryotic and archaeal LeuRSs is proposed to facilitate precise quality control of aminoacylation fidelity. These mechanistic distinctions could also be capitalized upon for development of the benzoxaboroles as a broad spectrum antibacterial. PMID:26172575

  1. Site-Directed RNA Editing in Vivo Can Be Triggered by the Light-Driven Assembly of an Artificial Riboprotein

    PubMed Central

    2015-01-01

    Site-directed RNA editing allows for the manipulation of RNA and protein function by reprogramming genetic information at the RNA level. For this we assemble artificial RNA-guided editases and demonstrate their transcript repair activity in cells and in developing embryos of the annelid Platynereis dumerilii. A hallmark of our assembly strategy is the covalent attachment of guideRNA and editing enzyme by applying the SNAP-tag technology, a process that we demonstrate here to be readily triggered by light in vitro, in mammalian cell culture, and also in P. dumerilii. Lacking both sophisticated chemistry and extensive genetic engineering, this technology provides a convenient route for the light-dependent switching of protein isoforms. The presented strategy may also serve as a blue-print for the engineering of addressable machineries that apply tailored nucleic acid analogues to manipulate RNA or DNA site-specifically in living organisms. PMID:26594902

  2. The majority of transcripts in the squid nervous system are extensively recoded by A-to-I RNA editing

    PubMed Central

    Alon, Shahar; Garrett, Sandra C; Levanon, Erez Y; Olson, Sara; Graveley, Brenton R; Rosenthal, Joshua J C; Eisenberg, Eli

    2015-01-01

    RNA editing by adenosine deamination alters genetic information from the genomic blueprint. When it recodes mRNAs, it gives organisms the option to express diverse, functionally distinct, protein isoforms. All eumetazoans, from cnidarians to humans, express RNA editing enzymes. However, transcriptome-wide screens have only uncovered about 25 transcripts harboring conserved recoding RNA editing sites in mammals and several hundred recoding sites in Drosophila. These studies on few established models have led to the general assumption that recoding by RNA editing is extremely rare. Here we employ a novel bioinformatic approach with extensive validation to show that the squid Doryteuthis pealeii recodes proteins by RNA editing to an unprecedented extent. We identify 57,108 recoding sites in the nervous system, affecting the majority of the proteins studied. Recoding is tissue-dependent, and enriched in genes with neuronal and cytoskeletal functions, suggesting it plays an important role in brain physiology. DOI: http://dx.doi.org/10.7554/eLife.05198.001 PMID:25569156

  3. Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase.

    PubMed Central

    O'Connell, M A; Krause, S; Higuchi, M; Hsuan, J J; Totty, N F; Jenny, A; Keller, W

    1995-01-01

    Double-stranded RNA (dsRNA)-specific adenosine deaminase converts adenosine to inosine in dsRNA. The protein has been purified from calf thymus, and here we describe the cloning of cDNAs encoding both the human and rat proteins as well as a partial bovine clone. The human and rat clones are very similar at the amino acid level except at their N termini and contain three dsRNA binding motifs, a putative nuclear targeting signal, and a possible deaminase motif. Antibodies raised against the protein encoded by the partial bovine clone specifically recognize the calf thymus dsRNA adenosine deaminase. Furthermore, the antibodies can immunodeplete a calf thymus extract of dsRNA adenosine deaminase activity, and the activity can be restored by addition of pure bovine deaminase. Staining of HeLa cells confirms the nuclear localization of the dsRNA-specific adenosine deaminase. In situ hybridization in rat brain slices indicates a widespread distribution of the enzyme in the brain. PMID:7862132

  4. Editing of the serotonin 2C receptor pre-mRNA: Effects of the Morris Water Maze.

    PubMed

    Du, Yunzhi; Stasko, Melissa; Costa, Alberto C; Davisson, Muriel T; Gardiner, Katheleen J

    2007-04-15

    The pre-mRNA encoding the serotonin 2C receptor, HTR2C (official mouse gene symbol, Htr2c), is subject to adenosine deamination that produces inosine at five sites within the coding region. Combinations of this site-specific A-to-I editing can produce 32 different mRNA sequences encoding 24 different protein isoforms with differing biochemical and pharmacological properties. Studies in humans have reported abnormalities in patterns of HTR2C editing in psychiatric disorders, and studies in rodents show altered patterns of editing in response to drug treatments and stressful situations. To further explore the biological significance of editing of the Htr2c mRNA and its regulation, we have examined patterns of Htr2c editing in C57BL/6J mice after exposure to the hidden platform version of the Morris Water Maze, a test of spatial learning that, in mice, is also associated with stress. In brains of both swimming controls and mice trained to find the platform, subtle time dependent changes in editing patterns are seen as soon as 1 h after a probe trial and typically last less than 24 h. Changes in whole brain with cerebellum removed differ from those seen in isolated hippocampus and cortex. Unexpectedly, in hippocampi from subsets of mice, abnormally low levels of editing were seen that were not correlated with behavior or with editing levels in cortex. These data implicate responses to spatial learning and stress, in addition to stochastic processes, in the generation of subtle changes in editing patterns of Htr2c. PMID:17307311

  5. Editing of the Serotonin 2C Receptor Pre-mRNA: Effects of the Morris Water Maze

    PubMed Central

    Du, Yunzhi; Stasko, Melissa; Costa, Alberto C.; Davisson, Muriel T.; Gardiner, Katheleen J.

    2007-01-01

    The pre-mRNA encoding the serotonin 2C receptor, HTR2C (official mouse gene symbol, Htr2c), is subject to adenosine deamination that produces inosine at five sites within the coding region. Combinations of this site-specific A-to-I editing can produce 32 different mRNA sequences encoding 24 different protein isoforms with differing biochemical and pharmacological properties. Studies in humans have reported abnormalities in patterns of HTR2C editing in psychiatric disorders, and studies in rodents show altered patterns of editing in response to drug treatments and stressful situations. To further explore the biological significance of editing of the Htr2c mRNA and its regulation, we have examined patterns of Htr2c editing in C57BL/6J mice after exposure to the hidden platform version of the Morris Water Maze, a test of spatial learning that, in mice, is also associated with stress. In brains of both swimming controls and mice trained to find the platform, subtle time dependent changes in editing patterns are seen as soon as one hour after a probe trial and typically last less than 24 hours. Changes in whole brain with cerebellum removed differ from those seen in isolated hippocampus and cortex. Unexpectedly, in hippocampi from subsets of mice, abnormally low levels of editing were seen that were not correlated with behavior or with editing levels in cortex. These data implicate responses to spatial learning and stress, in addition to stochastic processes, in the generation of subtle changes in editing patterns of Htr2c. PMID:17307311

  6. Sequence elements critical for efficient RNA editing of a tobacco chloroplast transcript in vivo and in vitro

    PubMed Central

    Hayes, Michael L.; Reed, Martha L.; Hegeman, Carla E.; Hanson, Maureen R.

    2006-01-01

    In tobacco chloroplast transcripts 34 nt are efficiently edited to U. No common consensus region is present around all editing sites; however, sites can be grouped in clusters that share short common sequences. Transgene transcripts carrying either the wild-type −31/+22 or −31/+60 sequence near NTrpoB C473, an editing site within tobacco rpoB transcripts, or three different mutated sequences, were all highly edited in vivo. Endogenous transcripts of rpoB, psbL and rps14, all of which contain common sequences S1, S2 and S3 5′ to NTrpoB C473, NTpsbL C2 and NTrps14 C80, were less edited in transgenic plants that over-express transcripts from NTrpoB C473 transgenes. Extent of reduction of endogenous editing differed between transgenic lines expressing mutated −31/+22 regions, depending on the abundance of the transgene transcripts. The −20/−5 sequence contains critical 5′ sequence elements. Synthetic RNA templates with alterations within this 5′ region were less efficiently edited in vitro than wild-type templates, by either tobacco or maize chloroplast extracts. The tobacco chloroplast extract supports both RNA editing and processing of 3′ transcript termini. We conclude that within the −20/−5 region, sequences common to editing sites in the transcripts of rpoB, psbL and rps14 are critical for efficient NTrpoB C473 editing. PMID:16893957

  7. Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module*

    PubMed Central

    Yan, Wei; Ye, Qing; Tan, Min; Chen, Xi; Eriani, Gilbert; Wang, En-Duo

    2015-01-01

    A conserved structural module following the KMSKS catalytic loop exhibits α-α-β-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNALeu, which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the β-strand enhance the editing activity of EcLeuRS and sense the size of the tRNALeu D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS. PMID:25817995

  8. p53-Dependent DNA damage response sensitive to editing-defective tRNA synthetase in zebrafish.

    PubMed

    Song, Youngzee; Shi, Yi; Carland, Tristan M; Lian, Shanshan; Sasaki, Tomoyuki; Schork, Nicholas J; Head, Steven R; Kishi, Shuji; Schimmel, Paul

    2016-07-26

    Brain and heart pathologies are caused by editing defects of transfer RNA (tRNA) synthetases, which preserve genetic code fidelity by removing incorrect amino acids misattached to tRNAs. To extend understanding of the broader impact of synthetase editing reactions on organismal homeostasis, and based on effects in bacteria ostensibly from small amounts of mistranslation of components of the replication apparatus, we investigated the sensitivity to editing of the vertebrate genome. We show here that in zebrafish embryos, transient overexpression of editing-defective valyl-tRNA synthetase (ValRS(ED)) activated DNA break-responsive H2AX and p53-responsive downstream proteins, such as cyclin-dependent kinase (CDK) inhibitor p21, which promotes cell-cycle arrest at DNA damage checkpoints, and Gadd45 and p53R2, with pivotal roles in DNA repair. In contrast, the response of these proteins to expression of ValRS(ED) was abolished in p53-deficient fish. The p53-activated downstream signaling events correlated with suppression of abnormal morphological changes caused by the editing defect and, in adults, reversed a shortened life span (followed for 2 y). Conversely, with normal editing activities, p53-deficient fish have a normal life span and few morphological changes. Whole-fish deep sequencing showed genomic mutations associated with the editing defect. We suggest that the sensitivity of p53 to expression of an editing-defective tRNA synthetase has a critical role in promoting genome integrity and organismal homeostasis. PMID:27402763

  9. Genome-wide identification and characterization of tissue-specific RNA editing events in D. melanogaster and their potential role in regulating alternative splicing

    PubMed Central

    Mazloomian, Alborz; Meyer, Irmtraud M

    2015-01-01

    RNA editing is a widespread mechanism that plays a crucial role in diversifying gene products. Its abundance and importance in regulating cellular processes were revealed using new sequencing technologies. The majority of these editing events, however, cannot be associated with regulatory mechanisms. We use tissue-specific high-throughput libraries of D. melanogaster to study RNA editing. We introduce an analysis pipeline that utilises large input data and explicitly captures ADAR's requirement for double-stranded regions. It combines probabilistic and deterministic filters and can identify RNA editing events with a low estimated false positive rate. Analyzing ten different tissue types, we predict 2879 editing sites and provide their detailed characterization. Our analysis pipeline accurately distinguishes genuine editing sites from SNPs and sequencing and mapping artifacts. Our editing sites are 3 times more likely to occur in exons with multiple splicing acceptor/donor sites than in exons with unique splice sites (p-value < 2.10−15). Furthermore, we identify 244 edited regions where RNA editing and alternative splicing are likely to influence each other. For 96 out of these 244 regions, we find evolutionary evidence for conserved RNA secondary-structures near splice sites suggesting a potential regulatory mechanism where RNA editing may alter splicing patterns via changes in local RNA structure. PMID:26512413

  10. Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase.

    PubMed

    Sasaki, Hiroshi M; Sekine, Shun-ichi; Sengoku, Toru; Fukunaga, Ryuya; Hattori, Motoyuki; Utsunomiya, Yukiko; Kuroishi, Chizu; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2006-10-01

    To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the "editing" mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the beta subunit. We report the crystal structure of an N-terminal fragment of the PheRS beta subunit (PheRS-beta(N)) from the archaeon, Pyrococcus horikoshii, at 1.94-A resolution. PheRS-beta(N) includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNA(Phe), indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3'-terminal adenosine, reduced Tyr-tRNA(Phe) deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNA(Phe) deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis. PMID:17003130

  11. Enhancement of alcohol drinking in mice depends on alterations in RNA editing of serotonin 2C receptors

    PubMed Central

    Watanabe, Yoshihisa; Yoshimoto, Kanji; Tatebe, Harutsugu; Kita, Masakazu; Nishikura, Kazuko; Kimura, Minoru; Tanaka, Masaki

    2014-01-01

    Serotonin 2C receptors (5-HT2CR) are G-protein-coupled receptors with various actions, including involvement in drug addiction. 5-HT2CR undergoes mRNA editing, converting genomically encoded adenosine residues to inosines via adenosine deaminases acting on RNA (ADARs). Here we show that enhanced alcohol drinking behaviour in mice is associated with the degree of 5-HT2CR mRNA editing in the nucleus accumbens and dorsal raphe nuceus, brain regions important for reward and addiction. Following chronic alcohol vapour exposure, voluntary alcohol intake increased in C57BL/6J mice, but remained unchanged in C3H/HeJ and DBA/2J mice. 5-HT2CR mRNA editing frequency in both regions increased significantly in C57BL/6J mice, as did expressions of 5-HT2CR, ADAR1 and ADAR2, but not in other strains. Moreover, mice that exclusively express the unedited isoform (INI) of 5-HT2CR mRNA on a C57BL/6J background did not exhibit increased alcohol intake compared with wild-type mice. Our results indicate that alterations in 5-HT2CR mRNA editing underlie alcohol preference in mice. PMID:24345557

  12. Extensive RNA editing of U to C in addition to C to U substitution in the rbcL transcripts of hornwort chloroplasts and the origin of RNA editing in green plants.

    PubMed Central

    Yoshinaga, K; Iinuma, H; Masuzawa, T; Uedal, K

    1996-01-01

    We cloned and sequenced a portion of chloroplast DNA from the hornwort Anthoceros formosae. A nucleotide sequence of 7556 bp contained structures similar to those of ndhK, ndhC, trnV, trnM, atpE, atpB, rbcL, trnR and accD. The arrangement of these was the same as that of other chloroplast DNA. However, two nonsense codons were located within the putative coding region of rbcL, although they were used as putative termination codons of the genes. RNA was extensively edited in the transcripts of rbcL when cDNA sequences were analyzed. The unusual nonsense codons of TGA and TAA became CGA and CAA respectively. These are examples of U to C type RNA editing, which was never been found before in chloroplast mRNA. In general, 13 Cs of genomic DNA were found as Ts in the cDNA sequence and seven Ts were found as Cs. This is the first finding of RNA editing on the transcripts of rbcL and also in bryophytes. This event had been thought to arise in land plants after the split of bryophytes. The origin of RNA editing is discussed in relation to the landing of green plants. PMID:8604330

  13. The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3

    PubMed Central

    Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal GP; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

    2015-01-01

    RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. PMID:26637431

  14. The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3.

    PubMed

    Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal G P; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

    2016-02-01

    RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. PMID:26637431

  15. Patterns of developmental expression of the RNA editing enzyme rADAR2.

    PubMed

    Paupard M-C; O'Connell, M A; Gerber, A P; Zukin, R S

    2000-01-01

    To date, two structurally related RNA-editing enzymes with adenosine deaminase activity have been identified in mammalian tissue: ADAR1 and ADAR2 [Bass B. I. et al. (1997) RNA 3, 947-949]. In rodents, ADAR2 undergoes alternative RNA splicing, giving rise to two splice variants that differ by the presence or absence of a 10-amino-acid insert in the carboxy-terminal catalytic domain. However, the physiological significance of the splicing and its regional and developmental regulation are as yet unknown. The present study examined spatial and temporal patterns of ADAR2 gene transcripts within specific neuronal populations of rat brain. The two rodent ADAR2 isoforms were expressed at comparable levels at all ages examined. rADAR2 messenger RNA expression was first detectable in the thalamic nuclei formation at embryonic day E19. The rADAR2b insert and rADAR2a splice probes produced images similar to that of the rADAR2 pan probe. At birth, rADAR2a messenger RNA splice variants were abundantly expressed in the thalamic nuclei. No signal for any probe was detectable in other brain regions, including neocortex, hippocampus, striatum and cerebellum at this stage of development. During the first week of postnatal life, rADAR2 messenger RNA expression (detected with the pan probe) increased gradually in several brain regions, with low expression detected at postnatal day P7 in the olfactory bulb, inferior colliculus, and within the pyramidal and granule cell layers of the hippocampus. Hybridization patterns of the rADAR2a variant probe reached peak expression at about the second week of life, while peak expression of the rADAR2b probe was reached at about the third week of life. At the end of the first week of life (P7), expression of both splice variants was strongest in the thalamic nuclei. By P14, rADAR2 messenger RNA expression was more consolidated in the deeper structures, including the thalamic nuclei and the granule cell layer of the cerebellum. By P21, maximal levels

  16. RED: A Java-MySQL Software for Identifying and Visualizing RNA Editing Sites Using Rule-Based and Statistical Filters

    PubMed Central

    Sun, Yongmei; Li, Xing; Wu, Di; Pan, Qi; Ji, Yuefeng; Ren, Hong; Ding, Keyue

    2016-01-01

    RNA editing is one of the post- or co-transcriptional processes that can lead to amino acid substitutions in protein sequences, alternative pre-mRNA splicing, and changes in gene expression levels. Although several methods have been suggested to identify RNA editing sites, there remains challenges to be addressed in distinguishing true RNA editing sites from its counterparts on genome and technical artifacts. In addition, there lacks a software framework to identify and visualize potential RNA editing sites. Here, we presented a software − ‘RED’ (RNA Editing sites Detector) − for the identification of RNA editing sites by integrating multiple rule-based and statistical filters. The potential RNA editing sites can be visualized at the genome and the site levels by graphical user interface (GUI). To improve performance, we used MySQL database management system (DBMS) for high-throughput data storage and query. We demonstrated the validity and utility of RED by identifying the presence and absence of C→U RNA-editing sites experimentally validated, in comparison with REDItools, a command line tool to perform high-throughput investigation of RNA editing. In an analysis of a sample data-set with 28 experimentally validated C→U RNA editing sites, RED had sensitivity and specificity of 0.64 and 0.5. In comparison, REDItools had a better sensitivity (0.75) but similar specificity (0.5). RED is an easy-to-use, platform-independent Java-based software, and can be applied to RNA-seq data without or with DNA sequencing data. The package is freely available under the GPLv3 license at http://github.com/REDetector/RED or https://sourceforge.net/projects/redetector. PMID:26930599

  17. RED: A Java-MySQL Software for Identifying and Visualizing RNA Editing Sites Using Rule-Based and Statistical Filters.

    PubMed

    Sun, Yongmei; Li, Xing; Wu, Di; Pan, Qi; Ji, Yuefeng; Ren, Hong; Ding, Keyue

    2016-01-01

    RNA editing is one of the post- or co-transcriptional processes that can lead to amino acid substitutions in protein sequences, alternative pre-mRNA splicing, and changes in gene expression levels. Although several methods have been suggested to identify RNA editing sites, there remains challenges to be addressed in distinguishing true RNA editing sites from its counterparts on genome and technical artifacts. In addition, there lacks a software framework to identify and visualize potential RNA editing sites. Here, we presented a software - 'RED' (RNA Editing sites Detector) - for the identification of RNA editing sites by integrating multiple rule-based and statistical filters. The potential RNA editing sites can be visualized at the genome and the site levels by graphical user interface (GUI). To improve performance, we used MySQL database management system (DBMS) for high-throughput data storage and query. We demonstrated the validity and utility of RED by identifying the presence and absence of C→U RNA-editing sites experimentally validated, in comparison with REDItools, a command line tool to perform high-throughput investigation of RNA editing. In an analysis of a sample data-set with 28 experimentally validated C→U RNA editing sites, RED had sensitivity and specificity of 0.64 and 0.5. In comparison, REDItools had a better sensitivity (0.75) but similar specificity (0.5). RED is an easy-to-use, platform-independent Java-based software, and can be applied to RNA-seq data without or with DNA sequencing data. The package is freely available under the GPLv3 license at http://github.com/REDetector/RED or https://sourceforge.net/projects/redetector. PMID:26930599

  18. Naturally occurring aminoacyl-tRNA synthetases editing-domain mutations that cause mistranslation in Mycoplasma parasites

    PubMed Central

    Li, Li; Boniecki, Michal T.; Jaffe, Jacob D.; Imai, Brian S.; Yau, Peter M.; Luthey-Schulten, Zaida A.; Martinis, Susan A.

    2011-01-01

    Mycoplasma parasites escape host immune responses via mechanisms that depend on remarkable phenotypic plasticity. Identification of these mechanisms is of great current interest. The aminoacyl-tRNA synthetases (AARSs) attach amino acids to their cognate tRNAs, but occasionally make errors that substitute closely similar amino acids. AARS editing pathways clear errors to avoid mistranslation during protein synthesis. We show here that AARSs in Mycoplasma parasites have point mutations and deletions in their respective editing domains. The deleterious effect on editing was confirmed with a specific example studied in vitro. In vivo mistranslation was determined by mass spectrometric analysis of proteins produced in the parasite. These mistranslations are uniform cases where the predicted closely similar amino acid replaced the correct one. Thus, natural AARS editing-domain mutations in Mycoplasma parasites cause mistranslation. We raise the possibility that these mutations evolved as a mechanism for antigen diversity to escape host defense systems. PMID:21606343

  19. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA

    PubMed Central

    Zhang, Yi; Liang, Zhen; Zong, Yuan; Wang, Yanpeng; Liu, Jinxing; Chen, Kunling; Qiu, Jin-Long; Gao, Caixia

    2016-01-01

    Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research. PMID:27558837

  20. Efficient and transgene-free genome editing in wheat through transient expression of CRISPR/Cas9 DNA or RNA.

    PubMed

    Zhang, Yi; Liang, Zhen; Zong, Yuan; Wang, Yanpeng; Liu, Jinxing; Chen, Kunling; Qiu, Jin-Long; Gao, Caixia

    2016-01-01

    Editing plant genomes is technically challenging in hard-to-transform plants and usually involves transgenic intermediates, which causes regulatory concerns. Here we report two simple and efficient genome-editing methods in which plants are regenerated from callus cells transiently expressing CRISPR/Cas9 introduced as DNA or RNA. This transient expression-based genome-editing system is highly efficient and specific for producing transgene-free and homozygous wheat mutants in the T0 generation. We demonstrate our protocol to edit genes in hexaploid bread wheat and tetraploid durum wheat, and show that we are able to generate mutants with no detectable transgenes. Our methods may be applicable to other plant species, thus offering the potential to accelerate basic and applied plant genome-engineering research. PMID:27558837

  1. ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity

    PubMed Central

    Robinson, J. E.; Paluch, J.; Dickman, D. K.; Joiner, W. J.

    2016-01-01

    It has been postulated that synaptic potentiation during waking is offset by a homoeostatic reduction in net synaptic strength during sleep. However, molecular mechanisms to support such a process are lacking. Here we demonstrate that deficiencies in the RNA-editing gene Adar increase sleep due to synaptic dysfunction in glutamatergic neurons in Drosophila. Specifically, the vesicular glutamate transporter is upregulated, leading to over-activation of NMDA receptors, and the reserve pool of glutamatergic synaptic vesicles is selectively expanded in Adar mutants. Collectively these changes lead to sustained neurotransmitter release under conditions that would otherwise result in synaptic depression. We propose that a shift in the balance from synaptic depression towards synaptic potentiation in sleep-promoting neurons underlies the increased sleep pressure of Adar-deficient animals. Our findings provide a plausible molecular mechanism linking sleep and synaptic plasticity. PMID:26813350

  2. RNA editing by ADARs is important for normal behavior in Caenorhabditis elegans.

    PubMed

    Tonkin, Leath A; Saccomanno, Lisa; Morse, Daniel P; Brodigan, Thomas; Krause, Michael; Bass, Brenda L

    2002-11-15

    Here we take advantage of the well-characterized and simple nervous system of Caenorhabditis elegans to further our understanding of the functions of RNA editing. We describe the two C.elegans ADAR genes, adr-1 and adr-2, and characterize strains containing homozygous deletions in each, or both, of these genes. We find that adr-1 is expressed in most, if not all, cells of the C.elegans nervous system and also in the developing vulva. Using chemotaxis assays, we show that both ADARs are important for normal behavior. Biochemical, molecular and phenotypic analyses indicate that ADR-1 and ADR-2 have distinct roles in C.elegans, but sometimes act together. PMID:12426375

  3. A method to convert mRNA into a gRNA library for CRISPR/Cas9 editing of any organism

    PubMed Central

    Arakawa, Hiroshi

    2016-01-01

    The clustered regularly interspersed palindromic repeats (CRISPR)/Cas9 (CRISPR-associated protein 9) system is a powerful tool for genome editing that can be used to construct a guide RNA (gRNA) library for genetic screening. For gRNA design, one must know the sequence of the 20-mer flanking the protospacer adjacent motif (PAM), which seriously impedes experimentally making gRNA. I describe a method to construct a gRNA library via molecular biology techniques without relying on bioinformatics. Briefly, one synthesizes complementary DNA from the mRNA sequence using a semi-random primer containing a PAM complementary sequence and then cuts out the 20-mer adjacent to the PAM using type IIS and type III restriction enzymes to create a gRNA library. The described approach does not require prior knowledge about the target DNA sequences, making it applicable to any species. PMID:27574704

  4. Affect-related Behaviors in Mice Misexpressing the RNA Editing Enzyme ADAR2

    PubMed Central

    Singh, Minati; Zimmerman, M. Bridget; Beltz, Terry G.; Johnson, Alan Kim

    2009-01-01

    Misediting of the serotonin (5HT) 2C receptor (5HT2CR) has been implicated in both depression and anxiety. The adenosine deaminases that act on double stranded RNAs (ADARs) are reported to modify the 5HT2CR by RNA editing. Transgenic mice misexpressing the RNA editing enzyme ADAR2 show an adult onset obese phenotype due to chronic hyperphagia, but little more than this is known about the behavior of these animals. The present experiments examined whether affect-associated behaviors are also altered in ADAR2 transgenic mice. Age- and weight-matched transgenic mice misexpressing ADAR2 were tested for signs of behavioral despair with the forced swim (FST) and tail suspension (TST) tests, and for anxiety by evaluating spontaneous exploration in a novel environment and by elevated plus maze performance. Plasma corticosterone was also determined by radioimmunoassay. Transgenic mice of both sexes displayed indications of increased behavioral despair on first exposures to the TST and the FST. Behavioral despair persisted in ADAR2 mice in that it was also observed in the FST in tests administered 24 hr and 1 week following the initial TST and FST. ADAR2 transgenic mice also displayed behaviors associated with anxiety as indicated by decreased entry into the open arms in an elevated plus maze test. Both sexes of ADAR2 transgenic mice displayed elevated plasma corticosterone. Taken together, the results suggest that ADAR2 transgenic mice represent a novel rodent model of endogenous behavioral despair and anxiety accompanied by elevated hypothalamo-pituitary adrenal axis activity. PMID:19361536

  5. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA.

    PubMed

    Svitashev, Sergei; Young, Joshua K; Schwartz, Christine; Gao, Huirong; Falco, S Carl; Cigan, A Mark

    2015-10-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future. PMID:26269544

  6. RNA Editing Genes Associated with Extreme Old Age in Humans and with Lifespan in C. elegans

    PubMed Central

    Puca, Annibale; Solovieff, Nadia; Kojima, Toshio; Wang, Meng C.; Melista, Efthymia; Meltzer, Micah; Fischer, Sylvia E. J.; Andersen, Stacy; Hartley, Stephen H.; Sedgewick, Amanda; Arai, Yasumichi; Bergman, Aviv; Barzilai, Nir; Terry, Dellara F.; Riva, Alberto; Anselmi, Chiara Viviani; Malovini, Alberto; Kitamoto, Aya; Sawabe, Motoji; Arai, Tomio; Gondo, Yasuyuki; Steinberg, Martin H.; Hirose, Nobuyoshi; Atzmon, Gil; Ruvkun, Gary; Baldwin, Clinton T.; Perls, Thomas T.

    2009-01-01

    Background The strong familiality of living to extreme ages suggests that human longevity is genetically regulated. The majority of genes found thus far to be associated with longevity primarily function in lipoprotein metabolism and insulin/IGF-1 signaling. There are likely many more genetic modifiers of human longevity that remain to be discovered. Methodology/Principal Findings Here, we first show that 18 single nucleotide polymorphisms (SNPs) in the RNA editing genes ADARB1 and ADARB2 are associated with extreme old age in a U.S. based study of centenarians, the New England Centenarian Study. We describe replications of these findings in three independently conducted centenarian studies with different genetic backgrounds (Italian, Ashkenazi Jewish and Japanese) that collectively support an association of ADARB1 and ADARB2 with longevity. Some SNPs in ADARB2 replicate consistently in the four populations and suggest a strong effect that is independent of the different genetic backgrounds and environments. To evaluate the functional association of these genes with lifespan, we demonstrate that inactivation of their orthologues adr-1 and adr-2 in C. elegans reduces median survival by 50%. We further demonstrate that inactivation of the argonaute gene, rde-1, a critical regulator of RNA interference, completely restores lifespan to normal levels in the context of adr-1 and adr-2 loss of function. Conclusions/Significance Our results suggest that RNA editors may be an important regulator of aging in humans and that, when evaluated in C. elegans, this pathway may interact with the RNA interference machinery to regulate lifespan. PMID:20011587

  7. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes

    PubMed Central

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  8. A survey of PPR proteins identifies DYW domains like those of land plant RNA editing factors in diverse eukaryotes.

    PubMed

    Schallenberg-Rüdinger, Mareike; Lenz, Henning; Polsakiewicz, Monika; Gott, Jonatha M; Knoop, Volker

    2013-01-01

    The pentatricopeptide repeat modules of PPR proteins are key to their sequence-specific binding to RNAs. Gene families encoding PPR proteins are greatly expanded in land plants where hundreds of them participate in RNA maturation, mainly in mitochondria and chloroplasts. Many plant PPR proteins contain additional carboxyterminal domains and have been identified as essential factors for specific events of C-to-U RNA editing, which is abundant in the two endosymbiotic plant organelles. Among those carboxyterminal domain additions to plant PPR proteins, the so-called DYW domain is particularly interesting given its similarity to cytidine deaminases. The frequency of organelle C-to-U RNA editing and the diversity of DYW-type PPR proteins correlate well in plants and both were recently identified outside of land plants, in the protist Naegleria gruberi. Here we present a systematic survey of PPR protein genes and report on the identification of additional DYW-type PPR proteins in the protists Acanthamoeba castellanii, Malawimonas jakobiformis, and Physarum polycephalum. Moreover, DYW domains were also found in basal branches of multi-cellular lineages outside of land plants, including the alga Nitella flexilis and the rotifers Adineta ricciae and Philodina roseola. Intriguingly, the well-characterized and curious patterns of mitochondrial RNA editing in the slime mold Physarum also include examples of C-to-U changes. Finally, we identify candidate sites for mitochondrial RNA editing in Malawimonas, further supporting a link between DYW-type PPR proteins and C-to-U editing, which may have remained hitherto unnoticed in additional eukaryote lineages. PMID:23899506

  9. Demonstration of mRNA editing and localization of guide RNA genes in kinetoplast-mitochondria of the plant trypanosomatid Phytomonas serpens.

    PubMed

    Maslov, D A; Hollar, L; Haghighat, P; Nawathean, P

    1998-06-01

    Maxicircle molecules of kDNA in several isolates of Phytomonas were detected by hybridization with the 12S rRNA gene probe from Leishmania tarentolae. The estimated size of maxicircles is isolate-specific and varies from 27 to 36 kb. Fully edited and polyadenylated mRNA for kinetoplast-encoded ribosomal protein S12 (RPS12) was found in the steady-state kinetoplast RNA isolated from Phytomonas serpens strain 1G. Two minicircles (1.45 kb) from this strain were also sequenced. Each minicircle contains two 120 bp conserved regions positioned 180 degrees apart, a region enriched with G and T bases and a variable region. One minicircle encodes a gRNA for the first block of editing of RPSl2 mRNA, and the other encodes a gRNA with unknown function. A gRNA gene for the second block of RPSl2 was found on a minicircle sequenced previously. On each minicircle, a gRNA gene is located in the variable region in a similar position and orientation with respect to the conserved regions. PMID:9662707

  10. The relationship between synthetic and editing functions of the active site of an aminoacyl-tRNA synthetase.

    PubMed Central

    Kim, H Y; Ghosh, G; Schulman, L H; Brunie, S; Jakubowski, H

    1993-01-01

    We have analyzed, by site-directed mutagenesis, the molecular basis of the editing function and its relation to the synthetic function of Escherichia coli methionyl-tRNA synthetase. The data obtained fit a model of the active site that partitions an amino acid substrate between synthetic and editing pathways. Hydrophobic and hydrogen bonding interactions direct the cognate substrate methionine through the synthetic pathway and prevent it from entering the editing pathway. Two hydrophobic interactions are proposed: between the side chain of Trp-305 and a methyl group of methionine and between the benzene ring of Tyr-15 and the beta- and gamma-CH2 groups of the substrate. An essential hydrogen bond forms between the OH of Tyr-15 and an electron pair of the sulfur atom of methionine. Consistent with these functions, side chains of Trp-305 and Tyr-15 are localized on opposite sides of the cavity forming a putative methionine binding pocket that is observed in the three-dimensional crystallographic structure of methionyl-tRNA synthetase. Enzymes W305A, Y15A, and Y15F have diminished ability to discriminate against homocysteine in the synthetic reaction, compared to the wild-type enzyme. At the same time, mutant enzymes have lost the ability to discriminate against methionine in the editing reaction and edited Met-AMP to a similar extent as Hcy-AMP. Interactions of residues Arg-233 and Asp-52 of methionyl-tRNA synthetase with the carboxyl and amino groups, respectively, of the substrate, which are essential for the synthetic function, were also essential for the editing function of the enzyme. Deacylation of Met-tRNA to S-methylhomocysteine thiolactone catalyzed by W305A, Y15A, and Y15F mutant enzymes was only slightly impaired relative to the wild-type enzyme. However, enzymes R233Q, R233A, and D52A did not deacylate Met-tRNA. The model also explains why the noncognate homocysteine is edited by methionyl-tRNA synthetase. PMID:8265588

  11. Region-specific alterations of A-to-I RNA editing of serotonin 2c receptor in the cortex of suicides with major depression.

    PubMed

    Weissmann, D; van der Laan, S; Underwood, M D; Salvetat, N; Cavarec, L; Vincent, L; Molina, F; Mann, J J; Arango, V; Pujol, J F

    2016-01-01

    Brain region-specific abnormalities in serotonergic transmission appear to underlie suicidal behavior. Alterations of RNA editing on the serotonin receptor 2C (HTR2C) pre-mRNA in the brain of suicides produce transcripts that attenuate 5-HT2CR signaling by impairing intracellular G-protein coupling and subsequent intracellular signal transduction. In brain, the distribution of RNA-editing enzymes catalyzing deamination (A-to-I modification) shows regional variation, including within the cerebral cortex. We tested the hypothesis that altered pre-mRNA 5-HT2CR receptor editing in suicide is region-specific. To this end, we investigated the complete 5-HT2CR mRNA-editing profile in two architectonically distinct cortical areas involved in mood regulation and decision-making in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicides. By using an original biochemical detection method, that is, capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), we corroborated the 5-HT2CR mRNA-editing profile previously described in the dorsolateral prefrontal cortex (Brodmann area 9 (BA9)). Editing of 5-HT2CR mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Compared with non-psychiatric control individuals, alterations of editing levels of 5-HT2CR mRNA were detected in both cortical areas of depressed suicides. A marked increase in editing on 5-HT2CR was especially observed in the anterior cingulate cortex in suicides, implicating this cortical area in suicide risk. The results suggest that region-specific changes in RNA editing of 5-HT2CR mRNA and deficient receptor function likely contribute to the etiology of major depressive disorder or suicide. PMID:27576167

  12. Leucyl-tRNA synthetase editing domain functions as a molecular rheostat to control codon ambiguity in Mycoplasma pathogens

    PubMed Central

    Li, Li; Palencia, Andrés; Lukk, Tiit; Li, Zhi; Luthey-Schulten, Zaida A.; Cusack, Stephen; Martinis, Susan A.; Boniecki, Michal T.

