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Sample records for alu rna editing

  1. 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

  2. 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.

  3. Alu Sequences in Undifferentiated Human Embryonic Stem Cells Display High Levels of A-to-I RNA Editing

    PubMed Central

    Osenberg, Sivan; Paz Yaacov, Nurit; Safran, Michal; Moshkovitz, Sharon; Shtrichman, Ronit; Sherf, Ofra; Jacob-Hirsch, Jasmine; Keshet, Gilmor; Amariglio, Ninette; Itskovitz-Eldor, Joseph; Rechavi, Gideon

    2010-01-01

    Adenosine to Inosine (A-to-I) RNA editing is a site-specific modification of RNA transcripts, catalyzed by members of the ADAR (Adenosine Deaminase Acting on RNA) protein family. RNA editing occurs in human RNA in thousands of different sites. Some of the sites are located in protein-coding regions but the majority is found in non-coding regions, such as 3′UTRs, 5′UTRs and introns - mainly in Alu elements. While editing is found in all tissues, the highest levels of editing are found in the brain. It was shown that editing levels within protein-coding regions are increased during embryogenesis and after birth and that RNA editing is crucial for organism viability as well as for normal development. In this study we characterized the A-to-I RNA editing phenomenon during neuronal and spontaneous differentiation of human embryonic stem cells (hESCs). We identified high editing levels of Alu repetitive elements in hESCs and demonstrated a global decrease in editing levels of non-coding Alu sites when hESCs are differentiating, particularly into the neural lineage. Using RNA interference, we showed that the elevated editing levels of Alu elements in undifferentiated hESCs are highly dependent on ADAR1. DNA microarray analysis showed that ADAR1 knockdown has a global effect on gene expression in hESCs and leads to a significant increase in RNA expression levels of genes involved in differentiation and development processes, including neurogenesis. Taken together, we speculate that A-to-I editing of Alu sequences plays a role in the regulation of hESC early differentiation decisions. PMID:20574523

  4. Consistent levels of A-to-I RNA editing across individuals in coding sequences and non-conserved Alu repeats

    PubMed Central

    2010-01-01

    Background Adenosine to inosine (A-to-I) RNA-editing is an essential post-transcriptional mechanism that occurs in numerous sites in the human transcriptome, mainly within Alu repeats. It has been shown to have consistent levels of editing across individuals in a few targets in the human brain and altered in several human pathologies. However, the variability across human individuals of editing levels in other tissues has not been studied so far. Results Here, we analyzed 32 skin samples, looking at A-to-I editing level in three genes within coding sequences and in the Alu repeats of six different genes. We observed highly consistent editing levels across different individuals as well as across tissues, not only in coding targets but, surprisingly, also in the non evolutionary conserved Alu repeats. Conclusions Our findings suggest that A-to-I RNA-editing of Alu elements is a tightly regulated process and, as such, might have been recruited in the course of primate evolution for post-transcriptional regulatory mechanisms. PMID:21029430

  5. REDIdb: the RNA editing database.

    PubMed

    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 http://biologia.unical.it/py_script/search.html.

  6. The crystal structure of the signal recognition particle Alu RNA binding heterodimer, SRP9/14.

    PubMed Central

    Birse, D E; Kapp, U; Strub, K; Cusack, S; Aberg, A

    1997-01-01

    The mammalian signal recognition particle (SRP) is an 11S cytoplasmic ribonucleoprotein that plays an essential role in protein sorting. SRP recognizes the signal sequence of the nascent polypeptide chain emerging from the ribosome, and targets the ribosome-nascent chain-SRP complex to the rough endoplasmic reticulum. The SRP consists of six polypeptides (SRP9, SRP14, SRP19, SRP54, SRP68 and SRP72) and a single 300 nucleotide RNA molecule. SRP9 and SRP14 proteins form a heterodimer that binds to the Alu domain of SRP RNA which is responsible for translation arrest. We report the first crystal structure of a mammalian SRP protein, that of the mouse SRP9/14 heterodimer, determined at 2.5 A resolution. SRP9 and SRP14 are found to be structurally homologous, containing the same alpha-beta-beta-beta-alpha fold. This we designate the Alu binding module (Alu bm), an additional member of the family of small alpha/beta RNA binding domains. The heterodimer has pseudo 2-fold symmetry and is saddle like, comprising a strongly curved six-stranded amphipathic beta-sheet with the four helices packed on the convex side and the exposed concave surface being lined with positively charged residues. PMID:9233785

  7. Alu-miRNA interactions modulate transcript isoform diversity in stress response and reveal signatures of positive selection.

    PubMed

    Pandey, Rajesh; Bhattacharya, Aniket; Bhardwaj, Vivek; Jha, Vineet; Mandal, Amit K; Mukerji, Mitali

    2016-09-02

    Primate-specific Alus harbor different regulatory features, including miRNA targets. In this study, we provide evidence for miRNA-mediated modulation of transcript isoform levels during heat-shock response through exaptation of Alu-miRNA sites in mature mRNA. We performed genome-wide expression profiling coupled with functional validation of miRNA target sites within exonized Alus, and analyzed conservation of these targets across primates. We observed that two miRNAs (miR-15a-3p and miR-302d-3p) elevated in stress response, target RAD1, GTSE1, NR2C1, FKBP9 and UBE2I exclusively within Alu. These genes map onto the p53 regulatory network. Ectopic overexpression of miR-15a-3p downregulates GTSE1 and RAD1 at the protein level and enhances cell survival. This Alu-mediated fine-tuning seems to be unique to humans as evident from the absence of orthologous sites in other primate lineages. We further analyzed signatures of selection on Alu-miRNA targets in the genome, using 1000 Genomes Phase-I data. We found that 198 out of 3177 Alu-exonized genes exhibit signatures of selection within Alu-miRNA sites, with 60 of them containing SNPs supported by multiple evidences (global-FST > 0.3, pair-wise-FST > 0.5, Fay-Wu's H < -20, iHS > 2.0, high ΔDAF) and implicated in p53 network. We propose that by affecting multiple genes, Alu-miRNA interactions have the potential to facilitate population-level adaptations in response to environmental challenges.

  8. Alu-miRNA interactions modulate transcript isoform diversity in stress response and reveal signatures of positive selection

    PubMed Central

    Pandey, Rajesh; Bhattacharya, Aniket; Bhardwaj, Vivek; Jha, Vineet; Mandal, Amit K.; Mukerji, Mitali

    2016-01-01

    Primate-specific Alus harbor different regulatory features, including miRNA targets. In this study, we provide evidence for miRNA-mediated modulation of transcript isoform levels during heat-shock response through exaptation of Alu-miRNA sites in mature mRNA. We performed genome-wide expression profiling coupled with functional validation of miRNA target sites within exonized Alus, and analyzed conservation of these targets across primates. We observed that two miRNAs (miR-15a-3p and miR-302d-3p) elevated in stress response, target RAD1, GTSE1, NR2C1, FKBP9 and UBE2I exclusively within Alu. These genes map onto the p53 regulatory network. Ectopic overexpression of miR-15a-3p downregulates GTSE1 and RAD1 at the protein level and enhances cell survival. This Alu-mediated fine-tuning seems to be unique to humans as evident from the absence of orthologous sites in other primate lineages. We further analyzed signatures of selection on Alu-miRNA targets in the genome, using 1000 Genomes Phase-I data. We found that 198 out of 3177 Alu-exonized genes exhibit signatures of selection within Alu-miRNA sites, with 60 of them containing SNPs supported by multiple evidences (global-FST > 0.3, pair-wise-FST > 0.5, Fay-Wu’s H < −20, iHS > 2.0, high ΔDAF) and implicated in p53 network. We propose that by affecting multiple genes, Alu-miRNA interactions have the potential to facilitate population-level adaptations in response to environmental challenges. PMID:27586304

  9. Alu-miRNA interactions modulate transcript isoform diversity in stress response and reveal signatures of positive selection.