    2013-01-01

    Mycoplasma leucyl-tRNA synthetases (LeuRSs) have been identified in which the connective polypeptide 1 (CP1) amino acid editing domain that clears mischarged tRNAs are missing (Mycoplasma mobile) or highly degenerate (Mycoplasma synoviae). Thus, these enzymes rely on a clearance pathway called pretransfer editing, which hydrolyzes misactivated aminoacyl-adenylate intermediate via a nebulous mechanism that has been controversial for decades. Even as the sole fidelity pathway for clearing amino acid selection errors in the pathogenic M. mobile, pretransfer editing is not robust enough to completely block mischarging of tRNALeu, resulting in codon ambiguity and statistical proteins. A high-resolution X-ray crystal structure shows that M. mobile LeuRS structurally overlaps with other LeuRS cores. However, when CP1 domains from different aminoacyl-tRNA synthetases and origins were fused to this common LeuRS core, surprisingly, pretransfer editing was enhanced. It is hypothesized that the CP1 domain evolved as a molecular rheostat to balance multiple functions. These include distal control of specificity and enzyme activity in the ancient canonical core, as well as providing a separate hydrolytic active site for clearing mischarged tRNA. PMID:23431144

  13. Structural and mutational studies of the amino acid-editing domain from archaeal/eukaryal phenylalanyl-tRNA synthetase

    PubMed Central

    Sasaki, Hiroshi M.; Sekine, Shun-ichi; Sengoku, Toru; Fukunaga, Ryuya; Hattori, Motoyuki; Utsunomiya, Yukiko; Kuroishi, Chizu; Kuramitsu, Seiki; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2006-01-01

    To achieve accurate aminoacylation of tRNAs with their cognate amino acids, errors in aminoacylation are corrected by the “editing” mechanism in several aminoacyl-tRNA synthetases. Phenylalanyl-tRNA synthetase (PheRS) hydrolyzes, or edits, misformed tyrosyl-tRNA with its editing domain in the β subunit. We report the crystal structure of an N-terminal fragment of the PheRS β subunit (PheRS-βN) from the archaeon, Pyrococcus horikoshii, at 1.94-Å resolution. PheRS-βN includes the editing domain B3/4, which has archaea/eukarya-specific insertions/deletions and adopts a different orientation relative to other domains, as compared with that of bacterial PheRS. Surprisingly, most residues constituting the editing active-site pocket were substituted between the archaeal/eukaryal and bacterial PheRSs. We prepared Ala-substituted mutants of P. horikoshii PheRS for 16 editing-pocket residues, of which 12 are archaea/eukarya-specific and four are more widely conserved. On the basis of their activities, Tyr-adenosine was modeled on the B3/4-domain structure. First, the mutations of Leu-202, Ser-211, Asp-234, and Thr-236 made the PheRS incorrectly hydrolyze the cognate Phe-tRNAPhe, indicating that these residues participate in the Tyr hydroxy group recognition and are responsible for discrimination against Phe. Second, the mutations of Leu-168 and Arg-223, which could interact with the tRNA 3′-terminal adenosine, reduced Tyr-tRNAPhe deacylation activity. Third, the mutations of archaea/eukarya-specific Gln-126, Glu-127, Arg-137, and Asn-217, which are proximal to the ester bond to be cleaved, also reduced Tyr-tRNAPhe deacylation activity. In particular, the replacement of Asn-217 abolished the activity, revealing its absolute requirement for the catalysis. PMID:17003130

  14. Structural and Kinetic Characterization of Escherichia coli TadA, the Wobble-Specific tRNA Deaminase

    SciTech Connect

    Kim,J.; Malashkevich, V.; Roday, S.; Lisbin, M.; Schramm, V.; Almo, S.

    2006-01-01

    The essential tRNA-specific adenosine deaminase catalyzes the deamination of adenosine to inosine at the wobble position of tRNAs. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C, or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. We report the first combined structural and kinetic characterization of a wobble-specific deaminase. The structure of the Escherichia coli enzyme clearly defines the dimer interface and the coordination of the catalytically essential zinc ion. The structure also identifies the nucleophilic water and highlights residues near the catalytic zinc likely to be involved in recognition and catalysis of polymeric RNA substrates. A minimal 19 nucleotide RNA stem substrate has permitted the first steady-state kinetic characterization of this enzyme (k{sub cat} = 13 {+-} 1 min{sup -1} and K{sub M} = 0.83 {+-} 0.22 {micro}M). A continuous coupled assay was developed to follow the reaction at high concentrations of polynucleotide substrates (>10 {micro}M). This work begins to define the chemical and structural determinants responsible for catalysis and substrate recognition and lays the foundation for detailed mechanistic analysis of this essential enzyme.

  15. The Epitranscriptome and Innate Immunity

    PubMed Central

    O’Connell, Mary A.; Mannion, Niamh M.; Keegan, Liam P.

    2015-01-01

    Our knowledge of the variety and abundances of RNA base modifications is rapidly increasing. Modified bases have critical roles in tRNAs, rRNAs, translation, splicing, RNA interference, and other RNA processes, and are now increasingly detected in all types of transcripts. Can new biological principles associated with this diversity of RNA modifications, particularly in mRNAs and long non-coding RNAs, be identified? This review will explore this question by focusing primarily on adenosine to inosine (A-to-I) RNA editing by the adenine deaminase acting on RNA (ADAR) enzymes that have been intensively studied for the past 20 years and have a wide range of effects. Over 100 million adenosine to inosine editing sites have been identified in the human transcriptome, mostly in embedded Alu sequences that form potentially innate immune-stimulating dsRNA hairpins in transcripts. Recent research has demonstrated that inosine in the epitranscriptome and ADAR1 protein establish innate immune tolerance for host dsRNA formed by endogenous sequences. Innate immune sensors that detect viral nucleic acids are among the readers of epitranscriptome RNA modifications, though this does preclude a wide range of other modification effects. PMID:26658668

  16. Striking differences in RNA editing requirements to express the rps4 gene in magnolia and sunflower mitochondria.

    PubMed

    Regina, Teresa M R; Lopez, Loredana; Picardi, Ernesto; Quagliariello, Carla

    2002-03-01

    The ribosomal protein S4 gene (rps4) has been identified as a single copy sequence in the mitochondrial genomes of two distant higher plants, Magnolia and Helianthus. Sequence analysis revealed that the rps4 genes present in the magnolia and sunflower mitochondrial genomes encode S4 polypeptides of 352 and 331 amino acids, respectively, longer than their counterparts in liverwort and bacteria. Expression of the rps4 genes in the investigated higher plant mitochondria was confirmed by Western blot analysis. In Helianthus, one of two short nucleotide insertions at the 3'-end introduces in the coding region a premature termination codon. Northern hybridizations and reverse transcription-polymerase chain reaction analysis demonstrated that the monocistronic RNA transcripts generated from the rps4 locus in Magnolia and Helianthus mitochondria are modified by RNA editing at 28 and 13 positions, respectively. Although evolutionarily conserved, RNA editing requirements of the rps4 appear more extensive in Magnolia than in Helianthus and in the other higher plants so far investigated. Furthermore, our analysis also suggests that selection of editing sites is RNA sequence-specific in a duplicated sequence context. PMID:11943458

  17. Extensive RNA editing in transcripts from the PsbB operon and RpoA gene of plastids from the enigmatic moss Takakia lepidozioides.

    PubMed

    Sugita, Mamoru; Miyata, Yuki; Maruyama, Kaori; Sugiura, Chika; Arikawa, Tomotsugu; Higuchi, Masanobu

    2006-09-01

    RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events. PMID:16960353

  18. ADAR2-Mediated Editing of miR-214 and miR-122 Precursor and Antisense RNA Transcripts in Liver Cancers

    PubMed Central

    Liu, Wan-Hsin; Chen, Chao-Hung; Yeh, Kun-Huei; Li, Chiao-Ling; Wu, Yi-Jinn; Chen, Ding-Shinn; Chen, Pei-Jer; Yeh, Shiou-Hwei

    2013-01-01

    A growing list of microRNAs (miRNAs) show aberrant expression patterns in hepatocellular carcinoma (HCC), but the regulatory mechanisms largely remain unclear. RNA editing catalyzed by members of the adenosine deaminase acting on the RNA (ADAR) family could target the miRNA precursors and affect the biogenesis process. Therefore, we investigate whether RNA editing could be one mechanism contributing to the deregulation of specific miRNAs in HCC. By overexpression of individual ADARs in hepatoma cells, RNA editing on the precursors of 16 miRNAs frequently deregulated in HCC was screened by a sensitive high-resolution melting platform. The results identified RNA precursors of miR-214 and miR-122 as potential targets edited by ADAR2. A subset of HCC showing elevated ADAR2 verified the major editings identified in ARAR2 overexpressed hepatoma cells, either with A-to-I or U-to-C changes. The unusual U-to-C editing at specific residues was demonstrated as being attributed to the A-to-I editing on the RNA transcripts complementary to the pri-miRNAs. The editing event caused a decrease of the RNA transcript complementary to pri-miR-214, which led to the decrease of pri-miR-214 and miR-214 and resulted in the increased protein level of its novel target gene Rab15. In conclusion, the current study discovered ADAR2-mediated editing of the complementary antisense transcripts as a novel mechanism for regulating the biogenesis of specific miRNAs during hepatocarcinogenesis. PMID:24386085

  19. A unique gene expression signature associated with serotonin 2C receptor RNA editing in the prefrontal cortex and altered in suicide

    PubMed Central

    Di Narzo, Antonio Fabio; Kozlenkov, Alexey; Roussos, Panos; Hao, Ke; Hurd, Yasmin; Lewis, David A.; Sibille, Etienne; Siever, Larry J.; Koonin, Eugene; Dracheva, Stella

    2014-01-01

    Editing of the pre-mRNA for the serotonin receptor 2C (5-HT2CR) by site-specific adenosine deamination (A-to-I pre-mRNA editing) substantially increases the functional plasticity of this key neurotransmitter receptor and is thought to contribute to homeostatic mechanisms in neurons. 5-HT2CR mRNA editing generates up to 24 different receptor isoforms. The extent of editing correlates with 5-HT2CR functional activity: more highly edited isoforms exhibit the least function. Altered 5-HT2CR editing has been reported in postmortem brains of suicide victims. We report a comparative analysis of the connections among 5-HT2CR editing, genome-wide gene expression and DNA methylation in suicide victims, individuals with major depressive disorder and non-psychiatric controls. The results confirm previous findings of an overrepresentation of highly edited mRNA variants (which encode hypoactive 5-HT2CR receptors) in the brains of suicide victims. A large set of genes for which the expression level is associated with editing was detected. This signature set of editing-associated genes is significantly enriched for genes that are involved in synaptic transmission, genes that are preferentially expressed in neurons, and genes whose expression is correlated with the level of DNA methylation. Notably, we report that the link between 5-HT2CR editing and gene expression is disrupted in suicide victims. The results suggest that the postulated homeostatic function of 5-HT2CR editing is dysregulated in individuals who committed suicide. PMID:24781207

  20. Substrate-Assisted and Enzymatic Pretransfer Editing of Nonstandard Amino Acids by Methionyl-tRNA Synthetase.

    PubMed

    Fortowsky, Grant B; Simard, Daniel J; Aboelnga, Mohamed M; Gauld, James W

    2015-09-22

    Aminoacyl-tRNA synthetases (aaRSs) are central to a number of physiological processes, including protein biosynthesis. In particular, they activate and then transfer their corresponding amino acid to the cognate tRNA. This is achieved with a generally remarkably high fidelity by editing against incorrect standard and nonstandard amino acids. Using docking, molecular dynamics (MD), and hybrid quantum mechanical/molecular mechanics methods, we have investigated mechanisms by which methionyl-tRNA synthetase (MetRS) may edit against the highly toxic, noncognate, amino acids homocysteine (Hcy) and its oxygen analogue, homoserine (Hse). Substrate-assisted editing of Hcy-AMP in which its own phosphate acts as the mechanistic base occurs with a rate-limiting barrier of 98.2 kJ mol(-1). This step corresponds to nucleophilic attack of the Hcy side-chain sulfur at its own carbonyl carbon (CCarb). In contrast, a new possible editing mechanism is identified in which an active site aspartate (Asp259) acts as the base. The rate-limiting step is now rotation about the substrate's aminoacyl Cβ-Cγ bond with a barrier of 27.5 kJ mol(-1), while for Hse-AMP, the rate-limiting step is cleavage of the CCarb-OP bond with a barrier of 30.9 kJ mol(-1). A similarly positioned aspartate or glutamate also occurs in the homologous enzymes LeuRS, IleRS, and ValRS, which also discriminate against Hcy. Docking and MD studies suggest that at least in the case of LeuRS and ValRS, a similar editing mechanism may be possible. PMID:26322377

  1. The mRNA-edited form of GABRA3 suppresses GABRA3-mediated Akt activation and breast cancer metastasis

    PubMed Central

    Gumireddy, Kiranmai; Li, Anping; Kossenkov, Andrew V.; Sakurai, Masayuki; Yan, Jinchun; Li, Yan; Xu, Hua; Wang, Jian; Zhang, Paul J.; Zhang, Lin; Showe, Louise C.; Nishikura, Kazuko; Huang, Qihong

    2016-01-01

    Metastasis is a critical event affecting breast cancer patient survival. To identify molecules contributing to the metastatic process, we analysed The Cancer Genome Atlas (TCGA) breast cancer data and identified 41 genes whose expression is inversely correlated with survival. Here we show that GABAA receptor alpha3 (Gabra3), normally exclusively expressed in adult brain, is also expressed in breast cancer, with high expression of Gabra3 being inversely correlated with breast cancer survival. We demonstrate that Gabra3 activates the AKT pathway to promote breast cancer cell migration, invasion and metastasis. Importantly, we find an A-to-I RNA-edited form of Gabra3 only in non-invasive breast cancers and show that edited Gabra3 suppresses breast cancer cell invasion and metastasis. A-to-I-edited Gabra3 has reduced cell surface expression and suppresses the activation of AKT required for cell migration and invasion. Our study demonstrates a significant role for mRNA-edited Gabra3 in breast cancer metastasis. PMID:26869349

  2. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing

    PubMed Central

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L.; Liu, Yule

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing. PMID:26450012

  3. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing.

    PubMed

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L; Liu, Yule

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing. PMID:26450012

  4. Strictly Conserved Lysine of Prolyl-tRNA Synthetase Editing Domain Facilitates Binding and Positioning of Misacylated tRNAPro

    PubMed Central

    2015-01-01

    To ensure high fidelity in translation, many aminoacyl-tRNA synthetases, enzymes responsible for attaching specific amino acids to cognate tRNAs, require proof-reading mechanisms. Most bacterial prolyl-tRNA synthetases (ProRSs) misactivate alanine and employ a post-transfer editing mechanism to hydrolyze Ala-tRNAPro. This reaction occurs in a second catalytic site (INS) that is distinct from the synthetic active site. The 2′-OH of misacylated tRNAPro and several conserved residues in the Escherichia coli ProRS INS domain are directly involved in Ala-tRNAPro deacylation. Although mutation of the strictly conserved lysine 279 (K279) results in nearly complete loss of post-transfer editing activity, this residue does not directly participate in Ala-tRNAPro hydrolysis. We hypothesized that the role of K279 is to bind the phosphate backbone of the acceptor stem of misacylated tRNAPro and position it in the editing active site. To test this hypothesis, we carried out pKa, charge neutralization, and free-energy of binding calculations. Site-directed mutagenesis and kinetic studies were performed to verify the computational results. The calculations revealed a considerably higher pKa of K279 compared to an isolated lysine and showed that the protonated state of K279 is stabilized by the neighboring acidic residue. However, substitution of this acidic residue with a positively charged residue leads to a significant increase in Ala-tRNAPro hydrolysis, suggesting that enhancement in positive charge density in the vicinity of K279 favors tRNA binding. A charge-swapping experiment and free energy of binding calculations support the conclusion that the positive charge at position 279 is absolutely necessary for tRNA binding in the editing active site. PMID:24450765

  5. The role of binding domains for dsRNA and Z-DNA in the in vivo editing of minimal substrates by ADAR1

    PubMed Central

    Herbert, Alan; Rich, Alexander

    2001-01-01

    RNA editing changes the read-out of genetic information, increasing the number of different protein products that can be made from a single gene. One form involves the deamination of adenosine to form inosine, which is subsequently translated as guanosine. The reaction requires a double-stranded RNA (dsRNA) substrate and is catalyzed by the adenosine deaminase that act on dsRNA (ADAR) family of enzymes. These enzymes possess dsRNA-binding domains (DRBM) and a catalytic domain. ADAR1 so far has been found only in vertebrates and is characterized by two Z-DNA-binding motifs, the biological function of which remains unknown. Here the role of the various functional domains of ADAR1 in determining the editing efficiency and specificity of ADAR1 is examined in cell-based assays. A variety of dsRNA substrates was tested. It was found that a 15-bp dsRNA stem with a single base mismatch was sufficient for editing. The particular adenosine modified could be varied by changing the position of the mismatch. Editing efficiency could be increased by placing multiple pyrimidines 5′ to the edited adenosine. With longer substrates, editing efficiency also increased and was partly due to the use of DRBMs. Additional editing sites were also observed that clustered on the complementary strand 11–15 bp from the first. An unexpected finding was that the DRBMs are not necessary for the editing of the shorter 15-bp substrates. However, mutation of the Z-DNA-binding domains of ADAR1 decreased the efficiency with which such a substrate was edited. PMID:11593027

  6. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing.

    PubMed

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75-98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing. PMID:27606350

  7. Sendai virus, an RNA virus with no risk of genomic integration, delivers CRISPR/Cas9 for efficient gene editing

    PubMed Central

    Park, Arnold; Hong, Patrick; Won, Sohui T; Thibault, Patricia A; Vigant, Frederic; Oguntuyo, Kasopefoluwa Y; Taft, Justin D; Lee, Benhur

    2016-01-01

    The advent of RNA-guided endonuclease (RGEN)-mediated gene editing, specifically via CRISPR/Cas9, has spurred intensive efforts to improve the efficiency of both RGEN delivery and targeted mutagenesis. The major viral vectors in use for delivery of Cas9 and its associated guide RNA, lentiviral and adeno-associated viral systems, have the potential for undesired random integration into the host genome. Here, we repurpose Sendai virus, an RNA virus with no viral DNA phase and that replicates solely in the cytoplasm, as a delivery system for efficient Cas9-mediated gene editing. The high efficiency of Sendai virus infection resulted in high rates of on-target mutagenesis in cell lines (75–98% at various endogenous and transgenic loci) and primary human monocytes (88% at the ccr5 locus) in the absence of any selection. In conjunction with extensive former work on Sendai virus as a promising gene therapy vector that can infect a wide range of cell types including hematopoietic stem cells, this proof-of-concept study opens the door to using Sendai virus as well as other related paramyxoviruses as versatile and efficient tools for gene editing. PMID:27606350

  8. Crystallization and X-ray diffraction analysis of the Trp/amber editing site of hepatitis delta virus (+)RNA: a case of rational design

    SciTech Connect

    MacElrevey, Celeste; Wedekind, Joseph E.

    2005-12-01

    Well diffracting decamer crystals of the hepatitis delta virus RNA-editing site were prepared, but exhibited merohedral twinning and base averaging owing to duplex symmetry. A longer asymmetric construct that includes additional flanking RNA sequences has been crystallized that does not appear to exhibit these defects. RNA editing by mammalian ADAR1 (Adenosine Deaminase Acting on RNA) is required for the life cycle of the hepatitis delta virus (HDV). Editing extends the single viral open reading frame to yield two protein products of alternate length. ADARs are believed to recognize double-stranded RNA substrates via a ‘structure-based’ readout mechanism. Crystals of 10-mer duplexes representing the HDV RNA-editing site diffracted to 1.35 Å resolution, but suffered from merohedral twinning and averaging of the base registry. Expansion of the construct to include two flanking 3 × 1 internal loops yielded crystals in the primitive tetragonal space group P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2. X-ray diffraction data were collected to 2.8 Å resolution, revealing a unit cell with parameters a = 62.5, c = 63.5 Å. The crystallization and X-ray analysis of multiple forms of the HDV RNA-editing substrate, encounters with common RNA crystal-growth defects and a strategy to overcome these problems are reported.

  9. Altered mRNA editing and expression of ionotropic glutamate receptors after kainic acid exposure in cyclooxygenase-2 deficient mice.

    PubMed

    Caracciolo, Luca; Barbon, Alessandro; Palumbo, Sara; Mora, Cristina; Toscano, Christopher D; Bosetti, Francesca; Barlati, Sergio

    2011-01-01

    Kainic acid (KA) binds to the AMPA/KA receptors and induces seizures that result in inflammation, oxidative damage and neuronal death. We previously showed that cyclooxygenase-2 deficient (COX-2(-/-)) mice are more vulnerable to KA-induced excitotoxicity. Here, we investigated whether the increased susceptibility of COX-2(-/-) mice to KA is associated with altered mRNA expression and editing of glutamate receptors. The expression of AMPA GluR2, GluR3 and KA GluR6 was increased in vehicle-injected COX-2(-/-) mice compared to wild type (WT) mice in hippocampus and cortex, whereas gene expression of NMDA receptors was decreased. KA treatment decreased the expression of AMPA, KA and NMDA receptors in the hippocampus, with a significant effect in COX-2(-/-) mice. Furthermore, we analyzed RNA editing levels and found that the level of GluR3 R/G editing site was selectively increased in the hippocampus and decreased in the cortex in COX-2(-/-) compared with WT mice. After KA, GluR4 R/G editing site, flip form, was increased in the hippocampus of COX-2(-/-) mice. Treatment of WT mice with the COX-2 inhibitor celecoxib for two weeks decreased the expression of AMPA/KA and NMDAR subunits after KA, as observed in COX-2(-/-) mice. After KA exposure, COX-2(-/-) mice showed increased mRNA expression of markers of inflammation and oxidative stress, such as cytokines (TNF-α, IL-1β and IL-6), inducible nitric oxide synthase (iNOS), microglia (CD11b) and astrocyte (GFAP). Thus, COX-2 gene deletion can exacerbate the inflammatory response to KA. We suggest that COX-2 plays a role in attenuating glutamate excitotoxicity by modulating RNA editing of AMPA/KA and mRNA expression of all ionotropic glutamate receptor subunits and, in turn, neuronal excitability. These changes may contribute to the increased vulnerability of COX-2(-/-) mice to KA. The overstimulation of glutamate receptors as a consequence of COX-2 gene deletion suggests a functional coupling between COX-2 and the

  10. The essential role of AMPA receptor GluR2 subunit RNA editing in the normal and diseased brain.

    PubMed

    Wright, Amanda; Vissel, Bryce

    2012-01-01

    α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are comprised of different combinations of GluA1-GluA4 (also known asGluR1-GluR4 and GluR-A to GluR-D) subunits. The GluA2 subunit is subject to RNA editing by the ADAR2 enzyme, which converts a codon for glutamine (Gln; Q), present in the GluA2 gene, to a codon for arginine (Arg; R) found in the mRNA. AMPA receptors are calcium (Ca(2+))-permeable if they contain the unedited GluA2(Q) subunit or if they lack the GluA2 subunit. While most AMPA receptors in the brain contain the edited GluA2(R) subunit and are therefore Ca(2+)-impermeable, recent evidence suggests that Ca(2+)-permeable AMPA receptors are important in synaptic plasticity, learning, and disease. Strong evidence supports the notion that Ca(2+)-permeable AMPA receptors are usually GluA2-lacking AMPA receptors, with little evidence to date for a significant role of unedited GluA2 in normal brain function. However, recent detailed studies suggest that Ca(2+)-permeable AMPA receptors containing unedited GluA2 do in fact occur in neurons and can contribute to excitotoxic cell loss, even where it was previously thought that there was no unedited GluA2.This review provides an update on the role of GluA2 RNA editing in the healthy and diseased brain and summarizes recent insights into the mechanisms that control this process. We suggest that further studies of the role of unedited GluA2 in normal brain function and disease are warranted, and that GluA2 editing should be considered as a possible contributing factor when Ca(2+)-permeable AMPA receptors are observed. PMID:22514516

  11. Accurate detection for a wide range of mutation and editing sites of microRNAs from small RNA high-throughput sequencing profiles.

    PubMed

    Zheng, Yun; Ji, Bo; Song, Renhua; Wang, Shengpeng; Li, Ting; Zhang, Xiaotuo; Chen, Kun; Li, Tianqing; Li, Jinyan

    2016-08-19

    Various types of mutation and editing (M/E) events in microRNAs (miRNAs) can change the stabilities of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles can contain many mutated and edited miRNAs. Systematic detection of miRNA mutation and editing sites from the huge volume of sRNA HTS profiles is computationally difficult, as high sensitivity and low false positive rate (FPR) are both required. We propose a novel method (named MiRME) for an accurate and fast detection of miRNA M/E sites using a progressive sequence alignment approach which refines sensitivity and improves FPR step-by-step. From 70 sRNA HTS profiles with over 1.3 billion reads, MiRME has detected thousands of statistically significant M/E sites, including 3'-editing sites, 57 A-to-I editing sites (of which 32 are novel), as well as some putative non-canonical editing sites. We demonstrated that a few non-canonical editing sites were not resulted from mutations in genome by integrating the analysis of genome HTS profiles of two human cell lines, suggesting the existence of new editing types to further diversify the functions of miRNAs. Compared with six existing studies or methods, MiRME has shown much superior performance for the identification and visualization of the M/E sites of miRNAs from the ever-increasing sRNA HTS profiles. PMID:27229138

  12. Transcriptome, genetic editing, and microRNA divergence substantiate sympatric speciation of blind mole rat, Spalax.

    PubMed

    Li, Kexin; Wang, Liuyang; Knisbacher, Binyamin A; Xu, Qinqin; Levanon, Erez Y; Wang, Huihua; Frenkel-Morgenstern, Milana; Tagore, Satabdi; Fang, Xiaodong; Bazak, Lily; Buchumenski, Ilana; Zhao, Yang; Lövy, Matěj; Li, Xiangfeng; Han, Lijuan; Frenkel, Zeev; Beiles, Avigdor; Cao, Yi Bin; Wang, Zhen Long; Nevo, Eviatar

    2016-07-01

    Incipient sympatric speciation in blind mole rat, Spalax galili, in Israel, caused by sharp ecological divergence of abutting chalk-basalt ecologies, has been proposed previously based on mitochondrial and whole-genome nuclear DNA. Here, we present new evidence, including transcriptome, DNA editing, microRNA, and codon usage, substantiating earlier evidence for adaptive divergence in the abutting chalk and basalt populations. Genetic divergence, based on the previous and new evidence, is ongoing despite restricted gene flow between the two populations. The principal component analysis, neighbor-joining tree, and genetic structure analysis of the transcriptome clearly show the clustered divergent two mole rat populations. Gene-expression level analysis indicates that the population transcriptome divergence is displayed not only by soil divergence but also by sex. Gene ontology enrichment of the differentially expressed genes from the two abutting soil populations highlights reproductive isolation. Alternative splicing variation of the two abutting soil populations displays two distinct splicing patterns. L-shaped FST distribution indicates that the two populations have undergone divergence with gene flow. Transcriptome divergent genes highlight neurogenetics and nutrition characterizing the chalk population, and energetics, metabolism, musculature, and sensory perception characterizing the abutting basalt population. Remarkably, microRNAs also display divergence between the two populations. The GC content is significantly higher in chalk than in basalt, and stress-response genes mostly prefer nonoptimal codons. The multiple lines of evidence of ecological-genomic and genetic divergence highlight that natural selection overrules the gene flow between the two abutting populations, substantiating the sharp ecological chalk-basalt divergence driving sympatric speciation. PMID:27339131

  13. Polyadenylation occurs at multiple sites in maize mitochondrial cox2 mRNA and is independent of editing status.

    PubMed Central

    Lupold, D S; Caoile, A G; Stern, D B

    1999-01-01

    Polyadenylation of nucleus-encoded transcripts has a well-defined role in gene expression. The extent and function of polyadenylation in organelles and prokaryotic systems, however, are less well documented. Recent reports of polyadenylation-mediated RNA destabilization in Escherichia coli and in vascular plant chloroplasts prompted us to look for polyadenylation in plant mitochondria. Here, we report the use of reverse transcription-polymerase chain reaction to map multiple polyadenylate addition sites in maize mitochondrial cox2 transcripts. The lack of sequence conservation surrounding these sites suggests that polyadenylation may occur at many 3' termini created by endoribonucleolytic and/or exoribonucleolytic activities, including those activities involved in 3' end maturation. Endogenous transcripts could be efficiently polyadenylated in vitro by using maize mitochondrial lysates with an activity that added AMP more efficiently than GMP. Polyadenylated substrates were tested for stability in maize mitochondrial S100 extracts, and we found that, compared with nonpolyadenylated RNAs, the polyadenylated substrates were less stable. Taken together with the low abundance of polyadenylated RNAs in maize mitochondria, our results are consistent with a degradation-related process. The fact that polyadenylation does not dramatically destabilize plant mitochondrial transcripts, at least in vitro, is in agreement with results obtained for animal mitochondria but differs from those obtained for chloroplasts and E. coli. Because fully edited, partially edited, and unedited transcripts were found among the cloned polyadenylated cox2 cDNAs, we conclude that RNA editing and polyadenylation are independent processes in maize mitochondria. PMID:10449588

  14. Changing blue fluorescent protein to green fluorescent protein using chemical RNA editing as a novel strategy in genetic restoration.

    PubMed

    Vu, Luyen T; Nguyen, Thanh T K; Alam, Shafiul; Sakamoto, Takashi; Fujimoto, Kenzo; Suzuki, Hitoshi; Tsukahara, Toshifumi

    2015-11-01

    Using the transition from cytosine of BFP (blue fluorescent protein) gene to uridine of GFP (green fluorescent protein) gene at position 199 as a model, we successfully controlled photochemical RNA editing to effect site-directed deamination of cytidine (C) to uridine (U). Oligodeoxynucleotides (ODNs) containing 5'-carboxyvinyl-2'-deoxyuridine ((CV) U) were used for reversible photoligation, and single-stranded 100-nt BFP DNA and in vitro-transcribed full-length BFP mRNA were the targets. Photo-cross-linking with the responsive ODNs was performed using UV (366 nm) irradiation, which was followed by heat treatment, and the cross-linked nucleotide was cleaved through photosplitting (UV, 312 nm). The products were analyzed using restriction fragment length polymorphism (RFLP) and fluorescence measurements. Western blotting and fluorescence-analysis results revealed that in vitro-translated proteins were synthesized from mRNAs after site-directed RNA editing. We detected substantial amounts of the target-base-substituted fragment using RFLP and observed highly reproducible spectra of the transition-GFP signal using fluorescence spectroscopy, which indicated protein stability. ODNc restored approximately 10% of the C-to-U transition. Thus, we successfully used non-enzymatic site-directed deamination for genetic restoration in vitro. In the near future, in vivo studies that include cultured cells and model animals will be conducted to treat genetic disorders. PMID:26031895

  15. EdiPy: a resource to simulate the evolution of plant mitochondrial genes under the RNA editing.

    PubMed

    Picardi, Ernesto; Quagliariello, Carla

    2006-02-01

    EdiPy is an online resource appropriately designed to simulate the evolution of plant mitochondrial genes in a biologically realistic fashion. EdiPy takes into account the presence of sites subjected to RNA editing and provides multiple artificial alignments corresponding to both genomic and cDNA sequences. Each artificial data set can successively be submitted to main and widespread evolutionary and phylogenetic software packages such as PAUP, Phyml, PAML and Phylip. As an online bioinformatic resource, EdiPy is available at the following web page: http://biologia.unical.it/py_script/index.html. PMID:16321571

  16. A population study of the minicircles in Trypanosoma cruzi: predicting guide RNAs in the absence of empirical RNA editing

    PubMed Central

    Thomas, Sean; Martinez, LL Isadora Trejo; Westenberger, Scott J; Sturm, Nancy R

    2007-01-01

    Background The structurally complex network of minicircles and maxicircles comprising the mitochondrial DNA of kinetoplastids mirrors the complexity of the RNA editing process that is required for faithful expression of encrypted maxicircle genes. Although a few of the guide RNAs that direct this editing process have been discovered on maxicircles, guide RNAs are mostly found on the minicircles. The nuclear and maxicircle genomes have been sequenced and assembled for Trypanosoma cruzi, the causative agent of Chagas disease, however the complement of 1.4-kb minicircles, carrying four guide RNA genes per molecule in this parasite, has been less thoroughly characterised. Results Fifty-four CL Brener and 53 Esmeraldo strain minicircle sequence reads were extracted from T. cruzi whole genome shotgun sequencing data. With these sequences and all published T. cruzi minicircle sequences, 108 unique guide RNAs from all known T. cruzi minicircle sequences and two guide RNAs from the CL Brener maxicircle were predicted using a local alignment algorithm and mapped onto predicted or experimentally determined sequences of edited maxicircle open reading frames. For half of the sequences no statistically significant guide RNA could be assigned. Likely positions of these unidentified gRNAs in T. cruzi minicircle sequences are estimated using a simple Hidden Markov Model. With the local alignment predictions as a standard, the HMM had an ~85% chance of correctly identifying at least 20 nucleotides of guide RNA from a given minicircle sequence. Inter-minicircle recombination was documented. Variable regions contain species-specific areas of distinct nucleotide preference. Two maxicircle guide RNA genes were found. Conclusion The identification of new minicircle sequences and the further characterization of all published minicircles are presented, including the first observation of recombination between minicircles. Extrapolation suggests a level of 4% recombinants in the population

  17. A conserved glutamate residue in the C-terminal deaminase domain of pentatricopeptide repeat proteins is required for RNA editing activity.

    PubMed

    Hayes, Michael L; Dang, Kim N; Diaz, Michael F; Mulligan, R Michael

    2015-04-17

    Many transcripts expressed from plant organelle genomes are modified by C-to-U RNA editing. Nuclear encoded pentatricopeptide repeat (PPR) proteins include an RNA binding domain that provides site specificity. In addition, many PPR proteins include a C-terminal DYW deaminase domain with characteristic zinc binding motifs (CXXC, HXE) and has recently been shown to bind zinc ions. The glutamate residue of the HXE motif is catalytically required in the reaction catalyzed by cytidine deaminase. In this work, we examine the activity of the DYW deaminase domain through truncation or mutagenesis of the HXE motif. OTP84 is required for editing three chloroplast sites, and transgenes expressing OTP84 with C-terminal truncations were capable of editing only one of the three cognate sites at high efficiency. These results suggest that the deaminase domain of OTP84 is required for editing two of the sites, but another deaminase is able to supply the deamination activity for the third site. OTP84 and CREF7 transgenes were mutagenized to replace the glutamate residue of the HXE motif, and transgenic plants expressing OTP84-E824A and CREF7-E554A were unable to efficiently edit the cognate editing sites for these genes. In addition, plants expressing CREF7-E554A exhibited substantially reduced capacity to edit a non-cognate site, rpoA C200. These results indicate that the DYW deaminase domains of PPR proteins are involved in editing their cognate editing sites, and in some cases may participate in editing additional sites in the chloroplast. PMID:25739442

  18. Hepatitis Delta Virus RNA Editing Is Highly Specific for the Amber/W Site and Is Suppressed by Hepatitis Delta Antigen

    PubMed Central

    Polson, Andrew G.; Ley, Herbert L.; Bass, Brenda L.; Casey, John L.

    1998-01-01

    RNA editing at adenosine 1012 (amber/W site) in the antigenomic RNA of hepatitis delta virus (HDV) allows two essential forms of the viral protein, hepatitis delta antigen (HDAg), to be synthesized from a single open reading frame. Editing at the amber/W site is thought to be catalyzed by one of the cellular enzymes known as adenosine deaminases that act on RNA (ADARs). In vitro, the enzymes ADAR1 and ADAR2 deaminate adenosines within many different sequences of base-paired RNA. Since promiscuous deamination could compromise the viability of HDV, we wondered if additional deamination events occurred within the highly base paired HDV RNA. By sequencing cDNAs derived from HDV RNA from transfected Huh-7 cells, we determined that the RNA was not extensively modified at other adenosines. Approximately 0.16 to 0.32 adenosines were modified per antigenome during 6 to 13 days posttransfection. Interestingly, all observed non-amber/W adenosine modifications, which occurred mostly at positions that are highly conserved among naturally occurring HDV isolates, were found in RNAs that were also modified at the amber/W site. Such coordinate modification likely limits potential deleterious effects of promiscuous editing. Neither viral replication nor HDAg was required for the highly specific editing observed in cells. However, HDAg was found to suppress editing at the amber/W site when expressed at levels similar to those found during HDV replication. These data suggest HDAg may regulate amber/W site editing during virus replication. PMID:9528763

  19. Multifunctional G-Rich and RRM-Containing Domains of TbRGG2 Perform Separate yet Essential Functions in Trypanosome RNA Editing

    PubMed Central

    Foda, Bardees M.; Downey, Kurtis M.; Fisk, John C.

    2012-01-01

    Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3′-to-5′ progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

  20. Multifunctional G-rich and RRM-containing domains of TbRGG2 perform separate yet essential functions in trypanosome RNA editing.

    PubMed

    Foda, Bardees M; Downey, Kurtis M; Fisk, John C; Read, Laurie K

    2012-09-01

    Efficient editing of Trypanosoma brucei mitochondrial RNAs involves the actions of multiple accessory factors. T. brucei RGG2 (TbRGG2) is an essential protein crucial for initiation and 3'-to-5' progression of editing. TbRGG2 comprises an N-terminal G-rich region containing GWG and RG repeats and a C-terminal RNA recognition motif (RRM)-containing domain. Here, we perform in vitro and in vivo separation-of-function studies to interrogate the mechanism of TbRGG2 action in RNA editing. TbRGG2 preferentially binds preedited mRNA in vitro with high affinity attributable to its G-rich region. RNA-annealing and -melting activities are separable, carried out primarily by the G-rich and RRM domains, respectively. In vivo, the G-rich domain partially complements TbRGG2 knockdown, but the RRM domain is also required. Notably, TbRGG2's RNA-melting activity is dispensable for RNA editing in vivo. Interactions between TbRGG2 and MRB1 complex proteins are mediated by both G-rich and RRM-containing domains, depending on the binding partner. Overall, our results are consistent with a model in which the high-affinity RNA binding and RNA-annealing activities of the G-rich domain are essential for RNA editing in vivo. The RRM domain may have key functions involving interactions with the MRB1 complex and/or regulation of the activities of the G-rich domain. PMID:22798390

  1. ADAR2-dependent RNA editing of GluR2 is involved in thiamine deficiency-induced alteration of calcium dynamics

    PubMed Central

    2010-01-01

    Background Thiamine (vitamin B1) deficiency (TD) causes mild impairment of oxidative metabolism and region-selective neuronal loss in the central nervous system (CNS). TD in animals has been used to model aging-associated neurodegeneration in the brain. The mechanisms of TD-induced neuron death are complex, and it is likely multiple mechanisms interplay and contribute to the action of TD. In this study, we demonstrated that TD significantly increased intracellular calcium concentrations [Ca2+]i in cultured cortical neurons. Results TD drastically potentiated AMPA-triggered calcium influx and inhibited pre-mRNA editing of GluR2, a Ca2+-permeable subtype of AMPA receptors. The Ca2+ permeability of GluR2 is regulated by RNA editing at the Q/R site. Edited GluR2 (R) subunits form Ca2+-impermeable channels, whereas unedited GluR2 (Q) channels are permeable to Ca2+ flow. TD inhibited Q/R editing of GluR2 and increased the ratio of unedited GluR2. The Q/R editing of GluR2 is mediated by adenosine deaminase acting on RNA 2 (ADAR2). TD selectively decreased ADAR2 expression and its self-editing ability without affecting ADAR1 in cultured neurons and in the brain tissue. Over-expression of ADAR2 reduced AMPA-mediated rise of [Ca2+]i and protected cortical neurons against TD-induced cytotoxicity, whereas down-regulation of ADAR2 increased AMPA-elicited Ca2+ influx and exacerbated TD-induced death of cortical neurons. Conclusions Our findings suggest that TD-induced neuronal damage may be mediated by the modulation of ADAR2-dependent RNA Editing of GluR2. PMID:21110885

  2. Missing Genes, Multiple ORFs, and C-to-U Type RNA Editing in Acrasis kona (Heterolobosea, Excavata) Mitochondrial DNA

    PubMed Central

    Fu, Cheng-Jie; Sheikh, Sanea; Miao, Wei; Andersson, Siv G.E.; Baldauf, Sandra L.

    2014-01-01

    Discoba (Excavata) is an ancient group of eukaryotes with great morphological and ecological diversity. Unlike the other major divisions of Discoba (Jakobida and Euglenozoa), little is known about the mitochondrial DNAs (mtDNAs) of Heterolobosea. We have assembled a complete mtDNA genome from the aggregating heterolobosean amoeba, Acrasis kona, which consists of a single circular highly AT-rich (83.3%) molecule of 51.5 kb. Unexpectedly, A. kona mtDNA is missing roughly 40% of the protein-coding genes and nearly half of the transfer RNAs found in the only other sequenced heterolobosean mtDNAs, those of Naegleria spp. Instead, over a quarter of A. kona mtDNA consists of novel open reading frames. Eleven of the 16 protein-coding genes missing from A. kona mtDNA were identified in its nuclear DNA and polyA RNA, and phylogenetic analyses indicate that at least 10 of these 11 putative nuclear-encoded mitochondrial (NcMt) proteins arose by direct transfer from the mitochondrion. Acrasis kona mtDNA also employs C-to-U type RNA editing, and 12 homologs of DYW-type pentatricopeptide repeat (PPR) proteins implicated in plant organellar RNA editing are found in A. kona nuclear DNA. A mapping of mitochondrial gene content onto a consensus phylogeny reveals a sporadic pattern of relative stasis and rampant gene loss in Discoba. Rampant loss occurred independently in the unique common lineage leading to Heterolobosea + Tsukubamonadida and later in the unique lineage leading to Acrasis. Meanwhile, mtDNA gene content appears to be remarkably stable in the Acrasis sister lineage leading to Naegleria and in their distant relatives Jakobida. PMID:25146648

  3. Mutations in ADAR1 cause Aicardi-Goutières syndrome associated with a type I interferon signature

    PubMed Central

    Rice, Gillian I; Kasher, Paul R; Forte, Gabriella M A; Mannion, Niamh M; Greenwood, Sam M; Szynkiewicz, Marcin; Dickerson, Jonathan E; Bhaskar, Sanjeev S; Zampini, Massimiliano; Briggs, Tracy A; Jenkinson, Emma M; Bacino, Carlos A; Battini, Roberta; Bertini, Enrico; Brogan, Paul A; Brueton, Louise A; Carpanelli, Marialuisa; Laet, Corinne De; de Lonlay, Pascale; del Toro, Mireia; Desguerre, Isabelle; Fazzi, Elisa; Garcia-Cazorla, Àngels; Heiberg, Arvid; Kawaguchi, Masakazu; Kumar, Ram; Lin, Jean-Pierre S-M; Lourenco, Charles M; Male, Alison M; Marques, Wilson; Mignot, Cyril; Olivieri, Ivana; Orcesi, Simona; Prabhakar, Prab; Rasmussen, Magnhild; Robinson, Robert A; Rozenberg, Flore; Schmidt, Johanna L; Steindl, Katharina; Tan, Tiong Y; van der Merwe, William G; Vanderver, Adeline; Vassallo, Grace; Wakeling, Emma L; Wassmer, Evangeline; Whittaker, Elizabeth; Livingston, John H; Lebon, Pierre; Suzuki, Tamio; McLaughlin, Paul J; Keegan, Liam P; O’Connell, Mary A; Lovell, Simon C; Crow, Yanick J

    2014-01-01

    Adenosine deaminases acting on RNA (ADARs) catalyze the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) and thereby potentially alter the information content and structure of cellular RNAs. Notably, although the overwhelming majority of such editing events occur in transcripts derived from Alu repeat elements, the biological function of non-coding RNA editing remains uncertain. Here, we show that mutations in ADAR1 (also known as ADAR) cause the autoimmune disorder Aicardi-Goutières syndrome (AGS). As in Adar1-null mice, the human disease state is associated with upregulation of interferon-stimulated genes, indicating a possible role for ADAR1 as a suppressor of type I interferon signaling. Considering recent insights derived from the study of other AGS-related proteins, we speculate that ADAR1 may limit the cytoplasmic accumulation of the dsRNA generated from genomic repetitive elements. PMID:23001123

  4. Direct-methods structure determination of a trypanosome RNA-editing substrate fragment with translational pseudosymmetry

    PubMed Central

    Mooers, Blaine H. M.

    2016-01-01

    Using direct methods starting from random phases, the crystal structure of a 32-base-pair RNA (675 non-H RNA atoms in the asymmetric unit) was determined using only the native diffraction data (resolution limit 1.05 Å) and the computer program SIR2014. The almost three helical turns of the RNA in the asymmetric unit introduced partial or imperfect translational pseudosymmetry (TPS) that modulated the intensities when averaged by the l Miller indices but still escaped automated detection. Almost six times as many random phase sets had to be tested on average to reach a correct structure compared with a similar-sized RNA hairpin (27 nucleotides, 580 non-H RNA atoms) without TPS. More sensitive methods are needed for the automated detection of partial TPS. PMID:27050127

  5. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

    PubMed Central

    Kim, Sojung; Kim, Daesik; Cho, Seung Woo; Kim, Jungeun; Kim, Jin-Soo

    2014-01-01

    RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection. PMID:24696461

  6. Global Analysis of Mouse Polyomavirus Infection Reveals Dynamic Regulation of Viral and Host Gene Expression and Promiscuous Viral RNA Editing

    PubMed Central

    Garren, Seth B.; Kondaveeti, Yuvabharath; Duff, Michael O.; Carmichael, Gordon G.

    2015-01-01

    Mouse polyomavirus (MPyV) lytically infects mouse cells, transforms rat cells in culture, and is highly oncogenic in rodents. We have used deep sequencing to follow MPyV infection of mouse NIH3T6 cells at various times after infection and analyzed both the viral and cellular transcriptomes. Alignment of sequencing reads to the viral genome illustrated the transcriptional profile of the early-to-late switch with both early-strand and late-strand RNAs being transcribed at all time points. A number of novel insights into viral gene expression emerged from these studies, including the demonstration of widespread RNA editing of viral transcripts at late times in infection. By late times in infection, 359 host genes were seen to be significantly upregulated and 857 were downregulated. Gene ontology analysis indicated transcripts involved in translation, metabolism, RNA processing, DNA methylation, and protein turnover were upregulated while transcripts involved in extracellular adhesion, cytoskeleton, zinc finger binding, SH3 domain, and GTPase activation were downregulated. The levels of a number of long noncoding RNAs were also altered. The long noncoding RNA MALAT1, which is involved in splicing speckles and used as a marker in many late-stage cancers, was noticeably downregulated, while several other abundant noncoding RNAs were strongly upregulated. We discuss these results in light of what is currently known about the MPyV life cycle and its effects on host cell growth and metabolism. PMID:26407100

  7. Prevention of 5-hydroxytryptamine2C receptor RNA editing and alternate splicing in C57BL/6 mice activates the hypothalamic-pituitary-adrenal axis and alters mood

    PubMed Central

    Bombail, Vincent; Qing, Wei; Chapman, Karen E; Holmes, Megan C

    2014-01-01

    The 5-hydroxytryptamine2C (5-HT)2C receptor is widely implicated in the aetiology of affective and eating disorders as well as regulation of the hypothalamo-pituitary-adrenal axis. Signalling through this receptor is regulated by A-to-I RNA editing, affecting three amino acids in the protein sequence, with unedited transcripts encoding a receptor (INI) that, in vitro, is hyperactive compared with edited isoforms. Targeted alteration (knock-in) of the Htr2c gene to generate ‘INI’ mice with no alternate splicing, solely expressing the full-length unedited isoform, did not produce an overt metabolic phenotype or altered anxiety behaviour, but did display reduced depressive-like and fear-associated behaviours. INI mice exhibited a hyperactive hypothalamo-pituitary-adrenal axis, with increased nadir plasma corticosterone and corticotrophin-releasing hormone expression in the hypothalamus but responded normally to chronic stress and showed normal circadian activity and activity in a novel environment. The circadian patterns of 5-HT2C receptor mRNA and mbii52, a snoRNA known to regulate RNA editing and RNA splicing of 5-HT2C receptor pre-mRNA, were altered in INI mice compared with wild-type control mice. Moreover, levels of 5-HT1A receptor mRNA were increased in the hippocampus of INI mice. These gene expression changes may underpin the neuroendocrine and behavioural changes observed in INI mice. However, the phenotype of INI mice was not consistent with a globally hyperactive INI receptor encoded by the unedited transcript in the absence of alternate splicing. Hence, the in vivo outcome of RNA editing may be neuronal cell type specific. PMID:25257581

  8. Targeted Mutagenesis, Precise Gene Editing, and Site-Specific Gene Insertion in Maize Using Cas9 and Guide RNA[OPEN

    PubMed Central

    Schwartz, Christine; Gao, Huirong; Falco, S. Carl; Cigan, A. Mark

    2015-01-01

    Targeted mutagenesis, editing of endogenous maize (Zea mays) genes, and site-specific insertion of a trait gene using clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas)-guide RNA technology are reported in maize. DNA vectors expressing maize codon-optimized Streptococcus pyogenes Cas9 endonuclease and single guide RNAs were cointroduced with or without DNA repair templates into maize immature embryos by biolistic transformation targeting five different genomic regions: upstream of the liguleless1 (LIG1) gene, male fertility genes (Ms26 and Ms45), and acetolactate synthase (ALS) genes (ALS1 and ALS2). Mutations were subsequently identified at all sites targeted, and plants containing biallelic multiplex mutations at LIG1, Ms26, and Ms45 were recovered. Biolistic delivery of guide RNAs (as RNA molecules) directly into immature embryo cells containing preintegrated Cas9 also resulted in targeted mutations. Editing the ALS2 gene using either single-stranded oligonucleotides or double-stranded DNA vectors as repair templates yielded chlorsulfuron-resistant plants. Double-strand breaks generated by RNA-guided Cas9 endonuclease also stimulated insertion of a trait gene at a site near LIG1 by homology-directed repair. Progeny showed expected Mendelian segregation of mutations, edits, and targeted gene insertions. The examples reported in this study demonstrate the utility of Cas9-guide RNA technology as a plant genome editing tool to enhance plant breeding and crop research needed to meet growing agriculture demands of the future. PMID:26269544

  9. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity

    PubMed Central

    Ran, F. Ann; Hsu, Patrick D.; Lin, Chie-Yu; Gootenberg, Jonathan S.; Konermann, Silvana; Trevino, Alexandro; Scott, David A.; Inoue, Azusa; Matoba, Shogo; Zhang, Yi; Zhang, Feng

    2013-01-01

    Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20-nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant with pairs of guide RNAs to introduce targeted double-strand breaks. Given that individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs effectively extends the number of specifically recognized bases in the target site. We demonstrate that paired nicking can be used to reduce off-target activity by 50–1,000 fold in cell lines and facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy thus enables a wide variety of genome editing applications with higher levels of specificity. PMID:23992846

  10. Comparative analysis of the DRADA A-to-I RNA editing gene from mammals, pufferfish and zebrafish.

    PubMed

    Slavov, D; Crnogorac-Jurcević, T; Clark, M; Gardiner, K

    2000-05-30

    The DRADA gene in mammals encodes an A-to-I RNA editase, an adenosine deaminase that acts on pre-mRNAs to produce site specific inosines. DRADA has been shown to deaminate specific adenosine residues in a subset of glutamate and serotonin receptors, and this editing results in proteins of altered sequences and functional properties. DRADA thus plays a role in creating protein diversity. To study the evolutionary significance of this gene, we have characterized the genomic structure of DRADA from Fugu rubripes, and compared the protein sequences of DRADA from mammals, pufferfish and zebrafish. The DRADA gene from Fugu is three-fold compacted with respect to the human gene, and contains a novel intron within the large second coding exon. DRADA cDNAs were isolated from zebrafish and a second pufferfish, Tetraodon fluviatilis. Comparisons among fish, and between fish and mammals, of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA binding domains vary within a single protein in their levels of conservation. Conservation within the Z DNA binding domain has also been assessed. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation. PMID:10854778

  11. The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing.

    PubMed

    de Solis, Christopher A; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E

    2016-01-01

    The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

  12. The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    PubMed Central

    de Solis, Christopher A.; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E.

    2016-01-01

    The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

  13. Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing

    PubMed Central

    Wyvekens, Nicolas; Topkar, Ved V.; Khayter, Cyd; Joung, J. Keith; Tsai, Shengdar Q.

    2015-01-01

    Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR/Cas nucleases and therefore provide a highly specific platform for performing genome editing. PMID:26068112

  14. Dimeric CRISPR RNA-Guided FokI-dCas9 Nucleases Directed by Truncated gRNAs for Highly Specific Genome Editing.

    PubMed

    Wyvekens, Nicolas; Topkar, Ved V; Khayter, Cyd; Joung, J Keith; Tsai, Shengdar Q

    2015-07-01

    Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing. PMID:26068112

  15. ADAR2 induces reproducible changes in sequence and abundance of mature microRNAs in the mouse brain.

    PubMed

    Vesely, Cornelia; Tauber, Stefanie; Sedlazeck, Fritz J; Tajaddod, Mansoureh; von Haeseler, Arndt; Jantsch, Michael F

    2014-10-29

    Adenosine deaminases that act on RNA (ADARs) deaminate adenosines to inosines in double-stranded RNAs including miRNA precursors. A to I editing is widespread and required for normal life. By comparing deep sequencing data of brain miRNAs from wild-type and ADAR2 deficient mouse strains, we detect editing sites and altered miRNA processing at high sensitivity. We detect 48 novel editing events in miRNAs. Some editing events reach frequencies of up to 80%. About half of all editing events depend on ADAR2 while some miRNAs are preferentially edited by ADAR1. Sixty-four percent of all editing events are located within the seed region of mature miRNAs. For the highly edited miR-3099, we experimentally prove retargeting of the edited miRNA to novel 3' UTRs. We show further that an abundant editing event in miR-497 promotes processing by Drosha of the corresponding pri-miRNA. We also detect reproducible changes in the abundance of specific miRNAs in ADAR2-deficient mice that occur independent of adjacent A to I editing events. This indicates that ADAR2 binding but not editing of miRNA precursors may influence their processing. Correlating with changes in miRNA abundance we find misregulation of putative targets of these miRNAs in the presence or absence of ADAR2. PMID:25260591

  16. Antiviral Chemistry & Chemotherapy's current antiviral agents FactFile 2008 (2nd edition): RNA viruses.

    PubMed

    De Clercq, Erik; Field, Hugh J

    2008-01-01

    Among the RNA viruses, other than the retroviruses (that is, HIV), which are dealt with separately in the current FactFile, the most important targets for the development of antiviral agents at the moment are the orthomyxoviruses (that is, influenza), the hepaciviruses (that is, hepatitis C virus [HCV]) and, to a lesser extent, the picornaviruses. Although the uncoating inhibitors amantadine and rimantadine were the first known inhibitors of influenza A, the neuraminidase inhibitors oseltamivir, zanamivir and peramivir have now become the prime antiviral drugs for the treatment of influenza A and B virus infections. For HCV infections, standard treatment consists of the combination of pegylated interferon-alpha with ribavirin, but several other antivirals targeted at specific viral functions such as the HCV protease and/ or polymerase may be expected to soon take an important share of this important market. Still untapped is the potential of a variety of uncoating inhibitors, as well as protease and/or polymerase inhibitors against the wide spectrum of picornaviruses. While ribavirin has been available for 35 years as a broad-spectrum anti-RNA virus agent, relatively new and unexplored is favipiravir (T-705) accredited with activity against influenza as well as flaviviruses, bunyaviruses and arenaviruses. PMID:18727441

  17. Decrease of mRNA Editing after Spinal Cord Injury is Caused by Down-regulation of ADAR2 that is Triggered by Inflammatory Response

    PubMed Central

    Narzo, Antonio Fabio Di; Kozlenkov, Alexey; Ge, Yongchao; Zhang, Bin; Sanelli, Leo; May, Zacnicte; Li, Yanqing; Fouad, Karim; Cardozo, Christopher; Koonin, Eugene V; Bennett, David J; Dracheva, Stella

    2015-01-01

    We recently showed that spinal cord injury (SCI) leads to a decrease in mRNA editing of serotonin receptor 2C (5-HT2CR) contributing to post-SCI spasticity. Here we study post-SCI mRNA editing and global gene expression using massively parallel sequencing. Evidence is presented that the decrease in 5-HT2CR editing is caused by down-regulation of adenosine deaminase ADAR2 and that editing of at least one other ADAR2 target, potassium channel Kv1.1, is decreased after SCI. Bayesian network analysis of genome-wide transcriptome data indicates that down-regulation of ADAR2 (1) is triggered by persistent inflammatory response to SCI that is associated with activation of microglia and (2) results in changes in neuronal gene expression that are likely to contribute both to post-SCI restoration of neuronal excitability and muscle spasms. These findings have broad implications for other diseases of the Central Nervous System and could open new avenues for developing efficacious antispastic treatments. PMID:26223940

  18. Phylogenetic comparison of the pre-mRNA adenosine deaminase ADAR2 genes and transcripts: conservation and diversity in editing site sequence and alternative splicing patterns.

    PubMed

    Slavov, D; Gardiner, K

    2002-10-16

    Adenosine deaminase that acts on RNA -2 (ADAR2) is a member of a family of vertebrate genes that encode adenosine (A)-to-inosine (I) RNA deaminases, enzymes that deaminate specific A residues in specific pre-mRNAs to produce I. Known substrates of ADAR2 include sites within the coding regions of pre-mRNAs of the ionotropic glutamate receptors, GluR2-6, and the serotonin receptor, 5HT2C. Mammalian ADAR2 expression is itself regulated by A-to-I editing and by several alternative splicing events. Because the biological consequences of ADAR2 function are significant, we have undertaken a phylogenetic comparison of these features. Here we report a comparison of cDNA sequences, genomic organization, editing site sequences and patterns of alternative splicing of ADAR2 genes from human, mouse, chicken, pufferfish and zebrafish. Coding sequences and intron/exon organization are highly conserved. All ADAR2 genes show evidence of transcript editing with required sequences and predicted secondary structures very highly conserved. Patterns and levels of editing and alternative splicing vary among organisms, and include novel N-terminal exons and splicing events. PMID:12459255

  19. AEF1/MPR25 is implicated in RNA editing of plastid atpF and mitochondrial nad5, and also promotes atpF splicing in Arabidopsis and rice.

    PubMed

    Yap, Aaron; Kindgren, Peter; Colas des Francs-Small, Catherine; Kazama, Tomohiko; Tanz, Sandra K; Toriyama, Kinya; Small, Ian

    2015-03-01

    RNA editing is an essential mechanism that modifies target cytidines to uridine in both mitochondrial and plastid mRNA. Target sites are recognized by pentatricopeptide repeat (PPR) proteins. Using bioinformatics predictions based on the code describing sequence recognition by PPR proteins, we have identified an Arabidopsis editing factor required for editing of atpF in plastids. A loss-of-function mutation in ATPF EDITING FACTOR 1 (AEF1, AT3G22150) results in severe variegation, presumably due to decreased plastid ATP synthase levels. Loss of editing at the atpF site is coupled with a large decrease in splicing of the atpF transcript, even though the editing site is within an exon and 53 nucleotides distant from the splice site. The rice orthologue of AEF1, MPR25, has been reported to be required for editing of a site in mitochondrial nad5 transcripts, and we confirm that editing of the same site is affected in the Arabidopsis aef1 mutant. We also show that splicing of chloroplast atpF transcripts is affected in the rice mpr25 mutant. AEF1 is thus highly unusual for an RNA editing specificity factor in that it has functions in both organelles. PMID:25585673

  20. High conservation of a 5' element required for RNA editing of a C target in chloroplast psbE transcripts.

    PubMed

    Hayes, Michael L; Hanson, Maureen R

    2008-09-01

    C-to-U editing modifies 30-40 distinct nucleotides within higher-plant chloroplast transcripts. Many C targets are located at the same position in homologous genes from different plants; these either could have emerged independently or could share a common origin. The 5' sequence GCCGUU, required for editing of C214 in tobacco psbE in vitro, is one of the few identified editing cis-elements. We investigated psbE sequences from many plant species to determine in what lineage(s) editing of psbE C214 emerged and whether the cis-element identified in tobacco is conserved in plants with a C214. The GCCGUU sequence is present at a high frequency in plants that carry a C214 in psbE. However, Sciadopitys verticillata (Pinophyta) edits C214 despite the presence of nucleotide differences compared to the conserved cis-element. The C214 site in psbE genes is represented in members of four branches of spermatophytes but not in gnetophytes, resulting in the parsimonious prediction that editing of psbE C214 was present in the ancestor of spermatophytes. Extracts from chloroplasts from a species that has a difference in the motif and lacks the C target are incapable of editing tobacco psbE C214 substrates, implying that the critical trans-acting protein factors were not retained without a C target. Because noncoding sequences are less constrained than coding regions, we analyzed sequences 5' to two C editing targets located within coding regions to search for possible editing-related conserved elements. Putative editing cis-elements were uncovered in the 5' UTRs near editing sites psbL C2 and ndhD C2. PMID:18696032

  1. Transcriptional Slippage and RNA Editing Increase the Diversity of Transcripts in Chloroplasts: Insight from Deep Sequencing of Vigna radiata Genome and Transcriptome.

    PubMed

    Lin, Ching-Ping; Ko, Chia-Yun; Kuo, Ching-I; Liu, Mao-Sen; Schafleitner, Roland; Chen, Long-Fang Oliver

    2015-01-01

    We performed deep sequencing of the nuclear and organellar genomes of three mungbean genotypes: Vigna radiata ssp. sublobata TC1966, V. radiata var. radiata NM92 and the recombinant inbred line RIL59 derived from a cross between TC1966 and NM92. Moreover, we performed deep sequencing of the RIL59 transcriptome to investigate transcript variability. The mungbean chloroplast genome has a quadripartite structure including a pair of inverted repeats separated by two single copy regions. A total of 213 simple sequence repeats were identified in the chloroplast genomes of NM92 and RIL59; 78 single nucleotide variants and nine indels were discovered in comparing the chloroplast genomes of TC1966 and NM92. Analysis of the mungbean chloroplast transcriptome revealed mRNAs that were affected by transcriptional slippage and RNA editing. Transcriptional slippage frequency was positively correlated with the length of simple sequence repeats of the mungbean chloroplast genome (R2=0.9911). In total, 41 C-to-U editing sites were found in 23 chloroplast genes and in one intergenic spacer. No editing site that swapped U to C was found. A combination of bioinformatics and experimental methods revealed that the plastid-encoded RNA polymerase-transcribed genes psbF and ndhA are affected by transcriptional slippage in mungbean and in main lineages of land plants, including three dicots (Glycine max, Brassica rapa, and Nicotiana tabacum), two monocots (Oryza sativa and Zea mays), two gymnosperms (Pinus taeda and Ginkgo biloba) and one moss (Physcomitrella patens). Transcript analysis of the rps2 gene showed that transcriptional slippage could affect transcripts at single sequence repeat regions with poly-A runs. It showed that transcriptional slippage together with incomplete RNA editing may cause sequence diversity of transcripts in chloroplasts of land plants. PMID:26076132

  2. Retrotransposons as regulators of gene expression.

    PubMed

    Elbarbary, Reyad A; Lucas, Bronwyn A; Maquat, Lynne E

    2016-02-12

    Transposable elements (TEs) are both a boon and a bane to eukaryotic organisms, depending on where they integrate into the genome and how their sequences function once integrated. We focus on two types of TEs: long interspersed elements (LINEs) and short interspersed elements (SINEs). LINEs and SINEs are retrotransposons; that is, they transpose via an RNA intermediate. We discuss how LINEs and SINEs have expanded in eukaryotic genomes and contribute to genome evolution. An emerging body of evidence indicates that LINEs and SINEs function to regulate gene expression by affecting chromatin structure, gene transcription, pre-mRNA processing, or aspects of mRNA metabolism. We also describe how adenosine-to-inosine editing influences SINE function and how ongoing retrotransposition is countered by the body's defense mechanisms. PMID:26912865

  3. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery

    PubMed Central

    Wang, Jianbin; DeClercq, Joshua J.; Hayward, Samuel B.; Li, Patrick Wai-Lun; Shivak, David A.; Gregory, Philip D.; Lee, Gary; Holmes, Michael C.

    2016-01-01

    The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8+ T cells and gene targeting of a GFP transgene cassette in >40% of CD8+ and CD4+ T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy. PMID:26527725

  4. Homology-driven genome editing in hematopoietic stem and progenitor cells using zinc finger nuclease mRNA and AAV6 donors

    PubMed Central

    Wang, Jianbin; Exline, Colin M.; DeClercq, Joshua J.; Llewellyn, G. Nicholas; Hayward, Samuel B.; Li, Patrick Wai-Lun; Shivak, David A.; Surosky, Richard T.; Gregory, Philip D.; Holmes, Michael C.; Cannon, Paula M

    2016-01-01

    Genome editing with targeted nucleases and DNA donor templates homologous to the break site has proven challenging in human hematopoietic stem and progenitor cells (HSPCs), and particularly in the most primitive, long-term repopulating cell population. Here we report that combining electroporation of zinc finger nuclease (ZFN) mRNA with donor template delivery by AAV serotype 6 vectors directs efficient genome editing in HSPCs, achieving site-specific insertion of a GFP cassette at the CCR5 and AAVS1 loci in mobilized peripheral blood CD34+ HSPCs at mean frequencies of 17% and 26%, respectively, and in fetal liver HSPCs at 19% and 43%, respectively. Notably, this approach modified the CD34+CD133+CD90+ cell population, a minor component of CD34+ cells that contains long-term repopulating hematopoietic stem cells (HSCs). Genome-edited HSPCs also engrafted in immune deficient mice long-term, confirming that HSCs are targeted by this approach. Our results provide a strategy for more robust application of genome editing technologies in HSPCs. PMID:26551060

  5. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.

    PubMed

    Wang, Jianbin; DeClercq, Joshua J; Hayward, Samuel B; Li, Patrick Wai-Lun; Shivak, David A; Gregory, Philip D; Lee, Gary; Holmes, Michael C

    2016-02-18

    The adoptive transfer of engineered T cells for the treatment of cancer, autoimmunity, and infectious disease is a rapidly growing field that has shown great promise in recent clinical trials. Nuclease-driven genome editing provides a method in which to precisely target genetic changes to further enhance T cell function in vivo. We describe the development of a highly efficient method to genome edit both primary human CD8 and CD4 T cells by homology-directed repair at a pre-defined site of the genome. Two different homology donor templates were evaluated, representing both minor gene editing events (restriction site insertion) to mimic gene correction, or the more significant insertion of a larger gene cassette. By combining zinc finger nuclease mRNA delivery with AAV6 delivery of a homologous donor we could gene correct 41% of CCR5 or 55% of PPP1R12C (AAVS1) alleles in CD8(+) T cells and gene targeting of a GFP transgene cassette in >40% of CD8(+) and CD4(+) T cells at both the CCR5 and AAVS1 safe harbor locus, potentially providing a robust genome editing tool for T cell-based immunotherapy. PMID:26527725

  6. Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper.

    PubMed

    Morris, C A; Owen, J R; Thomas, M C; El-Hiti, G A; Harwood, J L; Kille, P

    2014-01-01

    A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress. PMID:23738980

  7. Comparative analysis of the RED1 and RED2 A-to-I RNA editing genes from mammals, pufferfish and zebrafish.

    PubMed

    Slavov, D; Clark, M; Gardiner, K

    2000-05-30

    One type of RNA editing involves the deamination of adenosine (A) residues to inosines (I) at specific sites in specific pre-mRNAs. These inosines are subsequently read as guanosines by the ribosome, with potentially significant consequences for protein sequence. In mammals, two such A-to-I RNA editases are RED1, which edits some serotonin and glutamate receptors, and RED2, with unidentified substrates. To study the evolutionary conservation among these editases, we have isolated homologous genes from the Japanese pufferfish, Fugu rubripes. Fugu has two genes homologous to Red1 that are similar in size and organization and that show a fivefold compaction relative to the human gene; they differ, however, in their base compositional features. The Fugu gene for RED2 is unusually large, spanning more than 50kb; within the largest intron, there is evidence for a novel gene on the opposite strand. Because of these unusual features, the partial genomic structure was determined for the mouse RED2 gene. A partial cDNA for RED1 was also isolated from zebrafish. Comparisons between fish and between fish and mammals of the protein sequences show that the catalytic domains are highly conserved for each gene, while the RNA-binding domains vary within a single protein in their levels of conservation. Different levels of conservation among domains of different functional roles may reflect differences in editase substrate specificity and/or substrate sequence conservation. PMID:10854777

  8. Difference in microRNA expression and editing profile of lung tissues from different pig breeds related to immune responses to HP-PRRSV.

    PubMed

    Li, Jia; Chen, Zhisheng; Zhao, Junlong; Fang, Liurong; Fang, Rui; Xiao, Jiang; Chen, Xing; Zhou, Ao; Zhang, Yingyin; Ren, Liming; Hu, Xiaoxiang; Zhao, Yaofeng; Zhang, Shujun; Li, Ning

    2015-01-01

    Porcine reproductive and respiratory syndrome (PRRS) is one of the most devastating diseases for the pig industry. Our goal was to identify microRNAs involved in the host immune response to PRRS. We generated microRNA expression profiles of lung tissues from Tongcheng or Landrace pigs infected with a highly pathogenic PRRS virus (PRRSV) at 3, 5, 7 dpi (day post infection) and control individuals from these two breeds. Our data showed that 278 known and 294 novel microRNAs were expressed in these combined microRNA transcriptomes. Compared with control individuals, almost half of the known microRNAs (116 in Tongcheng and 153 in Landrace) showed significantly differential expression (DEmiRNAs) at least once. The numbers of down-regulated DEmiRNAs were larger than the corresponding number of up-regulated DEmiRNAs in both breeds. Interestingly, miR-2320-5p, which was predicted to bind to conserved sequences of the PRRSV genome, was down-regulated significantly at 3 dpi after PRRSV infection in both breeds. In addition, PRRSV infection induced a significant increase of microRNA editing level in both breeds. Our results provide novel insight into the role of microRNA in response to PRRSV infection in vivo, which will aid the research for developing novel therapies against PRRSV. PMID:25856272

  9. Synthetic RNA Polymerase III Promoters Facilitate High-Efficiency CRISPR-Cas9-Mediated Genome Editing in Yarrowia lipolytica.

    PubMed

    Schwartz, Cory M; Hussain, Murtaza Shabbir; Blenner, Mark; Wheeldon, Ian

    2016-04-15

    The oleaginous yeast Yarrowia lipolytica is a valuable microbial host for chemical production because it has a high capacity to synthesize, modify, and store intracellular lipids; however, rapid strain development has been hampered by the limited availability of genome engineering tools. We address this limitation by adapting the CRISPR-Cas9 system from Streptococcus pyogenes for markerless gene disruption and integration in Y. lipolytica. Single gene disruption efficiencies of 92% and higher were achieved when single guide RNAs (sgRNA) were transcribed with synthetic hybrid promoters that combine native RNA polymerase III (Pol III) promoters with tRNA. The Pol III-tRNA hybrid promoters exploit endogenous tRNA processing to produce mature sgRNA for Cas9 targeting. The highest efficiencies were achieved with a SCR1'-tRNA(Gly) promoter and Y. lipolytica codon-optimized Cas9 expressed from a UAS1B8-TEF promoter. Cotransformation of the Cas9 and sgRNA expressing plasmid with a homologous recombination donor plasmid resulted in markerless homologous recombination efficiency of over 64%. Homologous recombination was observed in 100% of transformants when nonhomologous end joining was disrupted. The end result of these studies was the development of pCRISPRyl, a modular tool for markerless gene disruption and integration in Y. lipolytica. PMID:26714206

  10. Rapid construction of multiple sgRNA vectors and knockout of the Arabidopsis IAA2 gene using the CRISPR/Cas9 genomic editing technology.