    PubMed

    Pandey, Rajesh; Bhattacharya, Aniket; Bhardwaj, Vivek; Jha, Vineet; Mandal, Amit K; Mukerji, Mitali

    2016-01-01

    Primate-specific Alus harbor different regulatory features, including miRNA targets. In this study, we provide evidence for miRNA-mediated modulation of transcript isoform levels during heat-shock response through exaptation of Alu-miRNA sites in mature mRNA. We performed genome-wide expression profiling coupled with functional validation of miRNA target sites within exonized Alus, and analyzed conservation of these targets across primates. We observed that two miRNAs (miR-15a-3p and miR-302d-3p) elevated in stress response, target RAD1, GTSE1, NR2C1, FKBP9 and UBE2I exclusively within Alu. These genes map onto the p53 regulatory network. Ectopic overexpression of miR-15a-3p downregulates GTSE1 and RAD1 at the protein level and enhances cell survival. This Alu-mediated fine-tuning seems to be unique to humans as evident from the absence of orthologous sites in other primate lineages. We further analyzed signatures of selection on Alu-miRNA targets in the genome, using 1000 Genomes Phase-I data. We found that 198 out of 3177 Alu-exonized genes exhibit signatures of selection within Alu-miRNA sites, with 60 of them containing SNPs supported by multiple evidences (global-FST > 0.3, pair-wise-FST > 0.5, Fay-Wu's H < -20, iHS > 2.0, high ΔDAF) and implicated in p53 network. We propose that by affecting multiple genes, Alu-miRNA interactions have the potential to facilitate population-level adaptations in response to environmental challenges. PMID:27586304

  10. Investigating RNA editing factors from trypanosome mitochondria

    PubMed Central

    Aphasizheva, Inna; Zhang, Liye; Aphasizhev, Ruslan

    2016-01-01

    Mitochondrial U-insertion/deletion mRNA editing is carried out by two principal multiprotein assemblies, enzymatic RNA editing core (RECC) and RNA editing substrate binding (RESC) complexes, and a plethora of auxiliary factors. An integral part of mitochondrial gene expression, editing receives inputs from primary mRNA and gRNA precursor processing pathways, and generates substrates for mRNA polyadenylation and translation. Although nearly all RECC-embedded enzymes have been implicated in specific editing reactions, the majority of proteins that populate the RESC are also essential for generating edited mRNAs. However, lack of recognizable motifs in RESC subunits limits the prowess of bioinformatics in guiding biochemical experiments and elucidating their specific biological functions. In this chapter, we describe a generic workflow for investigating mitochondrial mRNA editing in Trypanosoma brucei and focus on several methods that proved instrumental is assigning definitive functions to editing factors lacking known signature sequences. PMID:27020893

  11. RNA Editing in Plant Mitochondria

    NASA Astrophysics Data System (ADS)

    Hiesel, Rudolf; Wissinger, Bernd; Schuster, Wolfgang; Brennicke, Axel

    1989-12-01

    Comparative sequence analysis of genomic and complementary DNA clones from several mitochondrial genes in the higher plant Oenothera revealed nucleotide sequence divergences between the genomic and the messenger RNA-derived sequences. These sequence alterations could be most easily explained by specific post-transcriptional nucleotide modifications. Most of the nucleotide exchanges in coding regions lead to altered codons in the mRNA that specify amino acids better conserved in evolution than those encoded by the genomic DNA. Several instances show that the genomic arginine codon CGG is edited in the mRNA to the tryptophan codon TGG in amino acid positions that are highly conserved as tryptophan in the homologous proteins of other species. This editing suggests that the standard genetic code is used in plant mitochondria and resolves the frequent coincidence of CGG codons and tryptophan in different plant species. The apparently frequent and non-species-specific equivalency of CGG and TGG codons in particular suggests that RNA editing is a common feature of all higher plant mitochondria.

  12. 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

  13. 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

  14. Alu- and 7SL RNA Analogues Suppress MCF-7 Cell Viability through Modulating the Transcription of Endoplasmic Reticulum Stress Response Genes.