    PubMed

    Dingyuan, Liu; Ting, Qiu; Xiaohui, Ding; Miaomiao, Li; Muyuan, Zhu; Junhui, Wang

    2016-08-01

    IAA2 is a member of the Aux/IAA auxin responsive gene family in Arabidopsis thaliana. No iaa2 mutant has been reported until now, thus hindering its further mechanistic investigations. The normal genomic editing technology of CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) uses only a single guide RNA (sgRNA) to target one site in a specific gene, and the gene knockout efficiency is not high. Instead, multiple sgRNAs can target multiple sites; therefore, the efficiency may be improved. In the present investigation, we used the golden-gate cloning strategy and two rounds of PCR reactions to combine three sgRNAs in the same entry vector. The final expression vector was obtained by LR reactions with the destination vector containing the Cas9 expression cassette. Four out of the six sgRNAs were effective, and we also obtained a lot of insertion and deletion mutations. Compared with one sgRNA approach, multiple sgRNAs displayed higher gene-knockout efficiency and produced more germ-line mutants. Thus, we established a more rapid and efficient method and generated five mutants for further studies of IAA2 functions. PMID:27531614

  11. Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei

    PubMed Central

    McDermott, Suzanne M.; Carnes, Jason

    2015-01-01

    KREPB5 is an essential component of ∼20S editosomes in Trypanosoma brucei which contains a degenerate, noncatalytic RNase III domain. To explore the function of this protein, we used a novel approach to make and screen numerous conditional null T. brucei bloodstream form cell lines that express randomly mutagenized KREPB5 alleles. We identified nine single amino acid substitutions that could not complement the conditional loss of wild-type KREPB5. Seven of these were within the RNase III domain, and two were in the C-terminal region that has no homology to known motifs. Exclusive expression of these mutated KREPB5 alleles in the absence of wild-type allele expression resulted in growth inhibition, the loss of ∼20S editosomes, and inhibition of RNA editing in BF cells. Eight of these mutations were lethal in bloodstream form parasites but not in procyclic-form parasites, showing that multiple domains function in a life cycle-dependent manner. Amino acid changes at a substantial number of positions, including up to 7 per allele, allowed complementation and thus did not block KREPB5 function. Hence, the degenerate RNase III domain and a newly identified domain are critical for KREPB5 function and have differential effects between the life cycle stages of T. brucei that differentially edit mRNAs. PMID:26370513

  12. Carbon-proton scalar couplings in RNA. 3D heteronuclear and 2D isotope-edited NMR of a [sup 13]C-labeled extra-stable hairpin

    SciTech Connect

    Hines, J.V.; Landry, S.M.; Varani, G.; Tinoco, I. Jr. Lawrence Berkeley Lab., CA )

    1994-06-29

    Long range carbon-proton scalar couplings were measured for an RNA hairpin of 12 nucleotides using 3D and [sup 13]C-edited 2D NMR. The large one-bond carbon-proton scalar couplings ([sup 1]J[sub CH]) and small n-bond couplings ([sup 1]J[sub CH]) produce ECOSY type cross-peaks, thus facilitating the determination of the sign and magnitude of the smaller [sup 2]J[sub CH] or [sup 3]J[sub CH]. The UUCGRNA hairpin (5[prime]-rGGACUUCGGUCC-3[prime]), whose structure has been determined by our laboratory, was uniformly [sup 13]C-labeled at 30% isotopic enrichment. The observed [sup 1]J[sub CH] couplings were then correlated to the known structure. The signs of [sup 2]J[sub C4[prime]H5[prime

  13. CRISPR/CAS9-Mediated Genome Editing of miRNA-155 Inhibits Proinflammatory Cytokine Production by RAW264.7 Cells

    PubMed Central

    Jing, Weixia; Zhang, Xuewu; Sun, Wenyan; Hou, Xiujuan; Yao, Zhongqiang; Zhu, Yuelan

    2015-01-01

    MicroRNA 155 (miR-155) is a key proinflammatory regulator in clinical and experimental rheumatoid arthritis (RA). Here we generated a miR-155 genome knockout (GKO) RAW264.7 macrophage cell line using the clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CAS9) technology. While upregulating the Src homology-2 domain-containing inositol 5-phosphatase 1 (SHIP1), the miR-155 GKO line is severely impaired in producing proinflammatory cytokines but slightly increased in osteoclastogenesis upon treatment with receptor activator of nuclear factor-κB ligand (RANKL). Taken together, our results suggest that genome editing of miR-155 holds the potential as a therapeutic strategy in RA. PMID:26697483

  14. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  15. An AU-Rich Sequence Element (UUUN[A/U]U) Downstream of the Edited C in Apolipoprotein B mRNA Is a High-Affinity Binding Site for Apobec-1: Binding of Apobec-1 to This Motif in the 3′ Untranslated Region of c-myc Increases mRNA Stability

    PubMed Central

    Anant, Shrikant; Davidson, Nicholas O.

    2000-01-01

    Apobec-1, the catalytic subunit of the mammalian apolipoprotein B (apoB) mRNA-editing enzyme, is a cytidine deaminase with RNA binding activity for AU-rich sequences. This RNA binding activity is required for Apobec-1 to mediate C-to-U RNA editing. Filter binding assays, using immobilized Apobec-1, demonstrate saturable binding to a 105-nt apoB RNA with a Kd of ∼435 nM. A series of AU-rich templates was used to identify a high-affinity (∼50 nM) binding site of consensus sequence UUUN[A/U]U, with multiple copies of this sequence constituting the high-affinity binding site. In order to determine whether this consensus site could be functionally demonstrated from within an apoB RNA, circular-permutation analysis was performed, revealing one major (UUUGAU) and one minor (UU) site located 3 and 16 nucleotides, respectively, downstream of the edited base. Secondary-structure predictions reveal a stem-loop flanking the edited base with Apobec-1 binding to the consensus site(s) at an open loop. A similar consensus (AUUUA) is present in the 3′ untranslated regions of several mRNAs, including that of c-myc, that are known to undergo rapid degradation. In this context, it is presumed that the consensus motif acts as a destabilizing element. As an independent test of the ability of Apobec-1 to bind to this sequence, F442A cells were transfected with Apobec-1 and the half-life of c-myc mRNA was determined following actinomycin D treatment. These studies demonstrated an increase in the half-life of c-myc mRNA from 90 to 240 min in control versus Apobec-1-expressing cells. Apobec-1 expression mutants, in which RNA binding activity is eliminated, failed to alter c-myc mRNA turnover. Taken together, the data establish a consensus binding site for Apobec-1 embedded in proximity to the edited base in apoB RNA. Binding to this site in other target RNAs raises the possibility that Apobec-1 may be involved in other aspects of RNA metabolism, independent of its role as an apoB RNA

  16. A systematic study of genetic algorithms with genotype editing

    SciTech Connect

    Huang, C. F.; Rocha, L. M.

    2004-01-01

    This paper presents our systematic study on an RNA-editing computational model of Genetic Algorithms (GA). This model is constructed based on several genetic editing characteristics that are gleaned from the RNA editing system as observed in several organisms. We have expanded the traditional Genetic Algorithm with artificial editing mechanisms as proposed by [15]. The incorporation of editing mechanisms provides a means for artificial agents with genetic descriptions to gain greater phenotypic plasticity, which may be environmentally regulated. The systematic study of this RNA-editing model has shed some light into the evolutionary implications of RNA editing and how to select proper RNA editors for design of more robust GAS. The results will also show promising applications to complex real-world problems. We expect that the framework proposed will both facilitate determining the evolutionary role of RNA editing in biology, and advance the current state of research in Evolutionary Computation.

  17. To edit or not to edit: regulation of ADAR editing specificity and efficiency.

    PubMed

    Deffit, Sarah N; Hundley, Heather A

    2016-01-01

    Hundreds to millions of adenosine (A)-to-inosine (I) modifications are present in eukaryotic transcriptomes and play an essential role in the creation of proteomic and phenotypic diversity. As adenosine and inosine have different base-pairing properties, the functional consequences of these modifications or 'edits' include altering coding potential, splicing, and miRNA-mediated gene silencing of transcripts. However, rather than serving as a static control of gene expression, A-to-I editing provides a means to dynamically rewire the genetic code during development and in a cell-type specific manner. Interestingly, during normal development, in specific cells, and in both neuropathological diseases and cancers, the extent of RNA editing does not directly correlate with levels of the substrate mRNA or the adenosine deaminase that act on RNA (ADAR) editing enzymes, implying that cellular factors are required for spatiotemporal regulation of A-to-I editing. The factors that affect the specificity and extent of ADAR activity have been thoroughly dissected in vitro. Yet, we still lack a complete understanding of how specific ADAR family members can selectively deaminate certain adenosines while others cannot. Additionally, in the cellular environment, ADAR specificity and editing efficiency is likely to be influenced by cellular factors, which is currently an area of intense investigation. Data from many groups have suggested two main mechanisms for controlling A-to-I editing in the cell: (1) regulating ADAR accessibility to target RNAs and (2) protein-protein interactions that directly alter ADAR enzymatic activity. Recent studies suggest cis- and trans-acting RNA elements, heterodimerization and RNA-binding proteins play important roles in regulating RNA editing levels in vivo. WIREs RNA 2016, 7:113-127. doi: 10.1002/wrna.1319. PMID:26612708

  18. Composition of the editing complex of Trypanosoma brucei.

    PubMed

    Stuart, K; Panigrahi, A K; Schnaufer, A; Drozdz, M; Clayton, C; Salavati, R

    2002-01-29

    The RNA editing that produces most functional mRNAs in trypanosomes is catalysed by a multiprotein complex. This complex catalyses the endoribonucleolytic cleavage, uridylate addition and removal, and RNA ligation steps of the editing process. Enzymatic and in vitro editing analyses reveal that each catalytic step contributes to the specificity of the editing and, together with the interaction between gRNA and the mRNA, results in precisely edited mRNAs. Tandem mass spectrometric analysis was used to identify the genes for several components of biochemically purified editing complexes. Their identity and presence in the editing complex were confirmed using immunochemical analyses utilizing mAbs specific to the editing complex components. The genes for two RNA ligases were identified. Genetic studies show that some, but not all, of the components of the complex are essential for editing. The TbMP52 RNA ligase is essential for editing while the TbMP48 RNA ligase is not. Editing was found to be essential in bloodstream form trypanosomes. This is surprising because mutants devoid of genes encoding RNAs that become edited survive as bloodstream forms but encouraging since editing complex components may be targets for chemotherapy. PMID:11839184

  19. Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis1[OPEN

    PubMed Central

    Law, Yee-Song; Zhang, Renshan; Guan, Xiaoqian; Cheng, Shifeng; Sun, Feng; Duncan, Owen; Murcha, Monika W.; Whelan, James; Lim, Boon Leong

    2015-01-01

    The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated. PMID:26304849

  20. Fasting decreases apolipoprotein B mRNA editing and the secretion of small molecular weight apoB by rat hepatocytes: Evidence that the total amount of apoB secreted is regulated post-transcriptionally

    SciTech Connect

    Leighton, J.K.; Joyner, J.; Zamarripa, J.; Deines, M.; Davis, R.A. )

    1990-09-01

    Two different molecular weight forms of apoB are produced from a common initial transcript via editing of a Gln codon (CAA) to a stop codon (UAA), leading to a truncated translation product (apo BS) that consists of the amino terminal half of the larger form (apoBL). Previous studies have shown that fasting coordinately decreases lipogenesis and the secretion of very low density lipoprotein (VLDL) lipids and apoBS. Secretion of the apoBL is unaffected by fasting. We studied whether editing of apoB RNA is repressed by fasting, thus accounting for the selective decreased secretion of apoBS. Column chromatography of (35S)methionine-labeled lipoproteins secreted by hepatocytes from fed rats showed that essentially all of apoBL is secreted in the VLDL fraction, whereas a significant amount (15%) of apoBS is secreted associated as lipoproteins eluting in the HDL fractions. Fasting decreased the relative amount of apoBS that eluted in the VLDL fractions and increased the amount secreted in the HDL fractions. Consistent with previous results, hepatocytes from fasted rats show a selective twofold decrease in apoBS secretion. Fasting did not affect the relative abundance of apoB RNA, determined by slot blot hybridization assays using two different 32P-labeled cDNA probes coding either for both molecular weight forms or for only the large molecular weight form. However, quantitative of the editing of apoB RNA showed that fasting caused a 60% decrease in the amount of apoB RNA possessing the stop codon. These data show that the editing of apoB RNA is sensitive to metabolic state (i.e., fasting) resulting in a selective decrease in the secretion of apoBS. However, since the total secretion of apoB was decreased by fasting, while apoB mRNA levels remained constant, additional (post-transcriptional) mechanisms play a role in regulating apoB secretion.

  1. An agent based model of genotype editing

    SciTech Connect

    Rocha, L. M.; Huang, C. F.

    2004-01-01

    This paper presents our investigation on an agent-based model of Genotype Editing. This model is based on several characteristics that are gleaned from the RNA editing system as observed in several organisms. The incorporation of editing mechanisms in an evolutionary agent-based model provides a means for evolving agents with heterogenous post-transcriptional processes. The study of this agent-based genotype-editing model has shed some light into the evolutionary implications of RNA editing as well as established an advantageous evolutionary computation algorithm for machine learning. We expect that our proposed model may both facilitate determining the evolutionary role of RNA editing in biology, and advance the current state of research in agent-based optimization.

  2. ADAR1 Activation Drives Leukemia Stem Cell Self-Renewal by Impairing Let-7 Biogenesis.

    PubMed

    Zipeto, Maria Anna; Court, Angela C; Sadarangani, Anil; Delos Santos, Nathaniel P; Balaian, Larisa; Chun, Hye-Jung; Pineda, Gabriel; Morris, Sheldon R; Mason, Cayla N; Geron, Ifat; Barrett, Christian; Goff, Daniel J; Wall, Russell; Pellecchia, Maurizio; Minden, Mark; Frazer, Kelly A; Marra, Marco A; Crews, Leslie A; Jiang, Qingfei; Jamieson, Catriona H M

    2016-08-01

    Post-transcriptional adenosine-to-inosine RNA editing mediated by adenosine deaminase acting on RNA1 (ADAR1) promotes cancer progression and therapeutic resistance. However, ADAR1 editase-dependent mechanisms governing leukemia stem cell (LSC) generation have not been elucidated. In blast crisis chronic myeloid leukemia (BC CML), we show that increased JAK2 signaling and BCR-ABL1 amplification activate ADAR1. In a humanized BC CML mouse model, combined JAK2 and BCR-ABL1 inhibition prevents LSC self-renewal commensurate with ADAR1 downregulation. Lentiviral ADAR1 wild-type, but not an editing-defective ADAR1(E912A) mutant, induces self-renewal gene expression and impairs biogenesis of stem cell regulatory let-7 microRNAs. Combined RNA sequencing, qRT-PCR, CLIP-ADAR1, and pri-let-7 mutagenesis data suggest that ADAR1 promotes LSC generation via let-7 pri-microRNA editing and LIN28B upregulation. A small-molecule tool compound antagonizes ADAR1's effect on LSC self-renewal in stromal co-cultures and restores let-7 biogenesis. Thus, ADAR1 activation represents a unique therapeutic vulnerability in LSCs with active JAK2 signaling. PMID:27292188

  3. Substrate recognition by ADAR1 and ADAR2.

    PubMed Central

    Wong, S K; Sato, S; Lazinski, D W

    2001-01-01

    RNA editing catalyzed by ADAR1 and ADAR2 involves the site-specific conversion of adenosine to inosine within imperfectly duplexed RNA. ADAR1- and ADAR2-mediated editing occurs within transcripts of glutamate receptors (GluR) in the brain and in hepatitis delta virus (HDV) RNA in the liver. Although the Q/R site within the GluR-B premessage is edited more efficiently by ADAR2 than it is by ADAR1, the converse is true for the +60 site within this same transcript. ADAR1 and ADAR2 are homologs having two common functional regions, an N-terminal double-stranded RNA-binding domain and a C-terminal deaminase domain. It is neither understood why only certain adenosines within a substrate molecule serve as targets for ADARs, nor is it known which domain of an ADAR confers its specificity for particular editing sites. To assess the importance of several aspects of RNA sequence and structure on editing, we evaluated 20 different mutated substrates, derived from four editing sites, for their ability to be edited by either ADAR1 or ADAR2. We found that when these derivatives contained an A:C mismatch at the editing site, editing by both ADARs was enhanced compared to when A:A or A:G mismatches or A:U base pairs occurred at the same site. Hence substrate recognition and/or catalysis by ADARs could involve the base that opposes the edited adenosine. In addition, by using protein chimeras in which the deaminase domains were exchanged between ADAR1 and ADAR2, we found that this domain played a dominant role in defining the substrate specificity of the resulting enzyme. PMID:11421361

  4. Non-GMO genetically edited crop plants.

    PubMed

    Kanchiswamy, Chidananda Nagamangala; Malnoy, Mickael; Velasco, Riccardo; Kim, Jin-Soo; Viola, Roberto

    2015-09-01

    Direct delivery of purified Cas9 protein with guide RNA into plant cells, as opposed to plasmid-mediated delivery, displays high efficiency and reduced off-target effects. Following regeneration from edited cells, the ensuing plant is also likely to bypass genetically modified organism (GMO) legislation as the genome editing complex is degraded in the recipient cells. PMID:25978870

  5. High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing.

    PubMed

    Simpson, Rachel M; Bruno, Andrew E; Bard, Jonathan E; Buck, Michael J; Read, Laurie K

    2016-05-01

    Uridine insertion/deletion RNA editing in kinetoplastids entails the addition and deletion of uridine residues throughout the length of mitochondrial transcripts to generate translatable mRNAs. This complex process requires the coordinated use of several multiprotein complexes as well as the sequential use of noncoding template RNAs called guide RNAs. The majority of steady-state mitochondrial mRNAs are partially edited and often contain regions of mis-editing, termed junctions, whose role is unclear. Here, we report a novel method for sequencing entire populations of pre-edited partially edited, and fully edited RNAs and analyzing editing characteristics across populations using a new bioinformatics tool, the Trypanosome RNA Editing Alignment Tool (TREAT). Using TREAT, we examined populations of two transcripts, RPS12 and ND7-5', in wild-typeTrypanosoma brucei We provide evidence that the majority of partially edited sequences contain junctions, that intrinsic pause sites arise during the progression of editing, and that the mechanisms that mediate pausing in the generation of canonical fully edited sequences are distinct from those that mediate the ends of junction regions. Furthermore, we identify alternatively edited sequences that constitute plausible alternative open reading frames and identify substantial variability in the 5' UTRs of both canonical and alternatively edited sequences. This work is the first to use high-throughput sequencing to examine full-length sequences of whole populations of partially edited transcripts. Our method is highly applicable to current questions in the RNA editing field, including defining mechanisms of action for editing factors and identifying potential alternatively edited sequences. PMID:26908922

  6. Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing.

    PubMed

    Hashimoto, Masakazu; Takemoto, Tatsuya

    2015-01-01

    Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient, and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse. PMID:26066060

  7. Convergent Evolution of Fern-Specific Mitochondrial Group II Intron atp1i361g2 and Its Ancient Source Paralogue rps3i249g2 and Independent Losses of Intron and RNA Editing among Pteridaceae.

    PubMed

    Zumkeller, Simon Maria; Knoop, Volker; Knie, Nils

    2016-01-01

    Mitochondrial intron patterns are highly divergent between the major land plant clades. An intron in the atp1 gene, atp1i361g2, is an example for a group II intron specific to monilophytes (ferns). Here, we report that atp1i361g2 is lost independently at least 4 times in the fern family Pteridaceae. Such plant organelle intron losses have previously been found to be accompanied by loss of RNA editing sites in the flanking exon regions as a consequence of genomic recombination of mature cDNA. Instead, we now observe that RNA editing events in both directions of pyrimidine exchange (C-to-U and U-to-C) are retained in atp1 exons after loss of the intron in Pteris argyraea/biaurita and in Actiniopteris and Onychium We find that atp1i361g2 has significant similarity with intron rps3i249g2 present in lycophytes and gymnosperms, which we now also find highly conserved in ferns. We conclude that atp1i361g2 may have originated from the more ancestral rps3i249g2 paralogue by a reverse splicing copy event early in the evolution of monilophytes. Secondary structure elements of the two introns, most characteristically their domains III, show strikingly convergent evolution in the monilophytes. Moreover, the intron paralogue rps3i249g2 reveals relaxed evolution in taxa where the atp1i361g2 paralogue is lost. Our findings may reflect convergent evolution of the two related mitochondrial introns exerted by co-evolution with an intron-binding protein simultaneously acting on the two paralogues. PMID:27492234

  8. Convergent Evolution of Fern-Specific Mitochondrial Group II Intron atp1i361g2 and Its Ancient Source Paralogue rps3i249g2 and Independent Losses of Intron and RNA Editing among Pteridaceae

    PubMed Central

    Zumkeller, Simon Maria; Knoop, Volker; Knie, Nils

    2016-01-01

    Mitochondrial intron patterns are highly divergent between the major land plant clades. An intron in the atp1 gene, atp1i361g2, is an example for a group II intron specific to monilophytes (ferns). Here, we report that atp1i361g2 is lost independently at least 4 times in the fern family Pteridaceae. Such plant organelle intron losses have previously been found to be accompanied by loss of RNA editing sites in the flanking exon regions as a consequence of genomic recombination of mature cDNA. Instead, we now observe that RNA editing events in both directions of pyrimidine exchange (C-to-U and U-to-C) are retained in atp1 exons after loss of the intron in Pteris argyraea/biaurita and in Actiniopteris and Onychium. We find that atp1i361g2 has significant similarity with intron rps3i249g2 present in lycophytes and gymnosperms, which we now also find highly conserved in ferns. We conclude that atp1i361g2 may have originated from the more ancestral rps3i249g2 paralogue by a reverse splicing copy event early in the evolution of monilophytes. Secondary structure elements of the two introns, most characteristically their domains III, show strikingly convergent evolution in the monilophytes. Moreover, the intron paralogue rps3i249g2 reveals relaxed evolution in taxa where the atp1i361g2 paralogue is lost. Our findings may reflect convergent evolution of the two related mitochondrial introns exerted by co-evolution with an intron-binding protein simultaneously acting on the two paralogues. PMID:27492234

  9. Genome editing comes of age.

    PubMed

    Kim, Jin-Soo

    2016-09-01

    Genome editing harnesses programmable nucleases to cut and paste genetic information in a targeted manner in living cells and organisms. Here, I review the development of programmable nucleases, including zinc finger nucleases (ZFNs), TAL (transcription-activator-like) effector nucleases (TALENs) and CRISPR (cluster of regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) RNA-guided endonucleases (RGENs). I specifically highlight the key advances that set the foundation for the rapid and widespread implementation of CRISPR-Cas9 genome editing approaches that has revolutionized the field. PMID:27490630

  10. The Technique of Film Editing. Enlarged Edition.

    ERIC Educational Resources Information Center

    Reisz, Karel; Millar, Gavin

    Film editing is discussed from the point of view, not only of the person in the cutting room, but also of the person who has responsibility for the final film. Part I outlines the history of editing from the silent film to 1953. It discusses the practice of editing for action, dialogue, comedy, and montage sequences, as well as in documentaries,…

  11. The complete nucleotide sequence of the cassava (Manihot esculenta) chloroplast genome and the evolution of atpF in Malpighiales: RNA editing and multiple losses of a group II intron

    PubMed Central

    Wurdack, Kenneth J.; Kanagaraj, Anderson; Lee, Seung-Bum; Saski, Christopher; Jansen, Robert K.

    2008-01-01

    The complete sequence of the chloroplast genome of cassava (Manihot esculenta, Euphorbiaceae) has been determined. The genome is 161,453 bp in length and includes a pair of inverted repeats (IR) of 26,954 bp. The genome includes 128 genes; 96 are single copy and 16 are duplicated in the IR. There are four rRNA genes and 30 distinct tRNAs, seven of which are duplicated in the IR. The infA gene is absent; expansion of IRb has duplicated 62 amino acids at the 3′ end of rps19 and a number of coding regions have large insertions or deletions, including insertions within the 23S rRNA gene. There are 17 intron-containing genes in cassava, 15 of which have a single intron while two (clpP, ycf3) have two introns. The usually conserved atpF group II intron is absent and this is the first report of its loss from land plant chloroplast genomes. The phylogenetic distribution of the atpF intron loss was determined by a PCR survey of 251 taxa representing 34 families of Malpighiales and 16 taxa from closely related rosids. The atpF intron is not only missing in cassava but also from closely related Euphorbiaceae and other Malpighiales, suggesting that there have been at least seven independent losses. In cassava and all other sequenced Malphigiales, atpF gene sequences showed a strong association between C-to-T substitutions at nucleotide position 92 and the loss of the intron, suggesting that recombination between an edited mRNA and the atpF gene may be a possible mechanism for the intron loss. PMID:18214421

  12. Biology Today: Respect for RNA.

    ERIC Educational Resources Information Center

    Flannery, Maura C., Ed.

    1991-01-01

    The high points of the story of RNA are presented. The functions of RNA within the cell, how these functions are carried out, and how they evolved are described. The topics of splicing, self-splicing, RNA editing, transcription and translation, and antisense RNA are discussed. (KR)

  13. Genetic Architecture of Mitochondrial Editing in Arabidopsis thaliana

    PubMed Central

    Bentolila, Stéphane; Elliott, Leah E.; Hanson, Maureen R.

    2008-01-01

    We have analyzed the mitochondrial editing behavior of two Arabidopsis thaliana accessions, Landsberg erecta (Ler) and Columbia (Col). A survey of 362 C-to-U editing sites in 33 mitochondrial genes was conducted on RNA extracted from rosette leaves. We detected 67 new editing events in A. thaliana rosette leaves that had not been observed in a prior study of mitochondrial editing in suspension cultures. Furthermore, 37 of the 441 C-to-U editing events reported in A. thaliana suspension cultures were not observed in rosette leaves. Forty editing sites that are polymorphic in extent of editing were detected between Col and Ler. Silent editing sites, which do not change the encoded amino acid, were found in a large excess compared to nonsilent sites among the editing events that differed between accessions and between tissue types. Dominance relationships were assessed for 15 of the most polymorphic sites by evaluating the editing values of the reciprocal hybrids. Dominance is more common in nonsilent sites than in silent sites, while additivity was observed only in silent sites. A maternal effect was detected for 8 sites. QTL mapping with recombinant inbred lines detected 12 major QTL for 11 of the 13 editing traits analyzed, demonstrating that efficiency of editing of individual mitochondrial C targets is generally governed by a major factor. PMID:17565941

  14. Basic Wiring. Third Edition. Teacher Edition [and] Student Edition.

    ERIC Educational Resources Information Center

    Kaltwasser, Stan; Flowers, Gary; Blasingame, Don; Batson, Larry; Ipock, Dan; Carroll, Charles; Friesen, Wade; Fleming, Glenn

    This publication contains both a teacher edition and a student edition of materials for a foundation course in an electrical wiring program. The course introduces basic concepts and skills that are prerequisites to residential wiring and commercial and industrial wiring courses. The contents of the materials are tied to measurable and observable…

  15. Major Appliance Repair. Teacher Edition and Student Edition. Second Edition.

    ERIC Educational Resources Information Center

    Smreker, Gene; Calvert, King

    This second edition contains teacher and student guides for 14 units of instruction in major appliance repair. Each unit in the teacher edition includes some or all of the following basic components: objective sheet, suggested activities, answers to assignment sheets, answers to the written test, written test, a unit evaluation form, teacher…

  16. Diesel Technology: Introduction. Teacher Edition [and] Student Edition. Second Edition.

    ERIC Educational Resources Information Center

    Joerschke, John D.; Eichhorn, Lane

    This complete teacher edition of a diesel technology course consists of introductory pages, teacher pages, and the student edition. The introductory pages provide these tools: training and competency profile; National Automotive Technicians Education Foundation Crosswalk; instructional/task analysis; basic skills icons and classifications; basic…

  17. The levels of edit, second edition

    NASA Technical Reports Server (NTRS)

    Vanburen, R.; Buehler, M. F.

    1980-01-01

    The editorial process is analyzed, and five levels of edit are identified. These levels represent cumulative combinations of nine types of edit: Coordination, Policy, Integrity, Screening, Copy Clarification, Format, Mechanical Style, Language, and Substantive. The levels and types of edit, although developed for specific use with external reports at the Jet Propulsion Laboratory, cover the general range of technical editing, especially as it applies to an in-house technical publications organization. Each type of edit is set forth in terms of groups of actions to be performed by editor. The edit-level concept has enhanced understanding and communication among editors, authors, and publications managers concerning the specific editorial work to be done on each manuscript. It has also proved useful as a management tool for estimating and monitoring cost.

  18. Wikipedia editing dynamics

    NASA Astrophysics Data System (ADS)

    Gandica, Y.; Carvalho, J.; Sampaio dos Aidos, F.

    2015-01-01

    A model for the probabilistic function followed in editing Wikipedia is presented and compared with simulations and real data. It is argued that the probability of editing is proportional to the editor's number of previous edits (preferential attachment), to the editor's fitness, and to an aging factor. Using these simple ingredients, it is possible to reproduce the results obtained for Wikipedia editing dynamics for a collection of single pages as well as the averaged results. Using a stochastic process framework, a recursive equation was obtained for the average of the number of edits per editor that seems to describe the editing behavior in Wikipedia.

  19. Wikipedia editing dynamics.

    PubMed

    Gandica, Y; Carvalho, J; Sampaio Dos Aidos, F

    2015-01-01

    A model for the probabilistic function followed in editing Wikipedia is presented and compared with simulations and real data. It is argued that the probability of editing is proportional to the editor's number of previous edits (preferential attachment), to the editor's fitness, and to an aging factor. Using these simple ingredients, it is possible to reproduce the results obtained for Wikipedia editing dynamics for a collection of single pages as well as the averaged results. Using a stochastic process framework, a recursive equation was obtained for the average of the number of edits per editor that seems to describe the editing behavior in Wikipedia. PMID:25679673

  20. A minimalist mitochondrial threonyl-tRNA synthetase exhibits tRNA-isoacceptor specificity during proofreading.

    PubMed

    Zhou, Xiao-Long; Ruan, Zhi-Rong; Wang, Meng; Fang, Zhi-Peng; Wang, Yong; Chen, Yun; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2014-12-16

    Yeast mitochondria contain a minimalist threonyl-tRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNA(Thr)2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNA(Thr)1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNA(Thr)s during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pre-transfer editing activity. Remarkably, only the usual tRNA(Thr)2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNA(Thr)1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of a ScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNA(Thr)1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pre-transfer editing takes place in the aminoacylation catalytic core. PMID:25414329

  1. DNA-free genome editing methods for targeted crop improvement.

    PubMed

    Kanchiswamy, Chidananda Nagamangala

    2016-07-01

    Evolution of the next-generation clustered, regularly interspaced, short palindromic repeat/Cas9 (CRISPR/Cas9) genome editing tools, ribonucleoprotein (RNA)-guided endonuclease (RGEN) RNPs, is paving the way for developing DNA-free genetically edited crop plants. In this review, I discuss the various methods of RGEN RNPs tool delivery into plant cells and their limitations to adopt this technology to numerous crop plants. Furthermore, focus is given on the importance of developing DNA-free genome edited crop plants, including perennial crop plants. The possible regulation on the DNA-free, next-generation genome-edited crop plants is also highlighted. PMID:27100964

  2. Zinc-mediated binding of a low-molecular-weight stabilizer of the host anti-viral factor apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G.

    PubMed

    Radwan, Mohamed O; Sonoda, Sachiko; Ejima, Tomohiko; Tanaka, Ayumi; Koga, Ryoko; Okamoto, Yoshinari; Fujita, Mikako; Otsuka, Masami

    2016-09-15

    Apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G (APOBEC3G, A3G), is a human anti-virus restriction protein which works deaminase-dependently and -independently. A3G is known to be ubiquitinated by HIV-1 viral infectivity factor (Vif) protein, leading to proteasomal degradation. A3G contains two zinc ions at the N-terminal domain and the C-terminal domain. Four lysine residues, K(297), K(301), K(303), and K(334), are known to be required for Vif-mediated A3G ubiquitination and degradation. Previously, we reported compound SN-1, a zinc chelator that increases steady-state expression level of A3G in the presence of Vif. In this study, we prepared Biotin-SN-1, a biotinylated derivative of SN-1, to study the SN-1-A3G interaction. A pull-down assay revealed that Biotin-SN-1 bound A3G. A zinc-abstraction experiment indicated that SN-1 binds to the zinc site of A3G. We carried out a SN-1-A3G docking study using molecular operating environment. The calculations revealed that SN-1 binds to the C-terminal domain through Zn(2+), H(216), P(247), C(288), and Y(315). Notably, SN-1-binding covers the H(257), E(259), C(288), and C(291) residues that participate in zinc-mediated deamination, and the ubiquitination regions of A3G. The binding of SN-1 presumably perturbs the secondary structure between C(288) and Y(315), leading to less efficient ubiquitination. PMID:27475536

  3. Alignments of RNA structures.

    PubMed

    Blin, Guillaume; Denise, Alain; Dulucq, Serge; Herrbach, Claire; Touzet, Hélène

    2010-01-01

    We describe a theoretical unifying framework to express the comparison of RNA structures, which we call alignment hierarchy. This framework relies on the definition of common supersequences for arc-annotated sequences and encompasses the main existing models for RNA structure comparison based on trees and arc-annotated sequences with a variety of edit operations. It also gives rise to edit models that have not been studied yet. We provide a thorough analysis of the alignment hierarchy, including a new polynomial-time algorithm and an NP-completeness proof. The polynomial-time algorithm involves biologically relevant edit operations such as pairing or unpairing nucleotides. It has been implemented in a software, called gardenia, which is available at the Web server http://bioinfo.lifl.fr/RNA/gardenia. PMID:20431150

  4. Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing

    PubMed Central

    Chen, Zhiyuan; Hagen, Darren E.; Wang, Juanbin; Elsik, Christine G.; Ji, Tieming; Siqueira, Luiz G.; Hansen, Peter J.; Rivera, Rocío M.

    2016-01-01

    ABSTRACT Genomic imprinting is an epigenetic mechanism that leads to parental-allele-specific gene expression. Approximately 150 imprinted genes have been identified in humans and mice but less than 30 have been described as imprinted in cattle. For the purpose of de novo identification of imprinted genes in bovine, we determined global monoallelic gene expression in brain, skeletal muscle, liver, kidney and placenta of day ∼105 Bos taurus indicus × Bos taurus taurus F1 conceptuses using RNA sequencing. To accomplish this, we developed a bioinformatics pipeline to identify parent-specific single nucleotide polymorphism alleles after filtering adenosine to inosine (A-to-I) RNA editing sites. We identified 53 genes subject to monoallelic expression. Twenty three are genes known to be imprinted in the cow and an additional 7 have previously been characterized as imprinted in human and/or mouse that have not been reported as imprinted in cattle. Of the remaining 23 genes, we found that 10 are uncharacterized or unannotated transcripts located in known imprinted clusters, whereas the other 13 genes are distributed throughout the bovine genome and are not close to any known imprinted clusters. To exclude potential cis-eQTL effects on allele expression, we corroborated the parental specificity of monoallelic expression in day 86 Bos taurus taurus × Bos taurus taurus conceptuses and identified 8 novel bovine imprinted genes. Further, we identified 671 candidate A-to-I RNA editing sites and describe random X-inactivation in day 15 bovine extraembryonic membranes. Our results expand the imprinted gene list in bovine and demonstrate that monoallelic gene expression can be the result of cis-eQTL effects. PMID:27245094

  5. Global assessment of imprinted gene expression in the bovine conceptus by next generation sequencing.

    PubMed

    Chen, Zhiyuan; Hagen, Darren E; Wang, Juanbin; Elsik, Christine G; Ji, Tieming; Siqueira, Luiz G; Hansen, Peter J; Rivera, Rocío M

    2016-07-01

    Genomic imprinting is an epigenetic mechanism that leads to parental-allele-specific gene expression. Approximately 150 imprinted genes have been identified in humans and mice but less than 30 have been described as imprinted in cattle. For the purpose of de novo identification of imprinted genes in bovine, we determined global monoallelic gene expression in brain, skeletal muscle, liver, kidney and placenta of day ∼105 Bos taurus indicus × Bos taurus taurus F1 conceptuses using RNA sequencing. To accomplish this, we developed a bioinformatics pipeline to identify parent-specific single nucleotide polymorphism alleles after filtering adenosine to inosine (A-to-I) RNA editing sites. We identified 53 genes subject to monoallelic expression. Twenty three are genes known to be imprinted in the cow and an additional 7 have previously been characterized as imprinted in human and/or mouse that have not been reported as imprinted in cattle. Of the remaining 23 genes, we found that 10 are uncharacterized or unannotated transcripts located in known imprinted clusters, whereas the other 13 genes are distributed throughout the bovine genome and are not close to any known imprinted clusters. To exclude potential cis-eQTL effects on allele expression, we corroborated the parental specificity of monoallelic expression in day 86 Bos taurus taurus × Bos taurus taurus conceptuses and identified 8 novel bovine imprinted genes. Further, we identified 671 candidate A-to-I RNA editing sites and describe random X-inactivation in day 15 bovine extraembryonic membranes. Our results expand the imprinted gene list in bovine and demonstrate that monoallelic gene expression can be the result of cis-eQTL effects. PMID:27245094

  6. Developing new levels of edit

    SciTech Connect

    Prono, J.; DeLanoy, M.; Deupree, R.; Skiby, J.; Thompson, B.

    1998-07-01

    In 1985, the writing and editing group at Los Alamos National Laboratory established four levels of edit for technical reports. When a survey in 1994 showed that both authors and editors felt the levels were not meeting author needs, the authors set about revising them. Their goals were to simplify the editing process, focus editing on improving technical clarity, and ensure that value was added in editing. This paper describes the revision process and product -- three author-based levels of edit.

  7. Digital Video Editing

    ERIC Educational Resources Information Center

    McConnell, Terry

    2004-01-01

    Monica Adams, head librarian at Robinson Secondary in Fairfax country, Virginia, states that librarians should have the technical knowledge to support projects related to digital video editing. The process of digital video editing and the cables, storage issues and the computer system with software is described.

  8. A minimalist mitochondrial threonyl-tRNA synthetase exhibits tRNA-isoacceptor specificity during proofreading

    PubMed Central

    Zhou, Xiao-Long; Ruan, Zhi-Rong; Wang, Meng; Fang, Zhi-Peng; Wang, Yong; Chen, Yun; Liu, Ru-Juan; Eriani, Gilbert; Wang, En-Duo

    2014-01-01

    Yeast mitochondria contain a minimalist threonyl-tRNA synthetase (ThrRS) composed only of the catalytic core and tRNA binding domain but lacking the entire editing domain. Besides the usual tRNAThr2, some budding yeasts, such as Saccharomyces cerevisiae, also contain a non-canonical tRNAThr1 with an enlarged 8-nucleotide anticodon loop, reprograming the usual leucine CUN codons to threonine. This raises interesting questions about the aminoacylation fidelity of such ThrRSs and the possible contribution of the two tRNAThrs during editing. Here, we found that, despite the absence of the editing domain, S. cerevisiae mitochondrial ThrRS (ScmtThrRS) harbors a tRNA-dependent pre-transfer editing activity. Remarkably, only the usual tRNAThr2 stimulated pre-transfer editing, thus, establishing the first example of a synthetase exhibiting tRNA-isoacceptor specificity during pre-transfer editing. We also showed that the failure of tRNAThr1 to stimulate tRNA-dependent pre-transfer editing was due to the lack of an editing domain. Using assays of the complementation of a ScmtThrRS gene knockout strain, we showed that the catalytic core and tRNA binding domain of ScmtThrRS co-evolved to recognize the unusual tRNAThr1. In combination, the results provide insights into the tRNA-dependent editing process and suggest that tRNA-dependent pre-transfer editing takes place in the aminoacylation catalytic core. PMID:25414329

  9. CRISPR Genome Editing

    Cancer.gov

    A research article about a technique for gene editing known as CRISPR-Cas9. The technique has made it much easier and faster for cancer researchers to study mutations and test new therapeutic targets.

  10. Developing new levels of edit

    SciTech Connect

    Prono, J.K.

    1997-06-01

    Since 1985, Los Alamos National Laboratory (LANL) staff have had four levels of edit to choose from for technical reports. When a CQI survey showed that both authors and editors felt the levels were not meeting author needs, LANL set about revising them. The goals were to simplify the editing process, focus editing on improving technical clarity, and ensure value added in editing. This paper describes the revision process and product--three author-based levels of edit.

  11. The pivotal regulatory landscape of RNA modifications.

    PubMed

    Li, Sheng; Mason, Christopher E

    2014-01-01

    Posttranscriptionally modified nucleosides in RNA play integral roles in the cellular control of biological information that is encoded in DNA. The modifications of RNA span all three phylogenetic domains (Archaea, Bacteria, and Eukarya) and are pervasive across RNA types, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), and (less frequently) small nuclear RNA (snRNA) and microRNA (miRNA). Nucleotide modifications are also one of the most evolutionarily conserved properties of RNAs, and the sites of modification are under strong selective pressure. However, many of these modifications, as well as their prevalence and impact, have only recently been discovered. Here, we examine both labile and permanent modifications, from simple methylation to complex transcript alteration (RNA editing and intron retention); detail the models for their processing; and highlight remaining questions in the field of the epitranscriptome. PMID:24898039

  12. Engineered Viruses as Genome Editing Devices

    PubMed Central

    Chen, Xiaoyu; Gonçalves, Manuel A F V

    2016-01-01

    Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR−Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole. PMID:26336974

  13. Engineered Viruses as Genome Editing Devices.

    PubMed

    Chen, Xiaoyu; Gonçalves, Manuel A F V

    2016-03-01

    Genome editing based on sequence-specific designer nucleases, also known as programmable nucleases, seeks to modify in a targeted and precise manner the genetic information content of living cells. Delivering into cells designer nucleases alone or together with donor DNA templates, which serve as surrogate homologous recombination (HR) substrates, can result in gene knockouts or gene knock-ins, respectively. As engineered replication-defective viruses, viral vectors are having an increasingly important role as delivery vehicles for donor DNA templates and designer nucleases, namely, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated Cas9 (CRISPR-Cas9) nucleases, also known as RNA-guided nucleases (RGNs). We review this dual role played by engineered viral particles on genome editing while focusing on their main scaffolds, consisting of lentiviruses, adeno-associated viruses, and adenoviruses. In addition, the coverage of the growing body of research on the repurposing of viral vectors as delivery systems for genome editing tools is complemented with information regarding their main characteristics, pros, and cons. Finally, this information is framed by a concise description of the chief principles, tools, and applications of the genome editing field as a whole. PMID:26336974

  14. A-to-I editing of the 5HT2C receptor and behaviour.

    PubMed

    Gardiner, Katheleen; Du, Yunzhi

    2006-03-01

    Site-specific deamination of five adenosine residues in the pre-mRNA of the serotonin 2C receptor, 5HT2CR, alters the amino acid sequence of the encoded protein. Such RNA editing can produce 32 mRNA variants, encoding 24 protein isoforms that vary in biochemical and pharmacological properties. Because serotonin functions in the regulation of mood and behaviour, modulation of serotonin signalling by RNA editing may be relevant to such psychiatric disorders as anxiety and depression. Several recent human studies have reported changes in 5HT2CR editing in schizophrenia, major depression or suicide, but results are variable and not conclusive. Rodent studies have begun to examine effects of drug treatments and stress. Understanding the importance of 5HT2CR editing in mood and behaviour will be assisted by experiments designed to analyse multiple strains of mice, in different behavioural tests, with optimal evaluation of the time course of molecular changes. PMID:16769676

  15. Semiautomated improvement of RNA alignments

    PubMed Central

    Andersen, Ebbe S.; Lind-Thomsen, Allan; Knudsen, Bjarne; Kristensen, Susie E.; Havgaard, Jakob H.; Torarinsson, Elfar; Larsen, Niels; Zwieb, Christian; Sestoft, Peter; Kjems, Jørgen; Gorodkin, Jan

    2007-01-01

    We have developed a semiautomated RNA sequence editor (SARSE) that integrates tools for analyzing RNA alignments. The editor highlights different properties of the alignment by color, and its integrated analysis tools prevent the introduction of errors when doing alignment editing. SARSE readily connects to external tools to provide a flexible semiautomatic editing environment. A new method, Pcluster, is introduced for dividing the sequences of an RNA alignment into subgroups with secondary structure differences. Pcluster was used to evaluate 574 seed alignments obtained from the Rfam database and we identified 71 alignments with significant prediction of inconsistent base pairs and 102 alignments with significant prediction of novel base pairs. Four RNA families were used to illustrate how SARSE can be used to manually or automatically correct the inconsistent base pairs detected by Pcluster: the mir-399 RNA, vertebrate telomase RNA (vert-TR), bacterial transfer-messenger RNA (tmRNA), and the signal recognition particle (SRP) RNA. The general use of the method is illustrated by the ability to accommodate pseudoknots and handle even large and divergent RNA families. The open architecture of the SARSE editor makes it a flexible tool to improve all RNA alignments with relatively little human intervention. Online documentation and software are available at http://sarse.ku.dk. PMID:17804647

  16. Simple Genome Editing of Rodent Intact Embryos by Electroporation

    PubMed Central

    Kaneko, Takehito; Mashimo, Tomoji

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system is a powerful tool for genome editing in animals. Recently, new technology has been developed to genetically modify animals without using highly skilled techniques, such as pronuclear microinjection of endonucleases. Technique for animal knockout system by electroporation (TAKE) method is a simple and effective technology that produces knockout rats by introducing endonuclease mRNAs into intact embryos using electroporation. Using TAKE method and CRISPR/Cas system, the present study successfully produced knockout and knock-in mice and rats. The mice and rats derived from embryos electroporated with Cas9 mRNA, gRNA and single-stranded oligodeoxynucleotide (ssODN) comprised the edited targeted gene as a knockout (67% of mice and 88% of rats) or knock-in (both 33%). The TAKE method could be widely used as a powerful tool to produce genetically modified animals by genome editing. PMID:26556280

  17. Editing graphs for maximum effect

    SciTech Connect

    Murphy, P.W.; Rhiner, R.W.

    1991-01-08

    The paper contains over eighty rules for editing graphs, arranged under nine major headings in a logical sequence for editing all the graphs in a manuscript. It is excerpted from a monograph used at the Lawrence Livermore National Laboratory to train beginning technical editors in editing graphs; a corresponding Hypercard stack is also used in this training. 6 refs., 4 figs.

  18. MENTAL DEFICIENCY. SECOND EDITION.

    ERIC Educational Resources Information Center

    HILLIARD, L.T.; KIRMAN, BRIAN H.

    REVISED TO INCLUDE LEGISLATIVE AND ADMINISTRATIVE PROCEDURES NEW IN BRITAIN SINCE THE 1957 EDITION, THE TEXT INCLUDES RECENT ADVANCES IN ETIOLOGY, PATHOLOGY, AND TREATMENT OF MENTAL DEFICIENCY. CONSIDERATION OF THE BACKGROUND OF MENTAL DEFICIENCY INCLUDES HISTORICAL AND LEGAL ASPECTS, THE SOCIAL BACKGROUND OF MENTAL DEFECT, PRENATAL CAUSES OF…

  19. Behaviour Recovery. Second Edition

    ERIC Educational Resources Information Center

    Rogers, Bill

    2004-01-01

    This second edition of Behaviour Recovery puts emphasis on teaching behaviour concerning children with emotional and behavioural disorders (EBD). These children have many factors in their lives that affect their behaviour over which schools have limited control. This book acknowledges the challenge and explores the practical realities, options and…

  20. Beginning to edit physics

    SciTech Connect

    Murphy, P.W.

    1995-02-01

    A physicist-turned-editor shows you the basics required for copyediting physics papers (physical quantities, symbols, units, scientific notation, the structure of mathematical expressions, the nature of graphs), and points the way to learning enough ``editorial physics`` to begin substantive editing.

  1. Contaminant hydrogeology. 2. edition

    SciTech Connect

    Fetter, C.W.

    1999-08-01

    This book is a major revision of the 1993 edition and includes the important practical concepts that have since emerged. Contents include: mass transport in saturated media; transformation, retardation, and attenuation of solutes; flow and mass transport in the vadose zone; multiphase flow; inorganic chemicals in groundwater; organic compounds in groundwater; groundwater and soil monitoring; and site remediation.

  2. Editing Technical Writing.

    ERIC Educational Resources Information Center

    Samson, Donald C., Jr.

    Intended for students in upper-division technical communication courses and professionals in business and government who want to learn how to edit technical writing, this book describes what technical editors do and how they do it. Throughout the book are exercises that students can use as self-tests; answer keys are provided for checking work.…

  3. Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing

    PubMed Central

    Liang, Gang; Zhang, Huimin; Lou, Dengji; Yu, Diqiu

    2016-01-01

    The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers’ ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing. PMID:26891616

  4. Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes.

    PubMed

    Chen, Sean; Lee, Benjamin; Lee, Angus Yiu-Fai; Modzelewski, Andrew J; He, Lin

    2016-07-01

    The CRISPR/Cas9 system has been employed to efficiently edit the genomes of diverse model organisms. CRISPR-mediated mouse genome editing is typically accomplished by microinjection of Cas9 DNA/RNA and single guide RNA (sgRNA) into zygotes to generate modified animals in one step. However, microinjection is a technically demanding, labor-intensive, and costly procedure with poor embryo viability. Here, we describe a simple and economic electroporation-based strategy to deliver Cas9/sgRNA ribonucleoproteins into mouse zygotes with 100% efficiency for in vivo genome editing. Our methodology, designated as CRISPR RNP Electroporation of Zygotes (CRISPR-EZ), enables highly efficient and high-throughput genome editing in vivo, with a significant improvement in embryo viability compared with microinjection. Using CRISPR-EZ, we generated a variety of editing schemes in mouse embryos, including indel (insertion/deletion) mutations, point mutations, large deletions, and small insertions. In a proof-of-principle experiment, we used CRISPR-EZ to target the tyrosinase (Tyr) gene, achieving 88% bi-allelic editing and 42% homology-directed repair-mediated precise sequence modification in live mice. Taken together, CRISPR-EZ is simple, economic, high throughput, and highly efficient with the potential to replace microinjection for in vivo genome editing in mice and possibly in other mammals. PMID:27151215

  5. Caste-specific RNA editomes in the leaf-cutting ant Acromyrmex echinatior

    PubMed Central

    Li, Qiye; Wang, Zongji; Lian, Jinmin; Schiøtt, Morten; Jin, Lijun; Zhang, Pei; Zhang, Yanyan; Nygaard, Sanne; Peng, Zhiyu; Zhou, Yang; Deng, Yuan; Zhang, Wenwei; Boomsma, Jacobus J.; Zhang, Guojie

    2014-01-01

    Eusocial insects have evolved the capacity to generate adults with distinct morphological, reproductive and behavioural phenotypes from the same genome. Recent studies suggest that RNA editing might enhance the diversity of gene products at the post-transcriptional level, particularly to induce functional changes in the nervous system. Using head samples from the leaf-cutting ant Acromyrmex echinatior, we compare RNA editomes across eusocial castes, identifying ca. 11,000 RNA editing sites in gynes, large workers and small workers. Those editing sites map to 800 genes functionally enriched for neurotransmission, circadian rhythm, temperature response, RNA splicing and carboxylic acid biosynthesis. Most A. echinatior editing sites are species specific, but 8–23% are conserved across ant subfamilies and likely to have been important for the evolution of eusociality in ants. The level of editing varies for the same site between castes, suggesting that RNA editing might be a general mechanism that shapes caste behaviour in ants. PMID:25266559

  6. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes.

    PubMed

    Paix, Alexandre; Folkmann, Andrew; Rasoloson, Dominique; Seydoux, Geraldine

    2015-09-01

    Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins. PMID:26187122

  7. High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes

    PubMed Central

    Paix, Alexandre; Folkmann, Andrew; Rasoloson, Dominique; Seydoux, Geraldine

    2015-01-01

    Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro–assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins. PMID:26187122

  8. Genome Editing in Human Pluripotent Stem Cells.

    PubMed

    Smith, Cory; Ye, Zhaohui; Cheng, Linzhao

    2016-01-01

    Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities. PMID:27037079

  9. Video Editing System

    NASA Technical Reports Server (NTRS)

    Schlecht, Leslie E.; Kutler, Paul (Technical Monitor)

    1998-01-01

    This is a proposal for a general use system based, on the SGI IRIS workstation platform, for recording computer animation to videotape. In addition, this system would provide features for simple editing and enhancement. Described here are a list of requirements for the system, and a proposed configuration including the SGI VideoLab Integrator, VideoMedia VLAN animation controller and the Pioneer rewritable laserdisc recorder.

  10. Addition of uridines to edited RNAs in trypanosome mitochondria occurs independently of transcription

    SciTech Connect

    Harris, M.E.; Moore, D.R.; Hajduk, S.L. )

    1990-07-05

    RNA editing is a novel RNA processing event of unknown mechanism that results in the introduction of nucleotides not encoded in the DNA into specific RNA molecules. We have examined the post-transcriptional addition of nucleotides into the mitochondrial RNA of Trypanosoma brucei. Utilizing an isolated organelle system we have determined that addition of uridines to edited RNAs does not require ongoing transcription. Trypanosome mitochondria incorporate CTP, ATP, and UTP into RNA in the absence of transcription. GTP is incorporated into RNA only as a result of the transcription process. Post-transcriptional CTP and ATP incorporation can be ascribed to known enzymatic activities. CTP is incorporated into tRNAs as a result of synthesis or turnover of their 3{prime} CCA sequences. ATP is incorporated into the 3{prime} CCA of tRNAs and into mitochondrial messenger RNAs due to polyadenylation. In the absence of transcription, UTP is incorporated into transcripts known to undergo editing, and the degree of UTP incorporation is consistent with the degree of editing occurring in these transcripts. Cytochrome b mRNAs, which contain a single editing site near their 5{prime} ends, are initially transcribed unedited at that site. Post-transcriptional labeling of cytochrome b mRNAs in the organelle with (alpha-32P)UTP results in the addition of uridines near the 5{prime} end of the RNA but not in a 3{prime} region which lacks an editing site. These results indicate that RNA editing is a post-transcriptional process in the mitochondria of trypanosomes.

  11. Efficient Mitochondrial Genome Editing by CRISPR/Cas9

    PubMed Central

    Jo, Areum; Ham, Sangwoo; Lee, Gum Hwa; Lee, Yun-Il; Kim, SangSeong; Lee, Yun-Song; Shin, Joo-Ho; Lee, Yunjong

    2015-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 system has been widely used for nuclear DNA editing to generate mutations or correct specific disease alleles. Despite its flexible application, it has not been determined if CRISPR/Cas9, originally identified as a bacterial defense system against virus, can be targeted to mitochondria for mtDNA editing. Here, we show that regular FLAG-Cas9 can localize to mitochondria to edit mitochondrial DNA with sgRNAs targeting specific loci of the mitochondrial genome. Expression of FLAG-Cas9 together with gRNA targeting Cox1 and Cox3 leads to cleavage of the specific mtDNA loci. In addition, we observed disruption of mitochondrial protein homeostasis following mtDNA truncation or cleavage by CRISPR/Cas9. To overcome nonspecific distribution of FLAG-Cas9, we also created a mitochondria-targeted Cas9 (mitoCas9). This new version of Cas9 localizes only to mitochondria; together with expression of gRNA targeting mtDNA, there is specific cleavage of mtDNA. MitoCas9-induced reduction of mtDNA and its transcription leads to mitochondrial membrane potential disruption and cell growth inhibition. This mitoCas9 could be applied to edit mtDNA together with gRNA expression vectors without affecting genomic DNA. In this brief study, we demonstrate that mtDNA editing is possible using CRISPR/Cas9. Moreover, our development of mitoCas9 with specific localization to the mitochondria should facilitate its application for mitochondrial genome editing. PMID:26448933

  12. Optimization of genome editing through CRISPR-Cas9 engineering.

    PubMed

    Zhang, Jian-Hua; Adikaram, Poorni; Pandey, Mritunjay; Genis, Allison; Simonds, William F

    2016-04-01

    CRISPR (Clustered Regularly-Interspaced Short Palindromic Repeats)-Cas9 (CRISPR associated protein 9) has rapidly become the most promising genome editing tool with great potential to revolutionize medicine. Through guidance of a 20 nucleotide RNA (gRNA), CRISPR-Cas9 finds and cuts target protospacer DNA precisely 3 base pairs upstream of a PAM (Protospacer Adjacent Motif). The broken DNA ends are repaired by either NHEJ (Non-Homologous End Joining) resulting in small indels, or by HDR (Homology Directed Repair) for precise gene or nucleotide replacement. Theoretically, CRISPR-Cas9 could be used to modify any genomic sequences, thereby providing a simple, easy, and cost effective means of genome wide gene editing. However, the off-target activity of CRISPR-Cas9 that cuts DNA sites with imperfect matches with gRNA have been of significant concern because clinical applications require 100% accuracy. Additionally, CRISPR-Cas9 has unpredictable efficiency among different DNA target sites and the PAM requirements greatly restrict its genome editing frequency. A large number of efforts have been made to address these impeding issues, but much more is needed to fully realize the medical potential of CRISPR-Cas9. In this article, we summarize the existing problems and current advances of the CRISPR-Cas9 technology and provide perspectives for the ultimate perfection of Cas9-mediated genome editing. PMID:27340770

  13. ADAR2 editing activity in newly diagnosed versus relapsed pediatric high-grade astrocytomas

    PubMed Central

    2013-01-01

    Background High-grade (WHO grade III and IV) astrocytomas are aggressive malignant brain tumors affecting humans with a high risk of recurrence in both children and adults. To date, limited information is available on the genetic and molecular alterations important in the onset and progression of pediatric high-grade astrocytomas and, even less, on the prognostic factors that influence long-term outcome in children with recurrence. A-to-I RNA editing is an essential post-transcriptional mechanism that can alter the nucleotide sequence of several RNAs and is mediated by the ADAR enzymes. ADAR2 editing activity is particularly important in mammalian brain and is impaired in both adult and pediatric high-grade astrocytomas. Moreover, we have recently shown that the recovered ADAR2 activity in high-grade astrocytomas inhibits in vivo tumor growth. The aim of the present study is to investigate whether changes may occur in ADAR2-mediated RNA editing profiles of relapsed high-grade astrocytomas compared to their respective specimens collected at diagnosis, in four pediatric patients. Methods Total RNAs extracted from all tumor samples and controls were tested for RNA editing levels (by direct sequencing on cDNA pools) and for ADAR2 mRNA expression (by qRT-PCR). Results A significant loss of ADAR2-editing activity was observed in the newly diagnosed and recurrent astrocytomas in comparison to normal brain. Surprisingly, we found a substantial rescue of ADAR2 editing activity in the relapsed tumor of the only patient showing prolonged survival. Conclusions High-grade astrocytomas display a generalized loss of ADAR2-mediated RNA editing at both diagnosis and relapse. However, a peculiar Case, in complete remission of disease, displayed a total rescue of RNA editing at relapse, intriguingly suggesting ADAR2 activity/expression as a possible marker for long-term survival of patients with high-grade astrocytomas. PMID:23697632

  14. A third member of the RNA-specific adenosine deaminase gene family, ADAR3, contains both single- and double-stranded RNA binding domains.

    PubMed Central

    Chen, C X; Cho, D S; Wang, Q; Lai, F; Carter, K C; Nishikura, K

    2000-01-01

    Members of the double-stranded RNA- (dsRNA) specific adenosine deaminase gene family convert adenosine residues into inosines in dsRNA and are involved in A-to-I RNA editing of transcripts of glutamate receptor (GluR) subunits and serotonin receptor subtype 2C (5-HT(2C)R). We have isolated hADAR3, the third member of this class of human enzyme and investigated its editing site selectivity using in vitro RNA editing assay systems. As originally reported for rat ADAR3 or RED2, purified ADAR3 proteins could not edit GluR-B RNA at the "Q/R" site, the "R/G" site, and the intronic "hot spot" site. In addition, ADAR3 did not edit any of five sites discovered recently within the intracellular loop II region of 5-HT(2C)R RNAs, confirming its total lack of editing activity for currently known substrate RNAs. Filter-binding analyses revealed that ADAR3 is capable of binding not only to dsRNA but also to single-stranded RNA (ssRNA). Deletion mutagenesis identified a region rich in arginine residues located in the N-terminus that is responsible for binding of ADAR3 to ssRNA. The presence of this ssRNA-binding domain as well as its expression in restricted brain regions and postmitotic neurons make ADAR3 distinct from the other two ADAR gene family members, editing competent ADAR1 and ADAR2. ADAR3 inhibited in vitro the activities of RNA editing enzymes of the ADAR gene family, raising the possibility of a regulatory role in RNA editing. PMID:10836796

  15. Complete set of mitochondrial pan-edited mRNAs in Leishmania mexicana amazonensis LV78

    PubMed Central

    Maslov, Dmitri A.

    2010-01-01

    Editing of mRNA transcribed from the mitochondrial cryptogenes ND8 (G1), ND9 (G2), G3, G4, ND3 (G5), RPS12 (G6) was investigated in Leishmania mexicana amazonensis, strain LV78, by amplification of the cDNA, cloning and sequencing. For each of these genes, extensively and partially edited transcripts were found to be relatively abundant compared to the respective pre-edited molecules. Moreover, the editing patterns observed in a majority of transcripts of each gene were consistent among themselves which allowed for inferring consensus editing sequences. The open reading frames contained in the consensus sequences were predicted to encode polypeptides that were highly similar to their counterparts in other species of Trypanosomatidae. Several kinetoplast DNA minicircles from this species available in the public domain were found to contain genes for guide RNAs which mediate editing of some of the mRNAs. The results indicate that the investigated strain of L. m. amazonensis has preserved its full editing capacity in spite of the long-term maintenance in culture. This property differs drastically from the other Leishmania species which lost some or all of the G1–G5 mRNA editing ability in culture. PMID:20546801

  16. Discovery of new genes and deletion editing in Physarum mitochondria enabled by a novel algorithm for finding edited mRNAs

    PubMed Central

    Gott, Jonatha M.; Parimi, Neeta; Bundschuh, Ralf

    2005-01-01

    Gene finding is complicated in organisms that exhibit insertional RNA editing. Here, we demonstrate how our new algorithm Predictor of Insertional Editing (PIE) can be used to locate genes whose mRNAs are subjected to multiple frameshifting events, and extend the algorithm to include probabilistic predictions for sites of nucleotide insertion; this feature is particularly useful when designing primers for sequencing edited RNAs. Applying this algorithm, we successfully identified the nad2, nad4L, nad6 and atp8 genes within the mitochondrial genome of Physarum polycephalum, which had gone undetected by existing programs. Characterization of their mRNA products led to the unanticipated discovery of nucleotide deletion editing in Physarum. The deletion event, which results in the removal of three adjacent A residues, was confirmed by primer extension sequencing of total RNA. This finding is remarkable in that it comprises the first known instance of nucleotide deletion in this organelle, to be contrasted with nearly 500 sites of single and dinucleotide addition in characterized mitochondrial RNAs. Statistical analysis of this larger pool of editing sites indicates that there are significant biases in the 2 nt immediately upstream of editing sites, including a reduced incidence of nucleotide repeats, in addition to the previously identified purine-U bias. PMID:16147990

  17. Editing the Small Magazine. 2nd Edition Revised.

    ERIC Educational Resources Information Center

    Ferguson, Rowena

    Designed for the aspiring journalist and the professional editor, this revised second edition serves as a standard reference work as well as an ideal means of keeping abreast of new trends and developments in the editing of small magazines. It describes the latest technological developments in type-setting and production; discusses and evaluates…

  18. Genome-editing tools for stem cell biology

    PubMed Central

    Vasileva, E A; Shuvalov, O U; Garabadgiu, A V; Melino, G; Barlev, N A

    2015-01-01

    Human pluripotent stem cells provide a versatile platform for regenerative studies, drug testing and disease modeling. That the expression of only four transcription factors, Oct4, Klf4, Sox2 and c-Myc (OKSM), is sufficient for generation of induced pluripotent stem cells (iPSCs) from differentiated somatic cells has revolutionized the field and also highlighted the importance of OKSM as targets for genome editing. A number of novel genome-editing systems have been developed recently. In this review, we focus on successful applications of several such systems for generation of iPSCs. In particular, we discuss genome-editing systems based on zinc-finger fusion proteins (ZFs), transcription activator-like effectors (TALEs) and an RNA-guided DNA-specific nuclease, Cas9, derived from the bacterial defense system against viruses that utilizes clustered regularly interspaced short palindromic repeats (CRISPR). PMID:26203860

  19. Advances in therapeutic CRISPR/Cas9 genome editing.

    PubMed

    Savić, Nataša; Schwank, Gerald

    2016-02-01

    Targeted nucleases are widely used as tools for genome editing. Two years ago the clustered regularly interspaced short palindromic repeat (CRISPR)-associated Cas9 nuclease was used for the first time, and since then has largely revolutionized the field. The tremendous success of the CRISPR/Cas9 genome editing tool is powered by the ease design principle of the guide RNA that targets Cas9 to the desired DNA locus, and by the high specificity and efficiency of CRISPR/Cas9-generated DNA breaks. Several studies recently used CRISPR/Cas9 to successfully modulate disease-causing alleles in vivo in animal models and ex vivo in somatic and induced pluripotent stem cells, raising hope for therapeutic genome editing in the clinics. In this review, we will summarize and discuss such preclinical CRISPR/Cas9 gene therapy reports. PMID:26470680

  20. Genome editing and the next generation of antiviral therapy.

    PubMed

    Stone, Daniel; Niyonzima, Nixon; Jerome, Keith R

    2016-09-01

    Engineered endonucleases such as homing endonucleases (HEs), zinc finger nucleases (ZFNs), Tal-effector nucleases (TALENS) and the RNA-guided engineered nucleases (RGENs or CRISPR/Cas9) can target specific DNA sequences for cleavage, and are proving to be valuable tools for gene editing. Recently engineered endonucleases have shown great promise as therapeutics for the treatment of genetic disease and infectious pathogens. In this review, we discuss recent efforts to use the HE, ZFN, TALEN and CRISPR/Cas9 gene-editing platforms as antiviral therapeutics. We also discuss the obstacles facing gene-editing antiviral therapeutics as they are tested in animal models of disease and transition towards human application. PMID:27272125

  1. The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops

    PubMed Central

    Khatodia, Surender; Bhatotia, Kirti; Passricha, Nishat; Khurana, S. M. P.; Tuteja, Narendra

    2016-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system. It is an inexpensive, easy, most user friendly and rapidly adopted genome editing tool transforming to revolutionary paradigm. This technique enables precise genomic modifications in many different organisms and tissues. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double-stranded breaks with only a short RNA sequence to confer recognition of the target in animals and plants. Development of genetically edited (GE) crops similar to those developed by conventional or mutation breeding using this potential technique makes it a promising and extremely versatile tool for providing sustainable productive agriculture for better feeding of rapidly growing population in a changing climate. The emerging areas of research for the genome editing in plants include interrogating gene function, rewiring the regulatory signaling networks and sgRNA library for high-throughput loss-of-function screening. In this review, we have described the broad applicability of the Cas9 nuclease mediated targeted plant genome editing for development of designer crops. The regulatory uncertainty and social acceptance of plant breeding by Cas9 genome editing have also been described. With this powerful and innovative technique the designer GE non-GM plants could further advance climate resilient and sustainable agriculture in the future and maximizing yield by combating abiotic and biotic stresses. PMID:27148329

  2. The CRISPR/Cas Genome-Editing Tool: Application in Improvement of Crops.

    PubMed

    Khatodia, Surender; Bhatotia, Kirti; Passricha, Nishat; Khurana, S M P; Tuteja, Narendra

    2016-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats associated Cas9/sgRNA system is a novel targeted genome-editing technique derived from bacterial immune system. It is an inexpensive, easy, most user friendly and rapidly adopted genome editing tool transforming to revolutionary paradigm. This technique enables precise genomic modifications in many different organisms and tissues. Cas9 protein is an RNA guided endonuclease utilized for creating targeted double-stranded breaks with only a short RNA sequence to confer recognition of the target in animals and plants. Development of genetically edited (GE) crops similar to those developed by conventional or mutation breeding using this potential technique makes it a promising and extremely versatile tool for providing sustainable productive agriculture for better feeding of rapidly growing population in a changing climate. The emerging areas of research for the genome editing in plants include interrogating gene function, rewiring the regulatory signaling networks and sgRNA library for high-throughput loss-of-function screening. In this review, we have described the broad applicability of the Cas9 nuclease mediated targeted plant genome editing for development of designer crops. The regulatory uncertainty and social acceptance of plant breeding by Cas9 genome editing have also been described. With this powerful and innovative technique the designer GE non-GM plants could further advance climate resilient and sustainable agriculture in the future and maximizing yield by combating abiotic and biotic stresses. PMID:27148329

  3. Direct volume editing.

    PubMed

    Bürger, Kai; Krüger, Jens; Westermann, Rüdiger

    2008-01-01

    In this work we present basic methodology for interactive volume editing on GPUs, and we demonstrate the use of these methods to achieve a number of different effects. We present fast techniques to modify the appearance and structure of volumetric scalar fields given on Cartesian grids. Similar to 2D circular brushes as used in surface painting we present 3D spherical brushes for intuitive coloring of particular structures in such fields. This paint metaphor is extended to allow the user to change the data itself, and the use of this functionality for interactive structure isolation, hole filling, and artefact removal is demonstrated. Building on previous work in the field we introduce high-resolution selection volumes, which can be seen as a resolution-based focus+context metaphor. By utilizing such volumes we present a novel approach to interactive volume editing at sub-voxel accuracy. Finally, we introduce a fast technique to paste textures onto iso-surfaces in a 3D scalar field. Since the texture resolution is independent of the volume resolution, this technique allows structure-aligned textures containing appearance properties or textual information to be used for volume augmentation and annotation. PMID:18988988

  4. Natural Hazards, Second Edition

    NASA Astrophysics Data System (ADS)

    Rouhban, Badaoui

    Natural disaster loss is on the rise, and the vulnerability of the human and physical environment to the violent forces of nature is increasing. In many parts of the world, disasters caused by natural hazards such as earthquakes, floods, landslides, drought, wildfires, intense windstorms, tsunami, and volcanic eruptions have caused the loss of human lives, injury, homelessness, and the destruction of economic and social infrastructure. Over the last few years, there has been an increase in the occurrence, severity, and intensity of disasters, culminating with the devastating tsunami of 26 December 2004 in South East Asia.Natural hazards are often unexpected or uncontrollable natural events of varying magnitude. Understanding their mechanisms and assessing their distribution in time and space are necessary for refining risk mitigation measures. This second edition of Natural Hazards, (following a first edition published in 1991 by Cambridge University Press), written by Edward Bryant, associate dean of science at Wollongong University, Australia, grapples with this crucial issue, aspects of hazard prediction, and other issues. The book presents a comprehensive analysis of different categories of hazards of climatic and geological origin.

  5. The RNA WikiProject: community annotation of RNA families.

    PubMed

    Daub, Jennifer; Gardner, Paul P; Tate, John; Ramsköld, Daniel; Manske, Magnus; Scott, William G; Weinberg, Zasha; Griffiths-Jones, Sam; Bateman, Alex

    2008-12-01

    The online encyclopedia Wikipedia has become one of the most important online references in the world and has a substantial and growing scientific content. A search of Google with many RNA-related keywords identifies a Wikipedia article as the top hit. We believe that the RNA community has an important and timely opportunity to maximize the content and quality of RNA information in Wikipedia. To this end, we have formed the RNA WikiProject (http://en.wikipedia.org/wiki/Wikipedia:WikiProject_RNA) as part of the larger Molecular and Cellular Biology WikiProject. We have created over 600 new Wikipedia articles describing families of noncoding RNAs based on the Rfam database, and invite the community to update, edit, and correct these articles. The Rfam database now redistributes this Wikipedia content as the primary textual annotation of its RNA families. Users can, therefore, for the first time, directly edit the content of one of the major RNA databases. We believe that this Wikipedia/Rfam link acts as a functioning model for incorporating community annotation into molecular biology databases. PMID:18945806

  6. Children in Sport. Second Edition.

    ERIC Educational Resources Information Center

    Magill, Richard A., Ed.; And Others

    This anthology of papers is designed to serve both as a textbook and as a synthesis of research efforts in youth sport. Half of the 20 papers are reprinted from the first edition of this volume; of the remainder, some were written especially for this edition, while others are culled from journals and conference proceedings. Five subject sections…

  7. Drug Abuse Films, Second Edition.

    ERIC Educational Resources Information Center

    National Coordinating Council on Drug Education, Washington, DC.