    PubMed

    Baryakin, D N; Semenov, D V; Savelyeva, A V; Koval, O A; Rabinov, I V; Kuligina, E V; Richter, V A

    2013-10-01

    11% of the human genome is composed of Alu-retrotransposons, whose transcription by RNA polymerase III (Pol III) leads to the accumulation of several hundreds to thousands of Alu-RNA copies in the cytoplasm. Expression of Alu-RNA Pol III is significantly increased at various levels of stress, and the increase in the Alu-RNA level is accompanied by a suppression of proliferation, a decrease in viability, and induction of apoptotic processes in human cells. However, the question about the biological functions of Pol III Alu-transcripts, as well as their mechanism of action, remains open. In this work, analogues of Alu-RNA and its evolutionary ancestor, 7SL RNA, were synthesized. Transfection of human breast adenocarcinoma MCF-7 cells with the Alu-RNA and 7SL RNA analogues is accompanied by a decrease in viability and by induction of proapoptotic changes in these cells. The analysis of the combined action of these analogues and actinomycin D or tamoxifen revealed that the decreased viability of MCF-7 cells transfected with Alu-RNA and 7SL RNA was due to the modulation of transcription. A whole transcriptome analysis of gene expression revealed that increased gene expression of the transcription regulator NUPR1 (p8), as well as the transcription factor DDIT3 (CHOP), occurs under the action of both the Alu- and 7SL RNA analogues on MCF-7 cells. It has been concluded that induction of proapoptotic changes in human cells under the influence of the Alu-RNA and 7SL RNA analogues is related to the transcriptional activation of the genes of cellular stress factors, including the endoplasmic reticulum stress response factors.

  15. Iron toxicity in the retina requires Alu RNA and the NLRP3 inflammasome

    PubMed Central

    Gelfand, Bradley D.; Wright, Charles B.; Kim, Younghee; Yasuma, Tetsuhiro; Yasuma, Reo; Li, Shengjian; Fowler, Benjamin J.; Bastos-Carvalho, Ana; Kerur, Nagaraj; Uittenbogaard, Annette; Han, Youn Seon; Lou, Dingyuan; Kleinman, Mark E.; McDonald, W. Hayes; Núñez, Gabriel; Georgel, Philippe; Dunaief, Joshua L.; Ambati, Jayakrishna

    2015-01-01

    Excess iron induces tissue damage and is implicated in age-related macular degeneration (AMD). Iron toxicity is widely attributed to hydroxyl radical formation through Fenton's reaction. We report that excess iron, but not other Fenton catalytic metals, induces activation of the NLRP3 inflammasome, a pathway also implicated in AMD. Additionally, iron-induced degeneration of the retinal pigmented epithelium (RPE) is suppressed in mice lacking inflammasome components Caspase-1/11 or Nlrp3 or by inhibition of Caspase-1. Iron overload increases abundance of RNAs transcribed from short interspersed nuclear elements (SINEs): Alu RNAs and the rodent equivalent B1 and B2 RNAs, which are inflammasome agonists. Targeting Alu or B2 RNA prevents iron-induced inflammasome activation and RPE degeneration. Iron-induced SINE RNA accumulation is due to suppression of DICER1 via sequestration of the co-factor poly(C)-binding protein 2 (PCBP2). These findings reveal an unexpected mechanism of iron toxicity, with implications for AMD and neurodegenerative diseases associated with excess iron. PMID:26074074

  16. RNA Editing Dynamically Rewrites the Cancer Code

    PubMed Central

    Rayon-Estrada, Violeta; Papavasiliou, F. Nina; Harjanto, Dewi

    2016-01-01

    Global analyses of cancer transcriptomes demonstrate that ADAR (adenosine deaminase, RNA-specific)-mediated RNA editing dynamically contributes to genetic alterations in cancer, and directly correlates with progression and prognosis. RNA editing is abundant and frequently elevated in cancer, and affects functionally and clinically relevant sites in both coding and non-coding regions of the transcriptome. Therefore, ADAR and differentially edited transcripts may be promising biomarkers or targets for therapy.