    This second edition updates and expands a 1971 evaluation of films and audiovisuals related to drug education performed by the National Coordinating Council on Drug Education. Materials in this edition are evaluated both for accuracy and effectiveness as a communications tool. They are separated into two sections--films and other audiovisuals…

  8. RNA-SSPT: RNA Secondary Structure Prediction Tools.

    PubMed

    Ahmad, Freed; Mahboob, Shahid; Gulzar, Tahsin; Din, Salah U; Hanif, Tanzeela; Ahmad, Hifza; Afzal, Muhammad

    2013-01-01

    The prediction of RNA structure is useful for understanding evolution for both in silico and in vitro studies. Physical methods like NMR studies to predict RNA secondary structure are expensive and difficult. Computational RNA secondary structure prediction is easier. Comparative sequence analysis provides the best solution. But secondary structure prediction of a single RNA sequence is challenging. RNA-SSPT is a tool that computationally predicts secondary structure of a single RNA sequence. Most of the RNA secondary structure prediction tools do not allow pseudoknots in the structure or are unable to locate them. Nussinov dynamic programming algorithm has been implemented in RNA-SSPT. The current studies shows only energetically most favorable secondary structure is required and the algorithm modification is also available that produces base pairs to lower the total free energy of the secondary structure. For visualization of RNA secondary structure, NAVIEW in C language is used and modified in C# for tool requirement. RNA-SSPT is built in C# using Dot Net 2.0 in Microsoft Visual Studio 2005 Professional edition. The accuracy of RNA-SSPT is tested in terms of Sensitivity and Positive Predicted Value. It is a tool which serves both secondary structure prediction and secondary structure visualization purposes. PMID:24250115

  9. The Bacterial Origins of the CRISPR Genome-Editing Revolution.

    PubMed

    Sontheimer, Erik J; Barrangou, Rodolphe

    2015-07-01

    Like most of the tools that enable modern life science research, the recent genome-editing revolution has its biological roots in the world of bacteria and archaea. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci are found in the genomes of many bacteria and most archaea, and underlie an adaptive immune system that protects the host cell against invasive nucleic acids such as viral genomes. In recent years, engineered versions of these systems have enabled efficient DNA targeting in living cells from dozens of species (including humans and other eukaryotes), and the exploitation of the resulting endogenous DNA repair pathways has provided a route to fast, easy, and affordable genome editing. In only three years after RNA-guided DNA cleavage was first harnessed, the ability to edit genomes via simple, user-defined RNA sequences has already revolutionized nearly all areas of biological science. CRISPR-based technologies are now poised to similarly revolutionize many facets of clinical medicine, and even promise to advance the long-term goal of directly editing genomic sequences of patients with inherited disease. In this review, we describe the biological and mechanistic basis for these remarkable immune systems, and how their engineered derivatives are revolutionizing basic and clinical research. PMID:26078042

  10. Residential and Light Commercial HVAC. Teacher Edition and Student Edition. Second Edition.

    ERIC Educational Resources Information Center

    Stephenson, David

    This package contains teacher and student editions of a residential and light commercial heating, ventilation, and air conditioning (HVAC) course of study. The teacher edition contains information on the following: using the publication; national competencies; competency profile; related academic and workplace skills list; tools, equipment, and…

  11. Graphic Arts: Process Camera, Stripping, and Platemaking. Fourth Edition. Teacher Edition [and] Student Edition.

    ERIC Educational Resources Information Center

    Multistate Academic and Vocational Curriculum Consortium, Stillwater, OK.

    This publication contains both a teacher edition and a student edition of materials for a course in graphic arts that covers the process camera, stripping, and platemaking. The course introduces basic concepts and skills necessary for entry-level employment in a graphic communication occupation. The contents of the materials are tied to measurable…

  12. Oxyacetylene Welding and Oxyfuel Cutting. Third Edition. Teacher Edition [and] Student Edition [and] Student Workbook.

    ERIC Educational Resources Information Center

    Knapp, John; Harper, Eddie

    This Oklahoma curriculum guide, which includes a teacher edition, a student edition, and a student workbook, provides three units for a course on oxyacetylene welding, oxyfuel cutting, and cutting done with alternative fuels such as MAPP, propane, and natural gas. The three units are: "Oxyacetylene Welding"; "Oxyfuel Cutting"; and "Oxyacetylene…

  13. Fundamentals of Welding. Teacher Edition [and] Student Edition [and] Student Workbook. Second Edition.

    ERIC Educational Resources Information Center

    Fortney, Clarence; Gregory, Mike; New, Larry

    Teacher and student editions and a student workbook for fundamentals of welding comprise the first of six in a series of competency-based instructional materials for welding programs. Introductory pages in the teacher edition are training and competency profile, instructional/task analysis, basic skills icons and classifications, basic skills…

  14. Introduction to Surgical Technology. Third Edition. Teacher Edition [and] Student Edition.

    ERIC Educational Resources Information Center

    Bushey, Vicki; Hildebrand, Bob; Hildebrand, Dinah; Johnson, Dave; Sikes, John; Tahah, Ann; Walker, Susan; Zielsdorf, Lani

    These teacher and student editions provide instructional materials for an introduction to surgical technology course. Introductory materials in the teacher edition include information on use, instructional/task analysis, academic and workplace skill classifications and definitions, related academic and workplace skill list, and crosswalk to…

  15. RNA Interference

    MedlinePlus

    ... NIGMS Home > Science Education > RNA Interference Fact Sheet RNA Interference Fact Sheet Tagline (Optional) Middle/Main Content Area What is RNA interference? RNA interference (RNAi) is a natural process ...

  16. Ethics manual: fifth edition.

    PubMed

    Snyder, Lois; Leffler, Cathy

    2005-04-01

    Medicine, law, and social values are not static. Reexamining the ethical tenets of medical practice and their application in new circumstances is a necessary exercise. The fifth edition of the College's Ethics Manual covers emerging issues in medical ethics and revisits old ones. It reflects on many of the ethical tensions faced by internists and their patients and attempts to shed light on how existing principles extend to emerging concerns. In addition, by reiterating ethical principles that have provided guidance in resolving past ethical problems, the Manual may help physicians avert future problems. The Manual is not a substitute for the experience and integrity of individual physicians, but it may serve as a reminder of the shared obligations and duties of the medical profession. PMID:15809467

  17. Tool steels. 5. edition

    SciTech Connect

    Roberts, G.; Krauss, G.; Kennedy, R.

    1998-12-31

    The revision of this authoritative work contains a significant amount of new information from the past nearly two decades presented in an entirely new outline, making this a must have reference for engineers involved in tool-steel production, as well as in the selection and use of tool steels in metalworking and other materials manufacturing industries. The chapter on tool-steel manufacturing includes new production processes, such as electroslag refining, vacuum arc remelting, spray deposition processes (Osprey and centrifugal spray), and powder metal processing. The seven chapters covering tool-steel types in the 4th Edition have been expanded to 11 chapters covering nine main groups of tool steels as well as other types of ultrahigh strength steels sometimes used for tooling. Each chapter discusses in detail processing, composition, and applications specific to the particular group. In addition, two chapters have been added covering surface modification and trouble shooting production and performance problems.

  18. Volcanoes, Third Edition

    NASA Astrophysics Data System (ADS)

    Nye, Christopher J.

    It takes confidence to title a smallish book merely “Volcanoes” because of the impliction that the myriad facets of volcanism—chemistry, physics, geology, meteorology, hazard mitigation, and more—have been identified and addressed to some nontrivial level of detail. Robert and Barbara Decker have visited these different facets seamlessly in Volcanoes, Third Edition. The seamlessness comes from a broad overarching, interdisciplinary, professional understanding of volcanism combined with an exceptionally smooth translation of scientific jargon into plain language.The result is a book which will be informative to a very broad audience, from reasonably educated nongeologists (my mother loves it) to geology undergraduates through professional volcanologists. I bet that even the most senior professional volcanologists will learn at least a few things from this book and will find at least a few provocative discussions of subjects they know.

  19. RNA topology

    PubMed Central

    2013-01-01

    A new variety on non-coding RNA has been discovered by several groups: circular RNA (circRNA). This discovery raises intriguing questions about the possibility of the existence of knotted RNA molecules and the existence of a new class of enzymes changing RNA topology, RNA topoisomerases. PMID:23603781

  20. Effect of mismatch on binding of ADAR2/GluR-2 pre-mRNA complex.

    PubMed

    Yang, Junru; Song, Jianing; Zhang, John Z H; Ji, Changge

    2015-09-01

    RNA editing plays an important role in realizing the full potential of a given genome. Different from RNA splicing, RNA editing fine-tunes the sequence of RNA by changing only one or two nucleotides. A-I editing [deamination of adenosine (A) to create inosine (I)] is best characterized in mammals and occurs in the regions of double-stranded RNA (dsRNA). Adenosine deaminases acting on RNA (ADARs) are members of a family of enzymes involved in A-I deamination editing in numerous mRNA and pre-mRNA transcripts. Experimental study shows that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines. How ADAR2 selects specific sites for deamination is poorly understood. Mismatches have been suggested to be important factors that allow the ADAR2 to achieve specific deamination. Using molecular dynamic simulation, we studied the effect of mismatch on binding stability of the dsRNA/ADAR2 complex. By comparison of two binding domains of ADAR2, we found that ADAR2 dsRBM2 (second binding domain of ADAR2) does not bind well with mismatch reversed GluR-2 RNA. When mismatch is reversed, dsRBM2 of ADAR2 slides along the RNA duplex in the simulation. Detailed structural analysis indicates that the minor groove width of dsRNA and global shape of RNA may play an important role in the specific reading mechanism of ADAR2. PMID:26252972

  1. Practical writing and editing techniques

    SciTech Connect

    Hallinan, E.J.

    1984-01-01

    Technical writing and editing involve more than a knowledge of style and format. Often a draft must be prepared from existing documents and edited for content as well as style. A practical approach to writing and editing tasks includes increasing writer/editor exposure with cognizant personnel, researching source and legal documents, participating in project meetings, and reviewing the document by checking for inconsistencies and selectively questioning the contents. This approach will help the writer/editor prepare a reasonably accurate, politically acceptable document with a minimum of supervision.

  2. lncRNA-RNA Interactions across the Human Transcriptome

    PubMed Central

    Szcześniak, Michał Wojciech; Makałowska, Izabela

    2016-01-01

    Long non-coding RNAs (lncRNAs) represent a numerous class of non-protein coding transcripts longer than 200 nucleotides. There is possibility that a fraction of lncRNAs are not functional and represent mere transcriptional noise but a growing body of evidence shows they are engaged in a plethora of molecular functions and contribute considerably to the observed diversification of eukaryotic transcriptomes and proteomes. Still, however, only ca. 1% of lncRNAs have well established functions and much remains to be done towards decipherment of their biological roles. One of the least studied aspects of lncRNAs biology is their engagement in gene expression regulation through RNA-RNA interactions. By hybridizing with mate RNA molecules, lncRNAs could potentially participate in modulation of pre-mRNA splicing, RNA editing, mRNA stability control, translation activation, or abrogation of miRNA-induced repression. Here, we implemented a similarity-search based method for transcriptome-wide identification of RNA-RNA interactions, which enabled us to find 18,871,097 lncRNA-RNA base-pairings in human. Further analyses showed that the interactions could be involved in processing, stability control and functions of 57,303 transcripts. An extensive use of RNA-Seq data provided support for approximately one third of the interactions, at least in terms of the two RNA components being co-expressed. The results suggest that lncRNA-RNA interactions are broadly used to regulate and diversify the human transcriptome. PMID:26930590

  3. Edit while watching: home video editing made easy

    NASA Astrophysics Data System (ADS)

    Campanella, Marco; Weda, Hans; Barbieri, Mauro

    2007-01-01

    In recent years, more and more people capture their experiences in home videos. However, home video editing still is a difficult and time-consuming task. We present the Edit While Watching system that allows users to automatically create and change a summary of a home video in an easy, intuitive and lean-back way. Based on content analysis, video is indexed, segmented, and combined with proper music and editing effects. The result is an automatically generated home video summary that is shown to the user. While watching it, users can indicate whether they like certain content, so that the system will adapt the summary to contain more content that is similar or related to the displayed content. During the video playback users can also modify and enrich the content, seeing immediately the effects of their changes. Edit While Watching does not require a complex user interface: a TV and a few keys of a remote control are sufficient. A user study has shown that it is easy to learn and to use, even if users expressed the need for more control in the editing operations and in the editing process.

  4. Water quality management library. 2. edition

    SciTech Connect

    Eckenfelder, W.W.; Malina, J.F.; Patterson, J.W.

    1998-12-31

    A series of ten books offered in conjunction with Water Quality International, the Biennial Conference and Exposition of the International Association on Water Pollution Research and Control (IAWPRC). Volume 1, Activated Sludge Process, Design and Control, 2nd edition, 1998: Volume 2, Upgrading Wastewater Treatment Plants, 2nd edition, 1998: Volume 3, Toxicity Reduction, 2nd edition, 1998: Volume 4, Municipal Sewage Sludge Management, 2nd edition, 1998: Volume 5, Design and Retrofit of Wastewater Treatment Plants for Biological Nutrient Removal, 1st edition, 1992: Volume 6, Dynamics and Control of the Activated Sludge Process, 2nd edition, 1998: Volume 7: Design of Anaerobic Processes for the Treatment of Industrial and Municipal Wastes, 1st edition, 1992: Volume 8, Groundwater Remediation, 1st edition, 1992: Volume 9, Nonpoint Pollution and Urban Stormwater Management, 1st edition, 1995: Volume 10, Wastewater Reclamation and Reuse, 1st edition, 1998.

  5. Functional analysis of the Arabidopsis thaliana CHLOROPLAST BIOGENESIS 19 pentatricopeptide repeat editing protein.

    PubMed

    Ramos-Vega, Maricela; Guevara-García, Arturo; Llamas, Ernesto; Sánchez-León, Nidia; Olmedo-Monfil, Vianey; Vielle-Calzada, Jean Philippe; León, Patricia

    2015-10-01

    The Arabidopsis thaliana pentatricopeptide repeat (PPR) family of proteins contains several degenerate 35-aa motifs named PPR repeats. These proteins control diverse post-transcriptional regulatory mechanisms, including RNA editing. CLB19 belongs to the PLS subfamily of PPR proteins and is essential for the editing and functionality of the subunit A of plastid-encoded RNA polymerase (RpoA) and the catalytic subunit of the Clp protease (ClpP1). We demonstrate in vitro that CLB19 has a specific interaction with these two targets, in spite of their modest sequence similarity. Using site-directed mutagenesis of the rpoA target, we analyzed the essential nucleotides required for CLB19-rpoA interactions. We verified that, similar to other editing proteins, the C-terminal E domain of CLB19 is essential for editing but not for RNA binding. Using biomolecular fluorescence complementation, we demonstrated that the E domain of CLB19 interacts with the RNA-interacting protein MORF2/RIP2 but not with MORF9/RIP9. An interesting finding from this analysis was that overexpression of a truncated CLB19 protein lacking the E domain interferes with cell fate during megasporogenesis and the subsequent establishment of a female gametophyte, supporting an important role of plastids in female gametogenesis. Together these analyses provide important clues about the particularities of the CLB19 editing protein. PMID:25980341

  6. Marine botany. Second edition

    SciTech Connect

    Dawes, C.J.

    1998-12-01

    Marine plants are a diverse group that include unicellular algae, seaweeds, seagrasses, salt marshes, and mangrove forests. They carry out a variety of ecological functions and serve as the primary producers in coastal wetlands and oceanic waters. The theme that connects such a wide variety of plants is their ecology, which was also emphasized in the 1981 edition. The goal of this revision is to present taxonomic, physiological, chemical, and ecological aspects of marine plants, their adaptations, and how abiotic and biotic factors interact in their communities. The data are presented in a concise, comparative manner in order to identify similarities and differences between communities such as salt marsh and mangroves or subtidal seaweeds and seagrasses. To accomplish this, the text is organized into five chapters that introduce the marine habitats, consider abiotic and biotic factors, and anthropogenic influences on the communities followed by seven chapters that deal with microalgae, seaweeds, salt marshes, mangroves, seagrasses, and coral reefs. Two appendixes are included; one presents simple field techniques and the other is a summary of seaweed uses.

  7. RNA genetics

    SciTech Connect

    Domingo, E. ); Holland, J.J. . Dept. of Biology); Ahlquist, P. . Dept. of Plant Pathology)

    1988-01-01

    This book contains the proceedings on RNA genetics: Retroviruses, Viroids, and RNA recombination, Volume 2. Topics covered include: Replication of retrovirus genomes, Hepatitis B virus replication, and Evolution of RNA viruses.

  8. RNA Sociology: Group Behavioral Motifs of RNA Consortia

    PubMed Central

    Witzany, Guenther

    2014-01-01

    RNA sociology investigates the behavioral motifs of RNA consortia from the social science perspective. Besides the self-folding of RNAs into single stem loop structures, group building of such stem loops results in a variety of essential agents that are highly active in regulatory processes in cellular and non-cellular life. RNA stem loop self-folding and group building do not depend solely on sequence syntax; more important are their contextual (functional) needs. Also, evolutionary processes seem to occur through RNA stem loop consortia that may act as a complement. This means the whole entity functions only if all participating parts are coordinated, although the complementary building parts originally evolved for different functions. If complementary groups, such as rRNAs and tRNAs, are placed together in selective pressure contexts, new evolutionary features may emerge. Evolution initiated by competent agents in natural genome editing clearly contrasts with statistical error replication narratives. PMID:25426799

  9. Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression.

    PubMed

    Kennedy, Edward M; Bogerd, Hal P; Kornepati, Anand V R; Kang, Dong; Ghoshal, Delta; Marshall, Joy B; Poling, Brigid C; Tsai, Kevin; Gokhale, Nandan S; Horner, Stacy M; Cullen, Bryan R

    2016-05-11

    Covalent addition of a methyl group to adenosine N(6) (m(6)A) is an evolutionarily conserved and common RNA modification that is thought to modulate several aspects of RNA metabolism. While the presence of multiple m(6)A editing sites on diverse viral RNAs was reported starting almost 40 years ago, how m(6)A editing affects virus replication has remained unclear. Here, we used photo-crosslinking-assisted m(6)A sequencing techniques to precisely map several m(6)A editing sites on the HIV-1 genome and report that they cluster in the HIV-1 3' untranslated region (3' UTR). Viral 3' UTR m(6)A sites or analogous cellular m(6)A sites strongly enhanced mRNA expression in cis by recruiting the cellular YTHDF m(6)A "reader" proteins. Reducing YTHDF expression inhibited, while YTHDF overexpression enhanced, HIV-1 protein and RNA expression, and virus replication in CD4+ T cells. These data identify m(6)A editing and the resultant recruitment of YTHDF proteins as major positive regulators of HIV-1 mRNA expression. PMID:27117054

  10. CTD Writing and Editing Standards

    SciTech Connect

    Caruthers, C.M.

    1991-03-01

    The Computer and Telecommunication Division (CTD) recognizes that the communication of clear, accurate, reasonably complete information is essential to the success of its Laboratory mission. CTD therefore encourages all Division personnel to adhere to the principles of good writing and to the standards for grammar, usage, style, formats, and publication procedures that are described in CTD Writing and Editing Standards. We encourage CTD personnel to read CTD Writing and Editing Standards and to use it continually as a desktop reference. It will help CTD writers to produce better documents consistent with CTD standards in less time. Applying the principles specified in this document on how to write and organize technical information will speed up the editing, review, and revision processes. CTD Writing and Editing Standards complements the Argonne National Laboratory Technical Publications Guide, which serves as the basic Argonne documentation reference on issues concerning DOE orders and guidelines, NRC directives, and other sponsor requirements. However, this Laboratory-wide document does not address matters of grammar or style. Documents recommended in CTD Writing and Editing Standards are usually available for purchase at the Document Distribution Counter (Building 221, Room A-134) or through the mail (by calling extension 2-5405 and ordering copies).

  11. Newer Gene Editing Technologies toward HIV Gene Therapy

    PubMed Central

    Manjunath, N.; Yi, Guohua; Dang, Ying; Shankar, Premlata

    2013-01-01

    Despite the great success of highly active antiretroviral therapy (HAART) in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy. PMID:24284874

  12. In vivo genome editing using Staphylococcus aureus Cas9

    PubMed Central

    Ran, F. Ann; Cong, Le; Yan, Winston X.; Scott, David A.; Gootenberg, Jonathan S.; Kriz, Andrea J.; Zetsche, Bernd; Shalem, Ophir; Wu, Xuebing; Makarova, Kira S.; Koonin, Eugene; Sharp, Phillip A.; Zhang, Feng

    2015-01-01

    The RNA-guided endonuclease Cas9 has emerged as a versatile genome-editing platform. However, the size of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for basic research and therapeutic applications that employ the highly versatile adeno-associated virus (AAV) delivery vehicle. Here, we characterize six smaller Cas9 orthologs and show that Cas9 from Staphylococcus aureus (SaCas9) can edit the genome with efficiencies similar to those of SpCas9, while being >1kb shorter. We packaged SaCas9 and its sgRNA expression cassette into a single AAV vector and targeted the cholesterol regulatory gene Pcsk9 in the mouse liver. Within one week of injection, we observed >40% gene modification, accompanied by significant reductions in serum Pcsk9 and total cholesterol levels. We further demonstrate the power of using BLESS to assess the genome-wide targeting specificity of SaCas9 and SpCas9, and show that SaCas9 can mediate genome editing in vivo with high specificity. PMID:25830891

  13. Understanding Editing Behaviors in Multilingual Wikipedia.

    PubMed

    Kim, Suin; Park, Sungjoon; Hale, Scott A; Kim, Sooyoung; Byun, Jeongmin; Oh, Alice H

    2016-01-01

    Multilingualism is common offline, but we have a more limited understanding of the ways multilingualism is displayed online and the roles that multilinguals play in the spread of content between speakers of different languages. We take a computational approach to studying multilingualism using one of the largest user-generated content platforms, Wikipedia. We study multilingualism by collecting and analyzing a large dataset of the content written by multilingual editors of the English, German, and Spanish editions of Wikipedia. This dataset contains over two million paragraphs edited by over 15,000 multilingual users from July 8 to August 9, 2013. We analyze these multilingual editors in terms of their engagement, interests, and language proficiency in their primary and non-primary (secondary) languages and find that the English edition of Wikipedia displays different dynamics from the Spanish and German editions. Users primarily editing the Spanish and German editions make more complex edits than users who edit these editions as a second language. In contrast, users editing the English edition as a second language make edits that are just as complex as the edits by users who primarily edit the English edition. In this way, English serves a special role bringing together content written by multilinguals from many language editions. Nonetheless, language remains a formidable hurdle to the spread of content: we find evidence for a complexity barrier whereby editors are less likely to edit complex content in a second language. In addition, we find that multilinguals are less engaged and show lower levels of language proficiency in their second languages. We also examine the topical interests of multilingual editors and find that there is no significant difference between primary and non-primary editors in each language. PMID:27171158

  14. Understanding Editing Behaviors in Multilingual Wikipedia

    PubMed Central

    Hale, Scott A.; Kim, Sooyoung; Byun, Jeongmin; Oh, Alice H.

    2016-01-01

    Multilingualism is common offline, but we have a more limited understanding of the ways multilingualism is displayed online and the roles that multilinguals play in the spread of content between speakers of different languages. We take a computational approach to studying multilingualism using one of the largest user-generated content platforms, Wikipedia. We study multilingualism by collecting and analyzing a large dataset of the content written by multilingual editors of the English, German, and Spanish editions of Wikipedia. This dataset contains over two million paragraphs edited by over 15,000 multilingual users from July 8 to August 9, 2013. We analyze these multilingual editors in terms of their engagement, interests, and language proficiency in their primary and non-primary (secondary) languages and find that the English edition of Wikipedia displays different dynamics from the Spanish and German editions. Users primarily editing the Spanish and German editions make more complex edits than users who edit these editions as a second language. In contrast, users editing the English edition as a second language make edits that are just as complex as the edits by users who primarily edit the English edition. In this way, English serves a special role bringing together content written by multilinguals from many language editions. Nonetheless, language remains a formidable hurdle to the spread of content: we find evidence for a complexity barrier whereby editors are less likely to edit complex content in a second language. In addition, we find that multilinguals are less engaged and show lower levels of language proficiency in their second languages. We also examine the topical interests of multilingual editors and find that there is no significant difference between primary and non-primary editors in each language. PMID:27171158

  15. A non-inheritable maternal Cas9-based multiple-gene editing system in mice

    PubMed Central

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection–based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create “Cas9 transgene-free” gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice. PMID:26817415

  16. A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

    PubMed

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-01

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection-based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create "Cas9 transgene-free" gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice. PMID:26817415

  17. RNA genetics

    SciTech Connect

    Domingo, E.; Holland, J.J.; Ahlquist, P.

    1988-01-01

    These three volumes comprise reference on RNA genomes. The replication, mutation, recombination-assortment, and extreme evolutionary variability of RNA viruses and related RNA replicons is emphasized. The replication mechanisms of positive, negative, and double-stranded RNA viruses of animals and plants are featured.

  18. Genetic Changes at the Glycoprotein Editing Site Associated With Serial Passage of Sudan Virus.

    PubMed

    Alfson, Kendra J; Avena, Laura E; Beadles, Michael W; Menzie, Heather; Patterson, Jean L; Carrion, Ricardo; Griffiths, Anthony

    2015-10-01

    Sudan virus (SUDV), like the closely related Ebola virus (EBOV), is a filovirus that causes severe hemorrhagic disease. They both contain an RNA editing site in the glycoprotein gene that controls expression of soluble and full-length protein. We tested the consequences of cell culture passage on the genome sequence at the SUDV editing site locus and determined whether this affected virulence. Passage resulted in expansion of the SUDV editing site, similar to that observed with EBOV. We compared viruses possessing either the wild-type or expanded editing site, using a nonhuman primate model of disease. Despite differences in virus serum titer at one time point, there were no significant differences in time to death or any other measured parameter. These data imply that changes at this locus were not important for SUDV lethality. PMID:25920319

  19. Gas Metal Arc Welding and Flux-Cored Arc Welding. Third Edition. Teacher Edition [and] Student Edition [and] Student Workbook.

    ERIC Educational Resources Information Center

    Knapp, John; Harper, Eddie

    This packet, containing a teacher's edition, a student edition, and a student workbook, introduces students to high deposition welding and processes for "shielding" a weld. In addition to general information, the teacher edition consists of introductory pages and teacher pages, as well as unit information that corresponds to the materials in the…

  20. Gas Tungsten Arc Welding and Plasma Arc Cutting. Teacher Edition [and] Student Edition [and] Student Workbook. Second Edition.

    ERIC Educational Resources Information Center

    Harper, Eddie; Knapp, John

    This packet of instructional materials for a gas tungsten arc welding (GTAW) and plasma arc cutting course is comprised of a teacher edition, student edition, and student workbook. The teacher edition consists of introductory pages and teacher pages. Introductory pages include training and competency profile, state duty/task crosswalk,…

  1. RNA Crystallization

    NASA Technical Reports Server (NTRS)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  2. Language Editing at Astronomy & Astrophysics

    NASA Astrophysics Data System (ADS)

    Adams, J.

    2011-07-01

    In 2002, the A&A Board of Directors voted that all articles must be written in English and decided to improve the overall quality of the language in the articles with the help of a team of language editors. This article reviews the general advantages of editing the English expression and describes both the aims of this effort and its place in the full publication process. This is followed by the Guide to language editing that has been available on the Journal's website for several years now.

  3. Concepts of genetics: II edition

    SciTech Connect

    Klug, W.S.; Cummings, M.R.

    1986-01-01

    This book provides an introduction to the molecule, and progresses logically through cellular genetics and the genetics of organisms to the larger picture of population genetics. The Second Edition features new chapters on quantitative inheritance and recombinant DNA, a new appendix with a human gene map and coverage of gene disorders, expanded coverage of bacterial and viral genetics, and consolidated coverage of sex linkage, sex determination, sex chromosome abberations, and sex differentiation. Dozens of new figures are added in this edition. All diagrams, photographs, and tables work hand-in-hand with the text to explain important concepts. Practical exercises with answers at the back of the text provide immediate feedback.

  4. GENOME EDITING IN HUMAN CELLS USING CRISPR/CAS NUCLEASES

    PubMed Central

    Wyvekens, Nicolas; Tsai, Shengdar; Joung, J. Keith

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system has been broadly adopted for highly efficient genome editing in a variety of model organisms and human cell types. Unlike previous genome editing technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), the CRISPR/Cas technology does not require complex protein engineering and can be utilized by any researcher proficient in basic molecular biology and cell culture techniques. Here we describe protocols for design and cloning of vectors expressing single or multiplex gRNAs, for transient transfection of human cell lines, and for quantitation of mutation frequencies by T7 Endonuclease I assay. These protocols also include guidance for using two improvements that increase the specificity of CRISPR/Cas nucleases: truncated gRNAs and dimeric RNA-guided FokI nucleases. PMID:26423589

  5. DNA Editing by APOBECs: A Genomic Preserver and Transformer.

    PubMed

    Knisbacher, Binyamin A; Gerber, Doron; Levanon, Erez Y

    2016-01-01

    Information warfare is not limited to the cyber world because it is waged within our cells as well. The unique AID (activation-induced cytidine deaminase)/APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide) family comprises proteins that alter DNA sequences by converting deoxycytidines to deoxyuridines through deamination. This C-to-U DNA editing enables them to inhibit parasitic viruses and retrotransposons by disrupting their genomic content. In addition to attacking genomic invaders, APOBECs can target their host genome, which can be beneficial by initiating processes that create antibody diversity needed for the immune system or by accelerating the rate of evolution. AID can also alter gene regulation by removing epigenetic modifications from genomic DNA. However, when uncontrolled, these powerful agents of change can threaten genome stability and eventually lead to cancer. PMID:26608778

  6. Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity.

    PubMed

    Tycko, Josh; Myer, Vic E; Hsu, Patrick D

    2016-08-01

    Advances in the development of delivery, repair, and specificity strategies for the CRISPR-Cas9 genome engineering toolbox are helping researchers understand gene function with unprecedented precision and sensitivity. CRISPR-Cas9 also holds enormous therapeutic potential for the treatment of genetic disorders by directly correcting disease-causing mutations. Although the Cas9 protein has been shown to bind and cleave DNA at off-target sites, the field of Cas9 specificity is rapidly progressing, with marked improvements in guide RNA selection, protein and guide engineering, novel enzymes, and off-target detection methods. We review important challenges and breakthroughs in the field as a comprehensive practical guide to interested users of genome editing technologies, highlighting key tools and strategies for optimizing specificity. The genome editing community should now strive to standardize such methods for measuring and reporting off-target activity, while keeping in mind that the goal for specificity should be continued improvement and vigilance. PMID:27494557

  7. [An introduction on the editions of Zhang Gao's Yi shuo (Medical Narrations)].

    PubMed

    Wang, Xuguang; Lu, Xiang

    2014-11-01

    Zhang Gao's Yi shuo (Medical Narrations) of the Southern Song Dynasty had 2 kinds of editions: domestic editions and foreign editions. The former includes 1 Song edition, 14 Ming editions, 3 Qing editions and 25 editions after the Republic of China. The latter, mainly 2 classes, the Japanese edition and Korean printing type edition. In the Ming Dynasty, the editions of Yi shuo generated 2 branches: inherited edition and supplementary edition. The inherited editions include Gu Dingfang's edition, Zhang Yaode's edition, Wu Mianxue's edition, Wu Zhongheng's edition, Wang Kentang's edition, editions from Si ku quan shu (Imperial Collection of Four), stereotype edition of the 3th year of Xuantong reign (1911) from Shanghai Civilization Bookstore, edition of the 2(nd) year of Manji of Japan etc. The supplemental editions include Zhang Zili's edition, Shen Fan's edition, Fu Feng'ao's edition, transcript of the late Ming Dynasty preserved in the Library of Peking University, and Korean printing type edition etc. PMID:25620361

  8. Power Product Equipment Technician: Equipment Systems. Teacher Edition. Student Edition.

    ERIC Educational Resources Information Center

    Hilley, Robert

    This packet contains teacher and student editions on the topic of equipment systems, intended for the preparation of power product equipment technicians. This publication contains seven units: (1) principles of power transmission; (2) mechanical drive systems; (3) principles of fluid power; (4) hydraulic and pneumatic drive systems; (5) wheel and…

  9. Shielded Metal Arc Pipe Welding. Teacher Edition. Second Edition.

    ERIC Educational Resources Information Center

    Fortney, Clarence; And Others

    This second edition of the shielded metal arc pipe welding curriculum guide presents both basic and advanced pipe welding skills. All specifications for procedure and welder qualification are presented according to national standards. The standards also include the test position for both groove and fillet pipe welding. The guide contains three…

  10. Civil Technology Applications. Teacher Edition [and] Student Edition.

    ERIC Educational Resources Information Center

    Schertz, Karen

    Teacher and student editions of Civil Technology Applications are one in a series of competency-based instructional materials for drafting and civil technology programs. It includes the technical content and tasks necessary for a student to be employed as a drafter or civil technician in a civil engineering firm. Introductory pages in the teacher…

  11. Controlled flexibility in technical editing - The levels-of-edit concept at JPL

    NASA Technical Reports Server (NTRS)

    Buehler, M. F.

    1977-01-01

    The levels-of-edit concept, which can be used to specify the amount of editorial effort involved in the preparation of a manuscript for publication, is discussed. Nine types of editing are identified and described. These include coordination edit (preparing estimates, gathering cost data, monitoring production processes), policy edit, integrity edit (making sure that parts of a publication match in a physical or numerical sense), screening edit (ensuring that the quality of camera-ready copy is sufficient for external publication), copy clarification edit, format edit, mechanical style edit, language edit, and substantive edit (reviewing the manuscript for content coherence, emphasis, subordination and parallelism). These functions are grouped into five levels of edit. An edit-level number is assigned to each manuscript, providing a quantitative and qualitative indicator of the editing to be done which is clearly understood by authors, managers, and editors alike. In addition, clear boundaries are drawn between normal and extraordinary editing tasks. Individual organizations will group various edits in different ways to reflect their needs and priorities; the essential element of the system is unambiguous definition and coding of the types and amount of work to be done.

  12. RNA helicases

    PubMed Central

    Owttrim, George W.

    2013-01-01

    Similar to proteins, RNA molecules must fold into the correct conformation and associate with protein complexes in order to be functional within a cell. RNA helicases rearrange RNA secondary structure and RNA-protein interactions in an ATP-dependent reaction, performing crucial functions in all aspects of RNA metabolism. In prokaryotes, RNA helicase activity is associated with roles in housekeeping functions including RNA turnover, ribosome biogenesis, translation and small RNA metabolism. In addition, RNA helicase expression and/or activity are frequently altered during cellular response to abiotic stress, implying they perform defined roles during cellular adaptation to changes in the growth environment. Specifically, RNA helicases contribute to the formation of cold-adapted ribosomes and RNA degradosomes, implying a role in alleviation of RNA secondary structure stabilization at low temperature. A common emerging theme involves RNA helicases acting as scaffolds for protein-protein interaction and functioning as molecular clamps, holding RNA-protein complexes in specific conformations. This review highlights recent advances in DEAD-box RNA helicase association with cellular response to abiotic stress in prokaryotes. PMID:23093803

  13. New vectors for simple and streamlined CRISPR-Cas9 genome editing in Saccharomyces cerevisiae.

    PubMed

    Laughery, Marian F; Hunter, Tierra; Brown, Alexander; Hoopes, James; Ostbye, Travis; Shumaker, Taven; Wyrick, John J

    2015-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is an important tool for genome editing because the Cas9 endonuclease can induce targeted DNA double-strand breaks. Targeting of the DNA break is typically controlled by a single-guide RNA (sgRNA), a chimeric RNA containing a structural segment important for Cas9 binding and a 20mer guide sequence that hybridizes to the genomic DNA target. Previous studies have demonstrated that CRISPR-Cas9 technology can be used for efficient, marker-free genome editing in Saccharomyces cerevisiae. However, introducing the 20mer guide sequence into yeast sgRNA expression vectors often requires cloning procedures that are complex, time-consuming and/or expensive. To simplify this process, we have developed a new sgRNA expression cassette with internal restriction enzyme sites that permit rapid, directional cloning of 20mer guide sequences. Here we describe a flexible set of vectors based on this design for cloning and expressing sgRNAs (and Cas9) in yeast using different selectable markers. We anticipate that the Cas9-sgRNA expression vector with the URA3 selectable marker (pML104) will be particularly useful for genome editing in yeast, since the Cas9 machinery can be easily removed by counter-selection using 5-fluoro-orotic acid (5-FOA) following successful genome editing. The availability of new vectors that simplify and streamline the technical steps required for guide sequence cloning should help accelerate the use of CRISPR-Cas9 technology in yeast genome editing. PMID:26305040

  14. Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.