  17. RNA Editing Dynamically Rewrites the Cancer Code

    PubMed Central

    Rayon-Estrada, Violeta; Papavasiliou, F. Nina; Harjanto, Dewi

    2016-01-01

    Global analyses of cancer transcriptomes demonstrate that ADAR (adenosine deaminase, RNA-specific)-mediated RNA editing dynamically contributes to genetic alterations in cancer, and directly correlates with progression and prognosis. RNA editing is abundant and frequently elevated in cancer, and affects functionally and clinically relevant sites in both coding and non-coding regions of the transcriptome. Therefore, ADAR and differentially edited transcripts may be promising biomarkers or targets for therapy. PMID:27695712

  18. Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits.

    PubMed

    Ivanova, Elena; Berger, Audrey; Scherrer, Anne; Alkalaeva, Elena; Strub, Katharina

    2015-03-11

    The human genome contains about 1.5 million Alu elements, which are transcribed into Alu RNAs by RNA polymerase III. Their expression is upregulated following stress and viral infection, and they associate with the SRP9/14 protein dimer in the cytoplasm forming Alu RNPs. Using cell-free translation, we have previously shown that Alu RNPs inhibit polysome formation. Here, we describe the mechanism of Alu RNP-mediated inhibition of translation initiation and demonstrate its effect on translation of cellular and viral RNAs. Both cap-dependent and IRES-mediated initiation is inhibited. Inhibition involves direct binding of SRP9/14 to 40S ribosomal subunits and requires Alu RNA as an assembly factor but its continuous association with 40S subunits is not required for inhibition. Binding of SRP9/14 to 40S prevents 48S complex formation by interfering with the recruitment of mRNA to 40S subunits. In cells, overexpression of Alu RNA decreases translation of reporter mRNAs and this effect is alleviated with a mutation that reduces its affinity for SRP9/14. Alu RNPs also inhibit the translation of cellular mRNAs resuming translation after stress and of viral mRNAs suggesting a role of Alu RNPs in adapting the translational output in response to stress and viral infection.

  19. 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.

  20. 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.

  1. 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.

  2. TLR-Independent and P2X7-Dependent Signaling Mediate Alu RNA-Induced NLRP3 Inflammasome Activation in Geographic Atrophy

    PubMed Central

    Kerur, Nagaraj; Hirano, Yoshio; Tarallo, Valeria; Fowler, Benjamin J.; Bastos-Carvalho, Ana; Yasuma, Tetsuhiro; Yasuma, Reo; Kim, Younghee; Hinton, David R.; Kirschning, Carsten J.; Gelfand, Bradley D.; Ambati, Jayakrishna

    2013-01-01

    Purpose. Accumulation of Alu RNA transcripts due to DICER1 deficiency in the retinal pigmented epithelium (RPE) promotes geographic atrophy. Recently we showed that Alu RNA activated the NLRP3 inflammasome, leading to RPE cell death via interleukin-18 (IL-18)-mediated MyD88 signaling. However, the molecular basis for NLRP3 inflammasome activation by Alu RNA is not well understood. We sought to decipher the key signaling events triggered by Alu RNA that lead to priming and activation of the NLRP3 inflammasome and, ultimately, to RPE degeneration by investigating the roles of the purinoreceptor P2X7, the transcription factor NF-κB, and the Toll-like receptors (TLRs) in these processes. Methods. Human and mouse RPE cells were transfected with a plasmid encoding an Alu element (pAlu) or an in vitro-transcribed Alu RNA. Inflammasome priming was assessed by measuring NLRP3 and IL18 mRNA levels by real-time quantitative PCR. Using immunoblotting, we assessed NF-κB activation by monitoring phosphorylation of its p65 subunit, and inflammasome activation by monitoring caspase-1 cleavage into its active form. RPE degeneration was induced in mice by subretinal transfection of pAlu or Alu RNA. The NF-κB inhibitor BAY 11-7082, the P2X7 receptor antagonist A-740003, and the NLRP3 inflammasome inhibitor glyburide were delivered by intravitreous injections. We studied wild-type (WT) C57Bl/6J, P2rx7−/−, Nfkb1−/−, and Tlr23479−/− mice. RPE degeneration was assessed by fundus photography and zonula occludens-1 (ZO-1) staining of mouse RPE. Results. Alu RNA-induced NF-κB activation, independent of TLR-1, -2, -3, -4, -6, -7, and -9 signaling, was required for priming the NLRP3 inflammasome. Nfkb1−/− and P2rx7−/− mice and WT mice treated with the pharmacological inhibitors of NF-κB, P2X7, or NLRP3, were protected against Alu RNA-induced RPE degeneration. Conclusions. NF-κB and P2X7 are critical signaling intermediates in Alu RNA-induced inflammasome priming and