    PubMed

    Hendel, Ayal; Bak, Rasmus O; Clark, Joseph T; Kennedy, Andrew B; Ryan, Daniel E; Roy, Subhadeep; Steinfeld, Israel; Lunstad, Benjamin D; Kaiser, Robert J; Wilkens, Alec B; Bacchetta, Rosa; Tsalenko, Anya; Dellinger, Douglas; Bruhn, Laurakay; Porteus, Matthew H

    2015-09-01

    CRISPR-Cas-mediated genome editing relies on guide RNAs that direct site-specific DNA cleavage facilitated by the Cas endonuclease. Here we report that chemical alterations to synthesized single guide RNAs (sgRNAs) enhance genome editing efficiency in human primary T cells and CD34(+) hematopoietic stem and progenitor cells. Co-delivering chemically modified sgRNAs with Cas9 mRNA or protein is an efficient RNA- or ribonucleoprotein (RNP)-based delivery method for the CRISPR-Cas system, without the toxicity associated with DNA delivery. This approach is a simple and effective way to streamline the development of genome editing with the potential to accelerate a wide array of biotechnological and therapeutic applications of the CRISPR-Cas technology. PMID:26121415

  15. Strategies of Qualitative Inquiry. Third Edition

    ERIC Educational Resources Information Center

    Denzin, Norman K., Ed.; Lincoln, Yvonna S., Ed.

    2007-01-01

    "Strategies of Qualitative Inquiry, Third Edition," the second volume in the paperback version of "The SAGE Handbook of Qualitative Research, 3rd Edition," consists of Part III of the handbook ("Strategies of Inquiry"). "Strategies of Qualitative Inquiry, Third Edition" presents the major tactics--historically, the research methods--that…

  16. 48 CFR 53.102 - Current editions.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 2 2010-10-01 2010-10-01 false Current editions. 53.102... AND FORMS FORMS General 53.102 Current editions. The form prescriptions in subpart 53.2 and the illustrations in subpart 53.3 contain current edition dates. Contracting officers shall use the current...

  17. 48 CFR 53.102 - Current editions.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 2 2014-10-01 2014-10-01 false Current editions. 53.102... AND FORMS FORMS General 53.102 Current editions. The form prescriptions in subpart 53.2 and the illustrations in subpart 53.3 contain current edition dates. Contracting officers shall use the current...

  18. 48 CFR 53.102 - Current editions.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 2 2012-10-01 2012-10-01 false Current editions. 53.102... AND FORMS FORMS General 53.102 Current editions. The form prescriptions in subpart 53.2 and the illustrations in subpart 53.3 contain current edition dates. Contracting officers shall use the current...

  19. 48 CFR 53.102 - Current editions.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 2 2013-10-01 2013-10-01 false Current editions. 53.102... AND FORMS FORMS General 53.102 Current editions. The form prescriptions in subpart 53.2 and the illustrations in subpart 53.3 contain current edition dates. Contracting officers shall use the current...

  20. 48 CFR 53.102 - Current editions.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 48 Federal Acquisition Regulations System 2 2011-10-01 2011-10-01 false Current editions. 53.102... AND FORMS FORMS General 53.102 Current editions. The form prescriptions in subpart 53.2 and the illustrations in subpart 53.3 contain current edition dates. Contracting officers shall use the current...

  1. Transgene-free genome editing in Caenorhabditis elegans using CRISPR-Cas.

    PubMed

    Chiu, Hui; Schwartz, Hillel T; Antoshechkin, Igor; Sternberg, Paul W

    2013-11-01

    CRISPR-Cas is an efficient method for genome editing in organisms from bacteria to human cells. We describe a transgene-free method for CRISPR-Cas-mediated cleavage in nematodes, enabling RNA-homology-targeted deletions that cause loss of gene function; analysis of whole-genome sequencing indicates that the nuclease activity is highly specific. PMID:23979577

  2. Strengthening Family Resilience, Second Edition

    ERIC Educational Resources Information Center

    Walsh, Froma

    2006-01-01

    In a fully revised, updated, and expanded second edition, this informative clinical resource and text presents Froma Walsh's family resilience framework for intervention and prevention with clients dealing with adversity. Drawing on extensive research and clinical experience, the author describes key processes in resilience for practitioners to…

  3. Teaching Population Concepts. Revised Edition.

    ERIC Educational Resources Information Center

    King, Pat; Landahl, John

    This edition is designed to help teachers provide their students with some basic population concepts with stress placed on the elements of decision making. In the first section of the pamphlet, some of the basic concepts of population study are presented. These include populations, growth rates, birth and death rates, doubling time, migration, age…

  4. Veterinary Microbiology, 3rd Edition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Veterinary Microbiology, Third Edition is organized into four sections and begins with an updated and expanded introductory section on infectious disease pathogenesis, diagnosis and clinical management. The second section covers bacterial and fungal pathogens, and the third section describes viral d...

  5. Nuclear Electricity. 5th Edition.

    ERIC Educational Resources Information Center

    Hore-Lacy, Ian

    Educators must address the need for young people to be informed about both the scientific concepts and the reasons for controversy when dealing with controversial issues. Young people must be given the opportunity to form their own opinions when presented with evidence for conflicting arguments. Previous editions of "Nuclear Electricity" have…

  6. The Horizon Report. 2007 Edition

    ERIC Educational Resources Information Center

    New Media Consortium, 2007

    2007-01-01

    This fourth edition of the New Media Consortium's (NMC) annual "Horizon Report" describes the continuing work of the Horizon Project, a research-oriented effort that seeks to identify and describe emerging technologies likely to have a large impact on teaching, learning, or creative expression within higher education. Drawing on ongoing…

  7. Biology Teachers' Handbook, Second Edition.

    ERIC Educational Resources Information Center

    Klinckmann, Evelyn; And Others

    This is the second edition of the handbook written for teachers using the Biological Sciences Curriculum Study (BSCS) materials. Section one describes the historical background, aims and emphases of the BSCS materials and includes behavioral objectives for teaching inquiry processes. Section two includes 44 "Invitations to Enquiry." Section three,…

  8. A Writer's Reference. Third Edition.

    ERIC Educational Resources Information Center

    Hacker, Diana

    Designed to save the user time and packaged in a compact size which lies flat, this book is easy to consult while revising and editing a written draft. The book's "main menu," just inside the front cover, displays the contents as briefly and simply as possible. Each of the 12 sections in the book's main menu leads the user to a tabbed divider, on…

  9. Teaching Reading Sourcebook, Second Edition

    ERIC Educational Resources Information Center

    Honig, Bill; Diamond, Linda; Gutlohn, Linda

    2008-01-01

    The "Teaching Reading Sourcebook, Second Edition" is a comprehensive reference about reading instruction. Organized according to the elements of explicit instruction (what? why? when? and how?), the "Sourcebook" includes both a research-informed knowledge base and practical sample lesson models. It teaches the key elements of an effective reading…

  10. The Foundation Directory, Edition 4.

    ERIC Educational Resources Information Center

    Lewis, Marianna O., Ed.; Bowers, Patricia, Ed.

    The fourth edition of "The Foundation Directory" lists and describes 5,454 foundations and surveys their grants. The directory was prepared from foundation reports and government records. The foundations listed either have assets of $500.00 or made grants totally at least $25,000.00 in the year of record. Education is the leading beneficiary of…

  11. Promoting School Success. Third Edition

    ERIC Educational Resources Information Center

    Lovitt, Thomas C.

    2007-01-01

    Like its two predecessors, "Preventing School Dropouts" [C1991] and "Preventing School Failure" [C2000], this third edition is a book about teaching. Although primarily written for teachers, tutors and parents may also find this book helpful. It is a collection of carefully selected teaching techniques aimed at helping young adults learn important…

  12. Resident Care Guide. Third Edition.

    ERIC Educational Resources Information Center

    Woodbridge State School, NJ.

    The third edition of the Woodbridge State School Cottage Life Department Resident Care Guide is explained to be a developmental status scale devised in 1969 as part of a 5-year study for the purposes of measuring the entire population's self-help training abilities. The department is said to serve 954 residents; 424 are non-ambulatory and 530 are…

  13. Modern Indian Psychology. Revised Edition.

    ERIC Educational Resources Information Center

    Bryde, John F.

    Written on the basis of senior Indian verbal relatings collected over a 23-year span, this revised edition on modern Indian psychology incorporates suggestions from Indian students and their teachers, Indian and non-Indian social studies experts, and other Indian people. The book contains 6 major divisions: (1) "Culture and Indian Values" relates…

  14. Money and Schools. Fifth Edition

    ERIC Educational Resources Information Center

    Thompson, David C.; Crampton, Faith E.; Wood, R. Craig

    2012-01-01

    In the new edition of this essential, all-inclusive text, the authors provide more important research for future principals and others enrolled in graduate-level school finance courses. Written in a style that is highly readable, the book offers strong connections to real-world experiences. Readers get both a broad overview of funding concepts and…

  15. Unlocking Mathematics Teaching. Second Edition

    ERIC Educational Resources Information Center

    Koshy, Valsa, Ed.; Murray, Jean, Ed.

    2011-01-01

    Now in a fully updated second edition, "Unlocking Mathematics Teaching" is a comprehensive guide to teaching mathematics in the primary school. Combining theory and practice, selected experts outline the current context of mathematics education. They suggest strategies, activities and examples to help develop readers understanding and confidence…

  16. The Horizon Report. 2006 Edition

    ERIC Educational Resources Information Center

    New Media Consortium, 2006

    2006-01-01

    This third edition of the New Media Consortium's (NMC) annual "Horizon Report" describes the continuing work of the Horizon Project, a research-oriented effort that seeks to identify and describe emerging technologies likely to have a large impact on teaching, learning, or creative expression within higher education. Drawing on ongoing discussions…

  17. Writing Back: Revising and Editing.

    ERIC Educational Resources Information Center

    Johnstone, Velerie M.

    1990-01-01

    Discusses a "write-back" strategy for use when there is insufficient class time for conferencing with peers or the teacher. Notes that the teacher agrees to respond to students' drafts within 24 hours. Discusses 3 options available to students: (1) write back/respond; (2) edit; and (3) writing back. (RS)

  18. The Horizon Report. 2004 Edition

    ERIC Educational Resources Information Center

    New Media Consortium, 2004

    2004-01-01

    This first edition of the New Media Consortium's (NMC) annual "Horizon Report" details findings of the Horizon Project, a research-oriented effort that seeks to identify and describe emerging technologies likely to have a large impact on teaching, learning, or creative expression within higher education. Drawing on an ongoing series of interviews…

  19. The Horizon Report. 2005 Edition

    ERIC Educational Resources Information Center

    New Media Consortium, 2005

    2005-01-01

    This second edition of the New Media Consortium's (NMC) annual "Horizon Report" describes the continuing work of the Horizon Project, a research-oriented effort that seeks to identify and describe emerging technologies likely to have a large impact on teaching, learning, or creative expression within higher education. Drawing on an ongoing series…

  20. CRISPR/Cas9 in Genome Editing and Beyond.

    PubMed

    Wang, Haifeng; La Russa, Marie; Qi, Lei S

    2016-06-01

    The Cas9 protein (CRISPR-associated protein 9), derived from type II CRISPR (clustered regularly interspaced short palindromic repeats) bacterial immune systems, is emerging as a powerful tool for engineering the genome in diverse organisms. As an RNA-guided DNA endonuclease, Cas9 can be easily programmed to target new sites by altering its guide RNA sequence, and its development as a tool has made sequence-specific gene editing several magnitudes easier. The nuclease-deactivated form of Cas9 further provides a versatile RNA-guided DNA-targeting platform for regulating and imaging the genome, as well as for rewriting the epigenetic status, all in a sequence-specific manner. With all of these advances, we have just begun to explore the possible applications of Cas9 in biomedical research and therapeutics. In this review, we describe the current models of Cas9 function and the structural and biochemical studies that support it. We focus on the applications of Cas9 for genome editing, regulation, and imaging, discuss other possible applications and some technical considerations, and highlight the many advantages that CRISPR/Cas9 technology offers. PMID:27145843

  1. BRASERO: A Resource for Benchmarking RNA Secondary Structure Comparison Algorithms.

    PubMed

    Allali, Julien; Saule, Cédric; Chauve, Cédric; d'Aubenton-Carafa, Yves; Denise, Alain; Drevet, Christine; Ferraro, Pascal; Gautheret, Daniel; Herrbach, Claire; Leclerc, Fabrice; de Monte, Antoine; Ouangraoua, Aida; Sagot, Marie-France; Termier, Michel; Thermes, Claude; Touzet, Hélène

    2012-01-01

    The pairwise comparison of RNA secondary structures is a fundamental problem, with direct application in mining databases for annotating putative noncoding RNA candidates in newly sequenced genomes. An increasing number of software tools are available for comparing RNA secondary structures, based on different models (such as ordered trees or forests, arc annotated sequences, and multilevel trees) and computational principles (edit distance, alignment). We describe here the website BRASERO that offers tools for evaluating such software tools on real and synthetic datasets. PMID:22675348

  2. Diesel Technology: Steering and Suspension. Second Edition. Teacher Edition [and] Student Edition.

    ERIC Educational Resources Information Center

    Miller, Roger; Scarberry, Terry; Tesch, Carl; Kellum, Mary

    These teacher and student editions on steering and suspension are part of the diesel mechanics series of instructional materials. The series aligns with the medium/heavy duty truck task list developed by the National Automotive Technicians Education Foundation and used by the National Institute for Automotive Service Excellence in the…

  3. Diesel Technology: Safety Skills. Teacher Edition [and] Student Edition. Second Edition.

    ERIC Educational Resources Information Center

    Kellum, Mary

    Teacher and student editions of this document are one in a series of competency-based instructional materials for diesel technology programs. The series aligns with the medium/heavy diesel duty truck task list used by the National Institute for Automotive Service Excellence in the certification of medium/heavy duty truck technicians. Introductory…

  4. Moving RNA moves RNA forward.

    PubMed

    Peng, Lina; Li, Yujiao; Zhang, Lan; Yu, Wenqiang

    2013-10-01

    Cell communication affects all aspects of cell structure and behavior, such as cell proliferation, differentiation, division, and coordination of various physiological functions. The moving RNA in plants and mammalian cells indicates that nucleic acid could be one of the various types of messengers for cell communication. The microvesicle is a critical pathway that mediates RNA moving and keeps moving RNA stable in body fluids. When moving miRNA enters the target cell, it functions by altering the gene expression profile and significantly inhibiting mRNA translation in recipient cells. Thus, moving RNA may act as a long-range modulator during development, organogenesis, and tumor metastasis. PMID:24008386

  5. Recent Advances in Genome Editing Using CRISPR/Cas9

    PubMed Central

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

  6. RNA epigenetics

    PubMed Central

    Liu, Nian; Pan, Tao

    2014-01-01

    Summary Mammalian messenger and long non-coding RNA contain tens of thousands of post-transcriptional chemical modifications. Among these, the N6-methyl-adenosine (m6A) modification is the most abundant and can be removed by specific mammalian enzymes. M6A modification is recognized by families of RNA binding proteins that affect many aspects of mRNA function. mRNA/lncRNA modification represents another layer of epigenetic regulation of gene expression, analogous to DNA methylation and histone modification. PMID:24768686

  7. The emerging role of viral vectors as vehicles for DMD gene editing.

    PubMed

    Maggio, Ignazio; Chen, Xiaoyu; Gonçalves, Manuel A F V

    2016-01-01

    Duchenne muscular dystrophy (DMD) is a genetic disorder caused by mutations in the dystrophin-encoding DMD gene. The DMD gene, spanning over 2.4 megabases along the short arm of the X chromosome (Xp21.2), is the largest genetic locus known in the human genome. The size of DMD, combined with the complexity of the DMD phenotype and the extent of the affected tissues, begs for the development of novel, ideally complementary, therapeutic approaches. Genome editing based on the delivery of sequence-specific programmable nucleases into dystrophin-defective cells has recently enriched the portfolio of potential therapies under investigation. Experiments involving different programmable nuclease platforms and target cell types have established that the application of genome-editing principles to the targeted manipulation of defective DMD loci can result in the rescue of dystrophin protein synthesis in gene-edited cells. Looking towards translation into the clinic, these proof-of-principle experiments have been swiftly followed by the conversion of well-established viral vector systems into delivery agents for DMD editing. These gene-editing tools consist of zinc-finger nucleases (ZFNs), engineered homing endoculeases (HEs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs) based on clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 systems. Here, we succinctly review these fast-paced developments and technologies, highlighting their relative merits and potential bottlenecks, when used as part of in vivo and ex vivo gene-editing strategies. PMID:27215286

  8. Research progress of genome editing and derivative technologies in plants.

    PubMed

    Qiwei, Shan; Caixia, Gao

    2015-10-01

    Genome editing technologies using engineered nucleases have been widely used in many model organisms. Genome editing with sequence-specific nuclease (SSN) creates DNA double-strand breaks (DSBs) in the genomic target sites that are primarily repaired by the non-homologous end joining (NHEJ) or homologous recombination (HR) pathways, which can be employed to achieve targeted genome modifications such as gene mutations, insertions, replacements or chromosome rearrangements. There are three major SSNs─zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) system. In contrast to ZFN and TALEN, which require substantial protein engineering to each DNA target, the CRISPR/Cas9 system requires only a change in the guide RNA. For this reason, the CRISPR/Cas9 system is a simple, inexpensive and versatile tool for genome engineering. Furthermore, a modified version of the CRISPR/Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression, such as activation, depression and epigenetic regulation. In this review, we summarize the development and applications of genome editing technologies for basic research and biotechnology, as well as highlight challenges and future directions, with particular emphasis on plants. PMID:26496748

  9. The secondary structure of guide RNA molecules from Trypanosoma brucei.

    PubMed Central

    Schmid, B; Riley, G R; Stuart, K; Göringer, H U

    1995-01-01

    RNA editing in kinetoplastid organisms is a mitochondrial RNA processing phenomenon that is characterized by the insertion and deletion of uridine nucleotides into incomplete mRNAs. Key molecules in the process are guide RNAs which direct the editing reaction by virtue of their primary sequences in an RNA-RNA interaction with the pre-edited mRNAs. To understand the molecular details of this reaction, especially potential RNA folding and unfolding processes as well as assembly phenomena with mitochondrial proteins, we analyzed the secondary structure of four different guide RNAs from Trypanosoma brucei at physiological conditions. By using structure-sensitive chemical and enzymatic probes in combination with spectroscopic techniques we found that the four molecules despite their different primary sequences, fold into similar structures consisting of two imperfect hairpin loops of low thermodynamic stability. The molecules melt in two-state monomolecular transitions with Tms between 33 and 39 degrees C and transition enthalpies of -32 to -38 kcal/mol. Both terminal ends of the RNAs are single-stranded with the 3' ends possibly adopting a single-stranded, helical conformation. Thus, it appears that the gRNA structures are fine tuned to minimize stability for an optimal annealing reaction to the pre-mRNAs while at the same time maximizing higher order structural features to permit the assembly with other mitochondrial components into the editing machinery. Images PMID:7667084

  10. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system

    PubMed Central

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab′ fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  11. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system.

    PubMed

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM(+) mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab' fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  12. CRISPR-ERA: a comprehensive design tool for CRISPR-mediated gene editing, repression and activation

    PubMed Central

    Liu, Honglei; Wei, Zheng; Dominguez, Antonia; Li, Yanda; Wang, Xiaowo; Qi, Lei S.

    2015-01-01

    Summary: The CRISPR/Cas9 system was recently developed as a powerful and flexible technology for targeted genome engineering, including genome editing (altering the genetic sequence) and gene regulation (without altering the genetic sequence). These applications require the design of single guide RNAs (sgRNAs) that are efficient and specific. However, this remains challenging, as it requires the consideration of many criteria. Several sgRNA design tools have been developed for gene editing, but currently there is no tool for the design of sgRNAs for gene regulation. With accumulating experimental data on the use of CRISPR/Cas9 for gene editing and regulation, we implement a comprehensive computational tool based on a set of sgRNA design rules summarized from these published reports. We report a genome-wide sgRNA design tool and provide an online website for predicting sgRNAs that are efficient and specific. We name the tool CRISPR-ERA, for clustered regularly interspaced short palindromic repeat-mediated editing, repression, and activation (ERA). Availability and implementation: http://CRISPR-ERA.stanford.edu. Contact: stanley.qi@stanford.edu or xwwang@tsinghua.edu.cn Supplementary information: Supplementary data are available at Bioinformatics online. PMID:26209430

  13. PAM multiplicity marks genomic target sites as inhibitory to CRISPR-Cas9 editing

    PubMed Central

    Malina, Abba; Cameron, Christopher J. F.; Robert, Francis; Blanchette, Mathieu; Dostie, Josée; Pelletier, Jerry

    2015-01-01

    In CRISPR-Cas9 genome editing, the underlying principles for selecting guide RNA (gRNA) sequences that would ensure for efficient target site modification remain poorly understood. Here we show that target sites harbouring multiple protospacer adjacent motifs (PAMs) are refractory to Cas9-mediated repair in situ. Thus we refine which substrates should be avoided in gRNA design, implicating PAM density as a novel sequence-specific feature that inhibits in vivo Cas9-driven DNA modification. PMID:26644285

  14. Application of the genome editing tool CRISPR/Cas9 in non-human primates

    PubMed Central

    LUO, Xin; LI, Min; SU, Bing

    2016-01-01

    In the past three years, RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system has been used to facilitate efficient genome editing in many model and non-model animals. However, its application in nonhuman primates is still at the early stage, though in view of the similarities in anatomy, physiology, behavior and genetics, closely related nonhuman primates serve as optimal models for human biology and disease studies. In this review, we summarize the current proceedings of gene editing using CRISPR/Cas9 in nonhuman primates. PMID:27469252

  15. Potential pitfalls of CRISPR/Cas9-mediated genome editing.

    PubMed

    Peng, Rongxue; Lin, Guigao; Li, Jinming

    2016-04-01

    Recently, a novel technique named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas)9 system has been rapidly developed. This genome editing tool has improved our ability tremendously with respect to exploring the pathogenesis of diseases and correcting disease mutations, as well as phenotypes. With a short guide RNA, Cas9 can be precisely directed to target sites, and functions as an endonuclease to efficiently produce breaks in DNA double strands. Over the past 30 years, CRISPR has evolved from the 'curious sequences of unknown biological function' into a promising genome editing tool. As a result of the incessant development in the CRISPR/Cas9 system, Cas9 co-expressed with custom guide RNAs has been successfully used in a variety of cells and organisms. This genome editing technology can also be applied to synthetic biology, functional genomic screening, transcriptional modulation and gene therapy. However, although CRISPR/Cas9 has a broad range of action in science, there are several aspects that affect its efficiency and specificity, including Cas9 activity, target site selection and short guide RNA design, delivery methods, off-target effects and the incidence of homology-directed repair. In the present review, we highlight the factors that affect the utilization of CRISPR/Cas9, as well as possible strategies for handling any problems. Addressing these issues will allow us to take better advantage of this technique. In addition, we also review the history and rapid development of the CRISPR/Cas system from the time of its initial discovery in 2012. PMID:26535798

  16. Genome editing strategies: potential tools for eradicating HIV-1/AIDS

    PubMed Central

    Khalili, Kamel; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

    2015-01-01

    Current therapy for controlling HIV-1 infection and preventing AIDS progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or “sterile” cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including ZFNs, TALENs, and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS. PMID:25716921

  17. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum.

    PubMed

    Pohl, C; Kiel, J A K W; Driessen, A J M; Bovenberg, R A L; Nygård, Y

    2016-07-15

    CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions. PMID:27072635

  18. Image Editing Via Searching Source Image

    NASA Astrophysics Data System (ADS)

    Yu, Han; Deng, Liang-Jian

    Image editing has important applications by changing the image texture, illumination, target location, etc. As an important application of Poisson equation, Poisson image editing processes images on the gradient domain and has been applied to seamless clone, selection editing, image denoising, etc. In this paper, we present a new application of Poisson image editing, which is based on searching source image. The main feature of the new application is all modifying information comes from the source image. Experimental results show that the proposed application performs well.

  19. Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing.

    PubMed

    Kelley, Melissa L; Strezoska, Žaklina; He, Kaizhang; Vermeulen, Annaleen; Smith, Anja van Brabant

    2016-09-10

    The CRISPR-Cas9 system has become the most popular and efficient method for genome engineering in mammalian cells. The Streptococcus pyogenes Cas9 nuclease can function with two types of guide RNAs: the native dual crRNA and tracrRNA (crRNA:tracrRNA) or a chimeric single guide RNA (sgRNA). Although sgRNAs expressed from a DNA vector are predominant in the literature, guide RNAs can be rapidly generated by chemical synthesis and provide equivalent functionality in gene editing experiments. This review highlights the attributes and advantages of chemically synthesized guide RNAs including the incorporation of chemical modifications to enhance gene editing efficiencies in certain applications. The use of synthetic guide RNAs is also uniquely suited to genome-scale high throughput arrayed screening, particularly when using complex phenotypic assays for functional genomics studies. Finally, the use of synthetic guide RNAs along with DNA-free sources of Cas9 (mRNA or protein) allows for transient CRISPR-Cas9 presence in the cell, thereby resulting in a decreased probability of off-target events. PMID:27374403

  20. Evaluation of microRNA alignment techniques.

    PubMed

    Ziemann, Mark; Kaspi, Antony; El-Osta, Assam

    2016-08-01

    Genomic alignment of small RNA (smRNA) sequences such as microRNAs poses considerable challenges due to their short length (∼21 nucleotides [nt]) as well as the large size and complexity of plant and animal genomes. While several tools have been developed for high-throughput mapping of longer mRNA-seq reads (>30 nt), there are few that are specifically designed for mapping of smRNA reads including microRNAs. The accuracy of these mappers has not been systematically determined in the case of smRNA-seq. In addition, it is unknown whether these aligners accurately map smRNA reads containing sequence errors and polymorphisms. By using simulated read sets, we determine the alignment sensitivity and accuracy of 16 short-read mappers and quantify their robustness to mismatches, indels, and nontemplated nucleotide additions. These were explored in the context of a plant genome (Oryza sativa, ∼500 Mbp) and a mammalian genome (Homo sapiens, ∼3.1 Gbp). Analysis of simulated and real smRNA-seq data demonstrates that mapper selection impacts differential expression results and interpretation. These results will inform on best practice for smRNA mapping and enable more accurate smRNA detection and quantification of expression and RNA editing. PMID:27284164

  1. Revealing editing and SNPs of microRNAs in colon tissues by analyzing high-throughput sequencing profiles of small RNAs

    PubMed Central

    2014-01-01

    Background Editing and mutations in microRNAs (miRNAs) can change the stability of pre-miRNAs and/or complementarities between miRNAs and their targets. Small RNA (sRNA) high-throughput sequencing (HTS) profiles contain miRNAs that are originated from mutated DNAs or are edited during their biogenesis procedures. It is largely unknown whether miRNAs are edited in colon tissues since existing studies mainly focused their attention on the editing of miRNAs in brain tissues. Results Through comprehensive analysis of four high-throughput sequencing profiles of normal and cancerous colon tissues, we identified 548 editing and/or SNPs in miRNAs that are significant in at least one of the sequencing profiles used. Our results show that the most abundant editing events of miRNAs in colon tissues are 3'-A and 3'-U. In addition to four known A-to-I editing sites previously reported in brain tissues, four novel A-to-I editing sites are also identified in colon tissues. Conclusions This suggests that A-to-I editing of miRNAs potentially is a commonly existing mechanism in different tissues to diversify the possible functional roles of miRNAs, but only a small portion of different miRNAs are edited by the A-to-I mechanism at a significant level. Our results suggest that there are other types of editing in miRNAs through unknown mechanisms. Furthermore, several SNPs in miRNAs are also identified. PMID:25521855

  2. Fuel Cell Handbook, Fourth Edition

    SciTech Connect

    Stauffer, D.B; Hirschenhofer, J.H.; Klett, M.G.; Engleman, R.R.

    1998-11-01

    Robust progress has been made in fuel cell technology since the previous edition of the Fuel Cell Handbook was published in January 1994. This Handbook provides a foundation in fuel cells for persons wanting a better understanding of the technology, its benefits, and the systems issues that influence its application. Trends in technology are discussed, including next-generation concepts that promise ultra high efficiency and low cost, while providing exceptionally clean power plant systems. Section 1 summarizes fuel cell progress since the last edition and includes existing power plant nameplate data. Section 2 addresses the thermodynamics of fuel cells to provide an understanding of fuel cell operation at two levels (basic and advanced). Sections 3 through 6 describe the four major fuel cell types and their performance based on cell operating conditions. The section on polymer electrolyte membrane fuel cells has been added to reflect their emergence as a significant fuel cell technology. Phosphoric acid, molten carbonate, and solid oxide fuel cell technology description sections have been updated from the previous edition. New information indicates that manufacturers have stayed with proven cell designs, focusing instead on advancing the system surrounding the fuel cell to lower life cycle costs. Section 7, Fuel Cell Systems, has been significantly revised to characterize near-term and next-generation fuel cell power plant systems at a conceptual level of detail. Section 8 provides examples of practical fuel cell system calculations. A list of fuel cell URLs is included in the Appendix. A new index assists the reader in locating specific information quickly.

  3. Pediatric surgical pathology. Second edition

    SciTech Connect

    Dehner, L.P.

    1987-01-01

    The edition provides view of congenital, hereditary, infectious, and inflammatory neoplastic diseases occurring during the first two decades of life, with special reference to clinical, laboratory, and roentgenographic features. Material includes observations from some of the major national studies on Wilms' tumor and rhabdomyosarcomas, the new classification of pediatric malignant lymphomas, a discussion of the role of immunocytochemistry as it applies to the diagnosis of childhood infections and neoplasms, an examination of graft-versus-host disease in the liver and intestinal tract and more.

  4. NASA Pocket Statistics: 1997 Edition

    NASA Technical Reports Server (NTRS)

    1997-01-01

    POCKET STATISTICS is published by the NATIONAL AERONAUTICS AND SPACE ADMINISTRATION (NASA). Included in each edition is Administrative and Organizational information, summaries of Space Flight Activity including the NASA Major Launch Record, Aeronautics and Space Transportation and NASA Procurement, Financial and Workforce data. The NASA Major Launch Record includes all launches of Scout class and larger vehicles. Vehicle and spacecraft development flights are also included in the Major Launch Record. Shuttle missions are counted as one launch and one payload, where free flying payloads are not involved. All Satellites deployed from the cargo bay of the Shuttle and placed in a separate orbit or trajectory are counted as an additional payload.

  5. RNA helicases

    PubMed Central

    Ranji, Arnaz

    2010-01-01

    RNA helicases serve multiple roles at the virus-host interface. In some situations, RNA helicases are essential host factors to promote viral replication; however, in other cases they serve as a cellular sensor to trigger the antiviral state in response to viral infection. All family members share the conserved ATP-dependent catalytic core linked to different substrate recognition and protein-protein interaction domains. These flanking domains can be shuffled between different helicases to achieve functional diversity. This review summarizes recent studies, This review summarizes recent studies of RNA helicases in virus biology. First, RNA helicases are catalysts of progressive RNA-protein rearrangements that begin at gene transcription and culminate in release of infectious virus. Second, RNA helicases can act as a scaffold for alternative protein-protein interactions that can defeat the antiviral state. The mounting fundamental understanding of RNA helicases is being used to develop selective and efficacious drugs against human and animal pathogens. The analysis of RNA helicases in virus model systems continues to provide insights into virology, cell biology and immunology and has provided fresh perspective to continue unraveling the complexity of virus-host interactions. PMID:21173576

  6. RNA. Introduction.

    PubMed

    Bao, Marie Z; Kruger, Robert P; Rivas, Fabiola; Smith, Orla; Szewczak, Lara

    2009-02-20

    Two scientists walk into a bar. After a pint and an exchange of pleasantries, one says to the other, "Where do you come from? Scientifically, I mean." The queried scientist responds, "Out of the RNA world." "Don't we all," the asker responds chuckling. Fifteen years ago, the joke would have been made with a nod to the notion that life arose from an RNA-based precursor, the so-called "RNA world." Yet had this conversation happened last week, the scientists would also be grinning in appreciation of the extent to which contemporary cellular biology is steeped in all things RNA. Ours is truly an RNA world.In this year's special review issue, the Cell editorial team has brought together articles focused on RNA in the modern world, providing perspectives on classical and emerging areas of inquiry. We extend our thanks to the many distinguished experts who contributed their time and effort as authors and reviewers to make the issue informative, thought-provoking, and timely. We hope that this collection of articles, written as we stand on the verge of a new wave of RNA biology, edifies and inspires by revealing the inner workings of these versatile molecules and by highlighting the next key questions that need to be addressed as we strive to understand the full functional scope of RNA in cells. PMID:19263588

  7. The Technology of Human Editing (The Friendly Editor).

    ERIC Educational Resources Information Center

    Bush, Don

    1992-01-01

    Contrasts robotic editing with human editing (discussing descriptive grammar, periodic sentences, theme-rheme concept, right-branching, zeugma, and Irish bulls). Maintains that, for any editing that requires thinking, humans are always superior. (SR)

  8. CRISPR/Cas9-Mediated Genome Editing of Mouse Small Intestinal Organoids.

    PubMed

    Schwank, Gerald; Clevers, Hans

    2016-01-01

    The CRISPR/Cas9 system is an RNA-guided genome-editing tool that has been recently developed based on the bacterial CRISPR-Cas immune defense system. Due to its versatility and simplicity, it rapidly became the method of choice for genome editing in various biological systems, including mammalian cells. Here we describe a protocol for CRISPR/Cas9-mediated genome editing in murine small intestinal organoids, a culture system in which somatic stem cells are maintained by self-renewal, while giving rise to all major cell types of the intestinal epithelium. This protocol allows the study of gene function in intestinal epithelial homeostasis and pathophysiology and can be extended to epithelial organoids derived from other internal mouse and human organs. PMID:27246017

  9. Alternate rRNA secondary structures as regulators of translation.

    PubMed

    Feng, Shu; Li, Heng; Zhao, Jing; Pervushin, Konstantin; Lowenhaupt, Ky; Schwartz, Thomas U; Dröge, Peter

    2011-02-01

    Structural dynamics of large molecular assemblies are intricately linked to function. For ribosomes, macromolecular changes occur especially during mRNA translation and involve participation of ribosomal RNA. Without suitable probes specific to RNA secondary structure, however, elucidation of more subtle dynamic ribosome structure-function relationships, especially in vivo, remains challenging. Here we report that the Z-DNA- and Z-RNA-binding domain Zα, derived from the human RNA editing enzyme ADAR1-L, binds with high stability to specific rRNA segments of Escherichia coli and human ribosomes. Zα impaired in Z-RNA recognition does not associate with ribosomes. Notably, Zα(ADAR1)-ribosome interaction blocks translation in vitro and in vivo, with substantial physiological consequences. Our study shows that ribosomes can be targeted by a protein that specifically recognizes an alternate rRNA secondary structure, and suggests a new mechanism of translational regulation on the ribosome. PMID:21217697

  10. Lentivirus pre-packed with Cas9 protein for safer gene editing.

    PubMed

    Choi, J G; Dang, Y; Abraham, S; Ma, H; Zhang, J; Guo, H; Cai, Y; Mikkelsen, J G; Wu, H; Shankar, P; Manjunath, N

    2016-07-01

    The CRISPR/Cas9 system provides an easy way to edit specific site/s in the genome and thus offers tremendous opportunity for human gene therapy for a wide range of diseases. However, one major concern is off-target effects, particularly with long-term expression of Cas9 nuclease when traditional expression methods such as via plasmid/viral vectors are used. To overcome this limitation, we pre-packaged Cas9 protein (Cas9P LV) in lentiviral particles for transient exposure and showed its effectiveness for gene disruption in cells, including primary T cells expressing specific single guide RNAs (sgRNAs). We then constructed an 'all in one virus' to express sgRNAs in association with pre-packaged Cas9 protein (sgRNA/Cas9P LV). We successfully edited CCR5 in TZM-bl cells by this approach. Using an sgRNA-targeting HIV long terminal repeat, we also were able to disrupt HIV provirus in the J-LAT model of viral latency. Moreover, we also found that pre-packaging Cas9 protein in LV particle reduced off-target editing of chromosome 4:-29134166 locus by CCR5 sgRNA, compared with continued expression from the vector. These results show that sgRNA/Cas9P LV can be used as a safer approach for human gene therapy applications. PMID:27052803

  11. CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots.

    PubMed

    Cai, Yupeng; Chen, Li; Liu, Xiujie; Sun, Shi; Wu, Cunxiang; Jiang, Bingjun; Han, Tianfu; Hou, Wensheng

    2015-01-01

    As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean. PMID:26284791

  12. Chimeric human mitochondrial PheRS exhibits editing activity to discriminate nonprotein amino acids.

    PubMed

    Kartvelishvili, Ekaterine; Peretz, Moshe; Tworowski, Dmitry; Moor, Nina; Safro, Mark

    2016-03-01

    Mitochondria are considered as the primary source of reactive oxygen species (ROS) in nearly all eukaryotic cells during respiration. The harmful effects of these compounds range from direct neurotoxicity to incorporation into proteins producing aberrant molecules with multiple physiological problems. Phenylalanine exposure to ROS produces multiple oxidized isomers: tyrosine, Levodopa, ortho-Tyr, meta-Tyr (m-Tyr), and so on. Cytosolic phenylalanyl-tRNA synthetase (PheRS) exerts control over the translation accuracy, hydrolyzing misacylated products, while monomeric mitochondrial PheRS lacks the editing activity. Recently we showed that "teamwork" of cytosolic and mitochondrial PheRSs cannot prevent incorporation of m-Tyr and l-Dopa into proteins. Here, we present human mitochondrial chimeric PheRS with implanted editing module taken from EcPheRS. The monomeric mitochondrial chimera possesses editing activity, while in bacterial and cytosolic PheRSs this type of activity was detected for the (αβ)2 architecture only. The fusion protein catalyzes aminoacylation of tRNA(Phe) with cognate phenylalanine and effectively hydrolyzes the noncognate aminoacyl-tRNAs: Tyr-tRNA(Phe) and m-Tyr-tRNA(Phe) . PMID:26645192

  13. Site-specific genome editing for correction of induced pluripotent stem cells derived from dominant dystrophic epidermolysis bullosa.

    PubMed

    Shinkuma, Satoru; Guo, Zongyou; Christiano, Angela M

    2016-05-17

    Genome editing with engineered site-specific endonucleases involves nonhomologous end-joining, leading to reading frame disruption. The approach is applicable to dominant negative disorders, which can be treated simply by knocking out the mutant allele, while leaving the normal allele intact. We applied this strategy to dominant dystrophic epidermolysis bullosa (DDEB), which is caused by a dominant negative mutation in the COL7A1 gene encoding type VII collagen (COL7). We performed genome editing with TALENs and CRISPR/Cas9 targeting the mutation, c.8068_8084delinsGA. We then cotransfected Cas9 and guide RNA expression vectors expressed with GFP and DsRed, respectively, into induced pluripotent stem cells (iPSCs) generated from DDEB fibroblasts. After sorting, 90% of the iPSCs were edited, and we selected four gene-edited iPSC lines for further study. These iPSCs were differentiated into keratinocytes and fibroblasts secreting COL7. RT-PCR and Western blot analyses revealed gene-edited COL7 with frameshift mutations degraded at the protein level. In addition, we confirmed that the gene-edited truncated COL7 could neither associate with normal COL7 nor undergo triple helix formation. Our data establish the feasibility of mutation site-specific genome editing in dominant negative disorders. PMID:27143720

  14. REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes.

    PubMed

    Kumar, Vikas; Madina, Bhaskara R; Gulati, Shelly; Vashisht, Ajay A; Kanyumbu, Chiedza; Pieters, Brittany; Shakir, Afzal; Wohlschlegel, James A; Read, Laurie K; Mooers, Blaine H M; Cruz-Reyes, Jorge

    2016-03-11

    Mitochondrial mRNAs in Trypanosoma brucei undergo extensive insertion and deletion of uridylates that are catalyzed by the RNA editing core complex (RECC) and directed by hundreds of small guide RNAs (gRNAs) that base pair with mRNA. RECC is largely RNA-free, and accessory mitochondrial RNA-binding complex 1 (MRB1) variants serve as scaffolds for the assembly of mRNA-gRNA hybrids and RECC. However, the molecular steps that create higher-order holoenzymes ("editosomes") are unknown. Previously, we identified an RNA editing helicase 2-associated subcomplex (REH2C) and showed that REH2 binds RNA. Here we showed that REH2C is an mRNA-associated ribonucleoprotein (mRNP) subcomplex with editing substrates, intermediates, and products. We isolated this mRNP from mitochondria lacking gRNA-bound RNP (gRNP) subcomplexes and identified REH2-associated cofactors 1 and 2 ((H2)F1 and (H2)F2). (H2)F1 is an octa-zinc finger protein required for mRNP-gRNP docking, pre-mRNA and RECC loading, and RNP formation with a short synthetic RNA duplex. REH2 and other eukaryotic DEAH/RHA-type helicases share a conserved regulatory C-terminal domain cluster that includes an oligonucleotide-binding fold. Recombinant REH2 and (H2)F1 constructs associate in a purified complex in vitro. We propose a model of stepwise editosome assembly that entails controlled docking of mRNP and gRNP modules via specific base pairing between their respective mRNA and gRNA cargo and regulatory REH2 and (H2)F1 subunits of the novel mRNP that may control specificity checkpoints in the editing pathway. PMID:26769962

  15. The zebrafish genome editing toolkit.

    PubMed

    Ata, H; Clark, K J; Ekker, S C

    2016-01-01

    Zebrafish (Danio rerio) is a unique model organism at the functional intersection between a high fecundity and conserved vertebrate physiology while being amenable to a multitude of genome editing techniques. The genome engineering field has experienced an unprecedented rate of growth in the recent years since the introduction of designer endonucleases, such as zinc finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats-Cas9 systems. With the ever-evolving toolset available to the scientific community, the important question one should ask is not simply how to make a mutant line, but rather how best to do so. For this purpose, understanding the toolset is just one end of the equation; understanding how DNA is repaired once double-strand breaks are induced by designer endonucleases, as well as understanding proper fish handling and line maintenance techniques, are also essential to rapidly edit the zebrafish genome. This chapter is outlined to provide a bird's-eye view on each of these three components. The goal of this chapter is to facilitate the adoption of the zebrafish as a model to study human genetic disease and to rapidly analyze the function of the vertebrate genome. PMID:27443924

  16. Automatic data editing: a brief introduction

    SciTech Connect

    Liepins, G.E.

    1982-01-01

    This paper briefly discusses the automatic data editing process: (1) check the data records for consistency, (2) analyze the inconsistent records to determine the inconsistent variables. It is stated that the application of automatic data editing is broad, and two specific examples are cited. One example, that of a vehicle maintenance data base is used to illustrate the process.

  17. Managing the Incompetent Teacher. Second Edition.

    ERIC Educational Resources Information Center

    Bridges, Edwin M.

    Featuring the same practical guidelines for ridding schools of incompetent teachers as the 1984 edition, this new edition incorporates substantially revised material on three topics: criteria and information sources for evaluating teaching effectiveness, remediation procedures, and grounds for dismissal. The book presents an eight-step systematic,…

  18. Editing Technical Proposals (The Friendly Editor).

    ERIC Educational Resources Information Center

    Bush, Don

    1994-01-01

    Makes suggestions for editing technical proposals. Discusses the marketeers, the hierarchy of hype, how to save days, managing story boards, expediting a laborious process, teaching engineers to write, writing incrementally, the art group, and the editing task. Argues that the best proposals come from starting to write early. (SR)

  19. Sex Differences in Cognitive Abilities. Fourth Edition

    ERIC Educational Resources Information Center

    Halpern, Diane F.

    2011-01-01

    The fourth edition of "Sex Differences in Cognitive Abilities" critically examines the breadth of research on this complex and controversial topic, with the principal aim of helping the reader to understand where sex differences are found--and where they are not. Since the publication of the third edition, there have been many exciting and…

  20. Web Thermo Tables (WTT) - Professional Edition

    National Institute of Standards and Technology Data Gateway

    SRD 203 NIST/TRC Web Thermo Tables (WTT) - Professional Edition (Online Subscription)   WTT - Professional Edition, a Web version of the TRC Thermodynamic Tables, represents a complete collection of critically evaluated thermodynamic property data primarily for pure organic compounds. As of Nov. 2011, WTT contains information on 23999 compounds.

  1. Web Thermo Tables (WTT) - Lite Edition

    National Institute of Standards and Technology Data Gateway

    SRD 202 NIST/TRC Web Thermo Tables (WTT) - Lite Edition (Online Subscription)   WTT - Lite Edition, a Web version of the TRC Thermodynamic Tables, represents a collection of critically evaluated thermodynamic property data for 150 commonly-used (primarily organic) pure compounds.

  2. Michigan Library Statistical Report. 1997 Edition.

    ERIC Educational Resources Information Center

    Michigan Library, Lansing.

    This 1997 edition focuses on statistical data supplied by Michigan public libraries, public library cooperatives, and those public libraries which serve as regional or subregional outlets for blind and physically handicapped patrons. Statistics on academic libraries are also presented in this edition, and summary statistics for prior fiscal years…

  3. New Edition of Chinese Biochemistry Textbook.

    ERIC Educational Resources Information Center

    Jian-Chuan, Ma

    1988-01-01

    Discusses the four previous editions of the biochemistry medical textbooks called the "Nationwide Unified Textbooks." Notes the new (1989) edition is much smaller, is organized differently, has new material, has a reorganized Dynamic Biochemistry core, and shows great importance to clinical biochemistry. (MVL)

  4. Teaching Strategies for Ethnic Studies. Fifth Edition.

    ERIC Educational Resources Information Center

    Banks, James A.

    A major goal of this fifth edition is to help present and future teachers acquire the knowledge, concepts, strategies, and resources needed to integrate information about ethnic groups into the mainstream curriculum. This edition incorporates new terms and concepts that are emerging in the field of multicultural education. Information on ethnic…

  5. Handbook of Reference Sources. Third Edition.

    ERIC Educational Resources Information Center

    Nichols, Margaret Irby

    This third edition of popular and useful reference works, which emphasizes the needs of small libraries, contains 975 annotated entries and lists 201 additional titles (most with bibliographic and order information) in the annotations, representing an expansion of 30 percent over the second edition. The appendix lists 116 basic or core reference…

  6. Teaching Visually Impaired Children. 3rd Edition

    ERIC Educational Resources Information Center

    Bishop, Virginia E.

    2004-01-01

    In this exceptional new third edition, the author has retained much of the practical "how to" approach of the previous editions, but adds depth in two dimensions: learning theory and the educational process. This book is "so comprehensive in scope and complete in detail that it would be the most likely recommended" (from the foreword by Dr.…

  7. Exposure Factors Handbook 2011 Edition (Final)

    EPA Science Inventory

    edition_cover.jpg" vspace = "5" hspace="5" align="right" border="2" alt="Cover of the Exposure Factors Handbook: 2011 Edition"> The mission of National Center for Environmental Assessment (NCEA) of EPA's ...

  8. Applications of genome editing in insects

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Insect genome editing was first reported 1991 in Drosophila melanogaster but the technology used was not portable to other species. Not until the recent development of facile, engineered DNA endonuclease systems has gene editing become widely available to insect scientists. Most applications in inse...

  9. Accounting for Independent Schools. Second Edition.

    ERIC Educational Resources Information Center

    National Association of Independent Schools, Boston, MA.

    This is a thoroughly revised edition of the 1969 publication, "Accounting for Independent Schools," a guide that attempted to codify basic accounting principles and practices for specific application to independent schools. The focus of the second edition is more on refining practices than on initiating them, and more on extending the managerial…

  10. America's Children and the Environment, Third Edition

    EPA Science Inventory

    America's Children and the Environment is the U.S. EPA's report of children's environmental health indicators. Two editions of the report have been published, in 2000 and 2003, and a website is maintained with updated values for the indicators. The new Third Edition of America'...

  11. AV Instruction Media and Methods; Third Edition.

    ERIC Educational Resources Information Center

    Brown, James W., Ed.; And Others

    In addition to updating material covered in the second edition and providing more illustrative material, this edition attempts to offer a systematic approach to the integration of instructional aids into the curriculum. It discusses the use of significant new developments in instructional technology-multimedia packages or kits, programed…

  12. Self-assembled DNA nanoclews for the efficient delivery of CRISPR-Cas9 for genome editing.

    PubMed

    Sun, Wujin; Ji, Wenyan; Hall, Jordan M; Hu, Quanyin; Wang, Chao; Beisel, Chase L; Gu, Zhen

    2015-10-01

    CRISPR-Cas9 represents a promising platform for genome editing, yet means for its safe and efficient delivery remain to be fully realized. A novel vehicle that simultaneously delivers the Cas9 protein and single guide RNA (sgRNA) is based on DNA nanoclews, yarn-like DNA nanoparticles that are synthesized by rolling circle amplification. The biologically inspired vehicles were efficiently loaded with Cas9/sgRNA complexes and delivered the complexes to the nuclei of human cells, thus enabling targeted gene disruption while maintaining cell viability. Editing was most efficient when the DNA nanoclew sequence and the sgRNA guide sequence were partially complementary, offering a design rule for enhancing delivery. Overall, this strategy provides a versatile method that could be adapted for delivering other DNA-binding proteins or functional nucleic acids. PMID:26310292

  13. Exclusive Use of trans-Editing Domains Prevents Proline Mistranslation*

    PubMed Central

    Vargas-Rodriguez, Oscar; Musier-Forsyth, Karin

    2013-01-01

    Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to cognate tRNAs. Although the accuracy of this process is critical for overall translational fidelity, similar sizes of many amino acids provide a challenge to ARSs. For example, prolyl-tRNA synthetases (ProRSs) mischarge alanine and cysteine onto tRNAPro. Many bacterial ProRSs possess an alanine-specific proofreading domain (INS) but lack the capability to edit Cys-tRNAPro. Instead, Cys-tRNAPro is cleared by a single-domain homolog of INS, the trans-editing YbaK protein. A global bioinformatics analysis revealed that there are six types of “INS-like” proteins. In addition to INS and YbaK, four additional single-domain homologs are widely distributed throughout bacteria: ProXp-ala (formerly named PrdX), ProXp-x (annotated as ProX), ProXp-y (annotated as YeaK), and ProXp-z (annotated as PA2301). The last three are domains of unknown function. Whereas many bacteria encode a ProRS containing an INS domain in addition to YbaK, many other combinations of INS-like proteins exist throughout the bacterial kingdom. Here, we focus on Caulobacter crescentus, which encodes a ProRS with a truncated INS domain that lacks catalytic activity, as well as YbaK and ProXp-ala. We show that C. crescentus ProRS can readily form Cys- and Ala-tRNAPro, and deacylation studies confirmed that these species are cleared by C. crescentus YbaK and ProXp-ala, respectively. Substrate specificity of C. crescentus ProXp-ala is determined, in part, by elements in the acceptor stem of tRNAPro and further ensured through collaboration with elongation factor Tu. These results highlight the diversity of approaches used to prevent proline mistranslation and reveal a novel triple-sieve mechanism of editing that relies exclusively on trans-editing factors. PMID:23564458

  14. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.

    PubMed

    Komor, Alexis C; Kim, Yongjoo B; Packer, Michael S; Zuris, John A; Liu, David R

    2016-05-19

    Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction. Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus resulting from the cellular response to dsDNA breaks. Here we report the development of 'base editing', a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting 'base editors' convert cytidines within a window of approximately five nucleotides, and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor, and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favour desired base-editing outcomes, resulting in permanent correction of ~15-75% of total cellular DNA with minimal (typically ≤1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations. PMID:27096365

  15. In Vitro CRISPR/Cas9 System for Efficient Targeted DNA Editing

    PubMed Central

    Liu, Yunkun; Tao, Weixin; Wen, Shishi; Li, Zhengyuan; Yang, Anna; Deng, Zixin

    2015-01-01

    ABSTRACT The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, an RNA-guided nuclease for specific genome editing in vivo, has been adopted in a wide variety of organisms. In contrast, the in vitro application of the CRISPR/Cas9 system has rarely been reported. We present here a highly efficient in vitro CRISPR/Cas9-mediated editing (ICE) system that allows specific refactoring of biosynthetic gene clusters in Streptomyces bacteria and other large DNA fragments. Cleavage by Cas9 of circular pUC18 DNA was investigated here as a simple model, revealing that the 3′→5′ exonuclease activity of Cas9 generates errors with 5 to 14 nucleotides (nt) randomly missing at the editing joint. T4 DNA polymerase was then used to repair the Cas9-generated sticky ends, giving substantial improvement in editing accuracy. Plasmid pYH285 and cosmid 10A3, harboring a complete biosynthetic gene cluster for the antibiotics RK-682 and holomycin, respectively, were subjected to the ICE system to delete the rkD and homE genes in frame. Specific insertion of the ampicillin resistance gene (bla) into pYH285 was also successfully performed. These results reveal the ICE system to be a rapid, seamless, and highly efficient way to edit DNA fragments, and a powerful new tool for investigating and engineering biosynthetic gene clusters. PMID:26556277

  16. Delivery and therapeutic applications of gene editing technologies ZFNs, TALENs, and CRISPR/Cas9.

    PubMed

    LaFountaine, Justin S; Fathe, Kristin; Smyth, Hugh D C

    2015-10-15

    In recent years, several new genome editing technologies have been developed. Of these the zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 RNA-guided endonuclease system are the most widely described. Each of these technologies utilizes restriction enzymes to introduce a DNA double stranded break at a targeted location with the guide of homologous binding proteins or RNA. Such targeting is viewed as a significant advancement compared to current gene therapy methods that lack such specificity. Proof-of-concept studies have been performed to treat multiple disorders, including in vivo experiments in mammals and even early phase human trials. Careful consideration and investigation of delivery strategies will be required so that the therapeutic potential for gene editing is achieved. In this review, the mechanisms of each of these gene editing technologies and evidence of therapeutic potential will be briefly described and a comprehensive list of past studies will be provided. The pharmaceutical approaches of each of these technologies are discussed along with the current delivery obstacles. The topics and information reviewed herein provide an outline of the groundbreaking research that is being performed, but also highlights the potential for progress yet to be made using these gene editing technologies. PMID:26278489

  17. Fuel Cell Handbook, Fifth Edition

    SciTech Connect

    Energy and Environmental Solutions

    2000-10-31

    Progress continues in fuel cell technology since the previous edition of the Fuel Cell Handbook was published in November 1998. Uppermost, polymer electrolyte fuel cells, molten carbonate fuel cells, and solid oxide fuel cells have been demonstrated at commercial size in power plants. The previously demonstrated phosphoric acid fuel cells have entered the marketplace with more than 220 power plants delivered. Highlighting this commercial entry, the phosphoric acid power plant fleet has demonstrated 95+% availability and several units have passed 40,000 hours of operation. One unit has operated over 49,000 hours. Early expectations of very low emissions and relatively high efficiencies have been met in power plants with each type of fuel cell. Fuel flexibility has been demonstrated using natural gas, propane, landfill gas, anaerobic digester gas, military logistic fuels, and coal gas, greatly expanding market opportunities. Transportation markets worldwide have shown remarkable interest in fuel cells; nearly every major vehicle manufacturer in the U.S., Europe, and the Far East is supporting development. This Handbook provides a foundation in fuel cells for persons wanting a better understanding of the technology, its benefits, and the systems issues that influence its application. Trends in technology are discussed, including next-generation concepts that promise ultrahigh efficiency and low cost, while providing exceptionally clean power plant systems. Section 1 summarizes fuel cell progress since the last edition and includes existing power plant nameplate data. Section 2 addresses the thermodynamics of fuel cells to provide an understanding of fuel cell operation at two levels (basic and advanced). Sections 3 through 8 describe the six major fuel cell types and their performance based on cell operating conditions. Alkaline and intermediate solid state fuel cells were added to this edition of the Handbook. New information indicates that manufacturers have stayed

  18. RNA Research

    NASA Technical Reports Server (NTRS)

    1998-01-01

    It is generally believed that an RNA World existed at an early stage in the history of life. During this early period, RNA molecules are seen to be potentially involved in both catalysis and the storage of genetic information. It is widely believed that this RNA World was extensive and therefore a sophisticated nucleic acid replication machinery would presumably predate the translation machinery which would not be needed until later stages in the development of life. This view of an extended RNA World is not necessarily correct. From the point of view of exobiology, the difference in these two views mainly affects the significance of studies of the extent of catalysis possible by RNA- In either case, the origin of the translation machinery and the principles of RNA evolution remain central problems in exobiology. Translation presents several interrelated themes of inquiry for exobiology. First, it is essential, for understanding the very origin of life, how peptides and eventually proteins might have come to be made on the early Earth in a template directed manner. Second, it is necessary to understand how a machinery of similar complexity to that found in the ribosomes of modem organisms came to exist by the time of the last common ancestor (as detected by 16S RRNA sequence studies). Third, the RNAs that comprise the ribosome are themselves likely of very early origin and studies of their history may be very informative about the nature of the RNA World. Moreover, studies of these RNAs will contribute to a better understanding of the potential roles of RNA in early evolution.

  19. Efficient edit distance with duplications and contractions

    PubMed Central

    2013-01-01

    We propose three algorithms for string edit distance with duplications and contractions. These include an efficient general algorithm and two improvements which apply under certain constraints on the cost function. The new algorithms solve a more general problem variant and obtain better time complexities with respect to previous algorithms. Our general algorithm is based on min-plus multiplication of square matrices and has time and space complexities of O (|Σ|MP (n)) and O (|Σ|n2), respectively, where |Σ| is the alphabet size, n is the length of the strings, and MP (n) is the time bound for the computation of min-plus matrix multiplication of two n × n matrices (currently, MP(n)=On3log3lognlog2n due to an algorithm by Chan). For integer cost functions, the running time is further improved to O|Σ|n3log2n. In addition, this variant of the algorithm is online, in the sense that the input strings may be given letter by letter, and its time complexity bounds the processing time of the first n given letters. This acceleration is based on our efficient matrix-vector min-plus multiplication algorithm, intended for matrices and vectors for which differences between adjacent entries are from a finite integer interval D. Choosing a constant 1log|D|n<λ<1, the algorithm preprocesses an n × n matrix in On2+λ|D| time and On2+λ|D|λ2log|D|2n space. Then, it may multiply the matrix with any given n-length vector in On2λ2log|D|2n time. Under some discreteness assumptions, this matrix-vector min-plus multiplication algorithm applies to several problems from the domains of context-free grammar parsing and RNA folding and, in particular, implies the asymptotically fastest On3log2n time algorithm for single-strand RNA folding with discrete cost functions. Finally, assuming a different constraint on the cost function, we present another version of the algorithm that exploits the run-length encoding of the strings and runs in O|Σ|nMP(ñ)ñ time and O

  20. Exploiting CRISPR-Cas immune systems for genome editing in bacteria.

    PubMed

    Barrangou, Rodolphe; van Pijkeren, Jan-Peter

    2016-02-01

    The CRISPR-Cas immune system is a DNA-encoded, RNA-mediated, DNA-targeting defense mechanism, which provides sequence-specific targeting of DNA. This molecular machinery can be engineered into the sgRNA:Cas9 technology, for programmable cleavage of DNA. Following the genesis of double-stranded DNA breaks, the DNA repair machinery generates mutations at the cleavage site using various pathways. This technology has revolutionized eukaryotic genome editing, and we are at the cusp of full exploitation in bacteria. Here, we discuss the potential of CRISPR-based technologies for use in bacteria, and highlight the application of single stranded DNA recombineering combined with CRISPR-Cas selection to edit the genome of a probiotic organism. We envision that CRISPR-Cas technologies will play a key role in the development of next-generation industrial bacteria. PMID:26629846

  1. Deep sequencing of the tobacco mitochondrial transcriptome reveals expressed ORFs and numerous editing sites outside coding regions

    PubMed Central

    2014-01-01

    Background The purpose of this study was to sequence and assemble the tobacco mitochondrial transcriptome and obtain a genomic-level view of steady-state RNA abundance. Plant mitochondrial genomes have a small number of protein coding genes with large and variably sized intergenic spaces. In the tobacco mitogenome these intergenic spaces contain numerous open reading frames (ORFs) with no clear function. Results The assembled transcriptome revealed distinct monocistronic and polycistronic transcripts along with large intergenic spaces with little to no detectable RNA. Eighteen of the 117 ORFs were found to have steady-state RNA amounts above background in both deep-sequencing and qRT-PCR experiments and ten of those were found to be polysome associated. In addition, the assembled transcriptome enabled a full mitogenome screen of RNA C→U editing sites. Six hundred and thirty five potential edits were found with 557 occurring within protein-coding genes, five in tRNA genes, and 73 in non-coding regions. These sites were found in every protein-coding transcript in the tobacco mitogenome. Conclusion These results suggest that a small number of the ORFs within the tobacco mitogenome may produce functional proteins and that RNA editing occurs in coding and non-coding regions of mitochondrial transcripts. PMID:24433288

  2. Characterization of a novel trypanosomatid small nucleolar RNA.

    PubMed Central

    Levitan, A; Xu, Y x; Ben-Dov, C; Ben-Shlomo, H; Zhang, Y; Michaeli, S

    1998-01-01

    Trypanosomes possess unique RNA processing mechanisms including trans- splicing of pre-mRNA and RNA editing of mitochondrial transcripts. The previous finding of a trimethylguanosine (TMG) capped U3 homologue in trypanosomes suggests that rRNA processing may be related to the processing in other eukaryotes. In this study, we describe the first trypanosomatid snoRNA that belongs to the snoRNAs that were shown to guide ribose methylation of rRNA. The RNA, identified in the monogenetic trypanosomatid Leptomonas collosoma, was termed snoRNA-2 and is encoded by a multi-copy gene. SnoRNA-2 is 85 nt long, it lacks a 5' cap and possesses the C and D boxes characteristic to all snoRNAs that bind fibrillarin. Computer analysis indicates a potential for base-pairing between snoRNA-2 and 5.8S rRNA, and 18S rRNA. The putative interaction domains obey the rules suggested for the interaction of guide snoRNA with its rRNA target for directing ribose methylation on the rRNA. However, mapping the methylated sites on the 5.8S rRNA and 18S rRNA indicates that the expected site on the 5.8S is methylated, whereas the site on the 18S is not. The proposed interaction with 5.8S rRNA is further supported by the presence of psoralen cross-link sites on snoRNA-2. GenBank search suggests that snoRNA-2 is not related to any published snoRNAs. Because of the early divergence of the Trypanosomatidae from the eukaryotic lineage, the presence of a methylating snoRNA that is encoded by a multi-copy gene suggests that methylating snoRNAs may have evolved in evolution from self-transcribed genes. PMID:9512552

  3. Image editing with spatiograms transfer.

    PubMed

    Papadakis, Nicolas; Bugeau, Aurélie; Caselles, Vicent

    2012-05-01

    Histogram equalization is a well-known method for image contrast enhancement. Nevertheless, as histograms do not include any information on the spatial repartition of colors, their application to local image editing problems remains limited. To cope with this lack of spatial information, spatiograms have been recently proposed for tracking purposes. A spatiogram is an image descriptor that combines a histogram with the mean and the variance of the position of each color. In this paper, we address the problem of local retouching of images by proposing a variational method for spatiogram transfer. More precisely, a reference spatiogram is used to modify the color value of a given region of interest of the processed image. Experiments on shadow removal and inpainting demonstrate the strength of the proposed approach. PMID:22249712

  4. Analysis of the Trypanosoma brucei EATRO 164 Bloodstream Guide RNA Transcriptome

    PubMed Central

    Kirby, Laura E.; Sun, Yanni; Nowak, Scooter; Koslowsky, Donna

    2016-01-01

    The mitochondrial genome of Trypanosoma brucei contains many cryptogenes that must be extensively edited following transcription. The RNA editing process is directed by guide RNAs (gRNAs) that encode the information for the specific insertion and deletion of uridylates required to generate translatable mRNAs. We have deep sequenced the gRNA transcriptome from the bloodstream form of the EATRO 164 cell line. Using conventionally accepted fully edited mRNA sequences, ~1 million gRNAs were identified. In contrast, over 3 million reads were identified in our insect stage gRNA transcriptome. A comparison of the two life cycle transcriptomes show an overall ratio of procyclic to bloodstream gRNA reads of 3.5:1. This ratio varies significantly by gene and by gRNA populations within genes. The variation in the abundance of the initiating gRNAs for each gene, however, displays a trend that correlates with the developmental pattern of edited gene expression. A comparison of related major classes from each transcriptome revealed a median value of ten single nucleotide variations per gRNA. Nucleotide variations were much less likely to occur in the consecutive Watson-Crick anchor region, indicating a very strong bias against G:U base pairs in this region. This work indicates that gRNAs are expressed during both life cycle stages, and that differential editing patterns observed for the different mitochondrial mRNA transcripts are not due to the presence or absence of gRNAs. However, the abundance of certain gRNAs may be important in the developmental regulation of RNA editing. PMID:27399202

  5. NRP 7th Edition: Are You Prepared?

    PubMed

    Zaichkin, Jeanette; Mccarney, Linda; Weiner, Gary

    2016-01-01

    The seventh edition of the American Academy of Pediatrics/American Heart Association Neonatal Resuscitation Program (NRP) materials must be in use by January 1, 2017. As in previous editions, changes in resuscitation science are based on an international review and consensus of current resuscitation science. The seventh edition NRP materials also include enhancements to training materials aimed at improving the quality of NRP instruction and providing the opportunity for ongoing education. A standardized approach to instructor training, an online Instructor Toolkit, eSim cases, and a new learning management system are among the new resources. PMID:27461196

  6. Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants.

    PubMed

    Osakabe, Yuriko; Watanabe, Takahito; Sugano, Shigeo S; Ueta, Risa; Ishihara, Ryosuke; Shinozaki, Kazuo; Osakabe, Keishi

    2016-01-01

    Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems. PMID:27226176

  7. Optimization of CRISPR/Cas9 genome editing to modify abiotic stress responses in plants

    PubMed Central

    Osakabe, Yuriko; Watanabe, Takahito; Sugano, Shigeo S; Ueta, Risa; Ishihara, Ryosuke; Shinozaki, Kazuo; Osakabe, Keishi

    2016-01-01

    Genome editing using the CRISPR/Cas9 system can be used to modify plant genomes, however, improvements in specificity and applicability are still needed in order for the editing technique to be useful in various plant species. Here, using genome editing mediated by a truncated gRNA (tru-gRNA)/Cas9 combination, we generated new alleles for OST2, a proton pump in Arabidopsis, with no off-target effects. By following expression of Cas9 and the tru-gRNAs, newly generated mutations in CRIPSR/Cas9 transgenic plants were detected with high average mutation rates of up to 32.8% and no off-target effects using constitutive promoter. Reducing nuclear localization signals in Cas9 decreased the mutation rate. In contrast, tru-gRNA Cas9 cassettes driven by meristematic- and reproductive-tissue-specific promoters increased the heritable mutation rate in Arabidopsis, showing that high expression in the germ line can produce bi-allelic mutations. Finally, the new mutant alleles obtained for OST2 exhibited altered stomatal closing in response to environmental conditions. These results suggest further applications in molecular breeding to improve plant function using optimized plant CRISPR/Cas9 systems. PMID:27226176

  8. Marketing/Planning Library and Information Services. Second Edition.

    ERIC Educational Resources Information Center

    Weingand, Darlene E.

    In the first edition of this book, the concepts of marketing and planning library and information services were presented as effective managerial strategies. Several paragraphs from the introduction to the first edition are reproduced, with author commentary, in this edition as an affirmation that the message is still true. In this second edition,…

  9. CRISPR/Cas9 for plant genome editing: accomplishments, problems and prospects.

    PubMed

    Paul, Joseph W; Qi, Yiping

    2016-07-01

    The increasing burden of the world population on agriculture requires the development of more robust crops. Dissecting the basic biology that underlies plant development and stress responses will inform the design of better crops. One powerful tool for studying plants at the molecular level is the RNA-programmed genome editing system composed of a clustered regularly interspaced short palindromic repeats (CRISPR)-encoded guide RNA and the nuclease Cas9. Here, some of the recent advances in CRISPR/Cas9 technology that have profound implications for improving the study of plant biology are described. These tools are also paving the way towards new horizons for biotechnologies and crop development. PMID:27114166

  10. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

    PubMed Central

    Zhang, Wen-Wei

    2015-01-01

    ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. PMID:26199327

  11. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

    PubMed

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  12. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

    PubMed Central

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  13. CRISPR-Cas systems for genome editing, regulation and targeting

    PubMed Central

    Sander, Jeffry D.; Joung, J. Keith

    2014-01-01

    Targeted genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered regularly interspaced short palindromic repeat (CRISPR) technology, an important new platform for generating RNA-guided nucleases (RGNs), such as Cas9, with customizable specificities. RGN-mediated genome editing is facile, rapid and has enabled the efficient modification of endogenous genes in a wide variety of biomedically important cell types and novel organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the capabilities of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease. PMID:24584096

  14. Research Review: Magazine Editors and Editing Practices.

    ERIC Educational Resources Information Center

    Jolliffe, Lee

    1994-01-01

    Reviews and critiques literature in the subfield of magazine editing research, chiefly biographical studies of individual editors and various types of studies of editorial practices, including surveys, magazine content analyses, and close qualitative examinations of editors' relationships with others. (SR)

  15. ASTM standards on thermocouples, 2nd edition

    SciTech Connect

    Not Available

    1986-01-01

    This edition has been expanded to include sixteen selected ASTM methods and practices on thermocouples. It also contains a Temperature Electromotive Force (EMF) Table for the NICROSIL-NISIL Thermocouple System.

  16. An Automatic Editing Algorithm for GPS data

    NASA Astrophysics Data System (ADS)

    Blewitt, Geoffrey

    1990-03-01

    An algorithm has been developed to edit automatically Global Positioning System data such that outlier deletion, cycle slip identification and correction are independent of clock instability, selective availability, receiver-satellite kinematics, and tropospheric conditions. This algorithm, called TurboEdit, operates on undifferenced, dual frequency carrier phase data, and requires (1) the use of P code pseudorange data and (2) a smoothly varying ionospheric electron content. The latter requirement can be relaxed if the analysis software incorporates ambiguity resolution techniques to estimate unresolved cycle slip parameters. TurboEdit was tested on the large data set from the CASA Uno experiment, which contained over 2500 cycle slips. Analyst intervention was required on 1% of the station-satellite passes, almost all of these problems being due to difficulties in extrapolating variations in the ionospheric delay. The algorithm is presently being adapted for real time data editing in the Rogue receiver for continuous monitoring applications.

  17. A genome editing primer for the hematologist.

    PubMed

    Hoban, Megan D; Bauer, Daniel E

    2016-05-26

    Gene editing enables the site-specific modification of the genome. These technologies have rapidly advanced such that they have entered common use in experimental hematology to investigate genetic function. In addition, genome editing is becoming increasingly plausible as a treatment modality to rectify genetic blood disorders and improve cellular therapies. Genome modification typically ensues from site-specific double-strand breaks and may result in a myriad of outcomes. Even single-strand nicks and targeted biochemical modifications that do not permanently alter the DNA sequence (epigenome editing) may be powerful instruments. In this review, we examine the various technologies, describe their advantages and shortcomings for engendering useful genetic alterations, and consider future prospects for genome editing to impact hematology. PMID:27053532

  18. An automatic editing algorithm for GPS data

    NASA Technical Reports Server (NTRS)

    Blewitt, Geoffrey

    1990-01-01

    An algorithm has been developed to edit automatically Global Positioning System data such that outlier deletion, cycle slip identification, and correction are independent of clock instability, selective availability, receiver-satellite kinematics, and tropospheric conditions. This algorithm, called TurboEdit, operates on undifferenced, dual frequency carrier phase data, and requires the use of P code pseudorange data and a smoothly varying ionospheric electron content. TurboEdit was tested on the large data set from the CASA Uno experiment, which contained over 2500 cycle slips.Analyst intervention was required on 1 percent of the station-satellite passes, almost all of these problems being due to difficulties in extrapolating variations in the ionospheric delay. The algorithm is presently being adapted for real time data editing in the Rogue receiver for continuous monitoring applications.

  19. The New Grolier Multimedia Encyclopedia 1994 Edition.

    ERIC Educational Resources Information Center

    Online Libraries and Microcomputers, 1994

    1994-01-01

    Reviews the 1994 edition of "The New Grolier Multimedia Encyclopedia" which is available on CD-ROM. Topics addressed include video files; content; ease of use; system requirements; cost; and print capabilities. (LRW)

  20. Computing services writing and editing standards

    SciTech Connect

    Caruthers, C.M.; Heiberger, A.A.

    1987-11-01

    Computing Services Writing and Editing Standards explains how to write and organize technical information clearly and concisely; it also establishes Argonne National Laboratory Computing Services standards for grammar, usage, style, formats, and publication procedures. This manual will enable writers to produce better documents consistent with Computing Services standards in less time. Applying the specified principles will also speed up the editing, review, and revision processes.