  3. 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

  4. Evolution of RNA editing in trypanosome mitochondria.

    PubMed

    Simpson, L; Thiemann, O H; Savill, N J; Alfonzo, J D; Maslov, D A

    2000-06-20

    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 C(34) to U(34) modification in the anticodon of the imported tRNA(Trp), 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.

  5. 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

  6. Genetic Architectures of Quantitative Variation in RNA Editing Pathways.

    PubMed

    Gu, Tongjun; Gatti, Daniel M; Srivastava, Anuj; Snyder, Elizabeth M; Raghupathy, Narayanan; Simecek, Petr; Svenson, Karen L; Dotu, Ivan; Chuang, Jeffrey H; Keller, Mark P; Attie, Alan D; Braun, Robert E; Churchill, Gary A

    2016-02-01

    RNA editing refers to post-transcriptional processes that alter the base sequence of RNA. Recently, hundreds of new RNA editing targets have been reported. However, the mechanisms that determine the specificity and degree of editing are not well understood. We examined quantitative variation of site-specific editing in a genetically diverse multiparent population, Diversity Outbred mice, and mapped polymorphic loci that alter editing ratios globally for C-to-U editing and at specific sites for A-to-I editing. An allelic series in the C-to-U editing enzyme Apobec1 influences the editing efficiency of Apob and 58 additional C-to-U editing targets. We identified 49 A-to-I editing sites with polymorphisms in the edited transcript that alter editing efficiency. In contrast to the shared genetic control of C-to-U editing, most of the variable A-to-I editing sites were determined by local nucleotide polymorphisms in proximity to the editing site in the RNA secondary structure. Our results indicate that RNA editing is a quantitative trait subject to genetic variation and that evolutionary constraints have given rise to distinct genetic architectures in the two canonical types of RNA editing.

  7. 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

  8. RNA editing of nuclear transcripts in Arabidopsis thaliana

    PubMed Central

    2010-01-01

    Background RNA editing is a transcript-based layer of gene regulation. To date, no systemic study on RNA editing of plant nuclear genes has been reported. Here, a transcriptome-wide search for editing sites in nuclear transcripts of Arabidopsis (Arabidopsis thaliana) was performed. Results MPSS (massively parallel signature sequencing) and PARE (parallel analysis of RNA ends) data retrieved from public databases were utilized, focusing on one-base-conversion editing. Besides cytidine (C)-to-uridine (U) editing in mitochondrial transcripts, many nuclear transcripts were found to be diversely edited. Interestingly, a sizable portion of these nuclear genes are involved in chloroplast- or mitochondrion-related functions, and many editing events are tissue-specific. Some editing sites, such as adenosine (A)-to-U editing loci, were found to be surrounded by peculiar elements. The editing events of some nuclear transcripts are highly enriched surrounding the borders between coding sequences (CDSs) and 3′ untranslated regions (UTRs), suggesting site-specific editing. Furthermore, RNA editing is potentially implicated in new start or stop codon generation, and may affect alternative splicing of certain protein-coding transcripts. RNA editing in the precursor microRNAs (pre-miRNAs) of ath-miR854 family, resulting in secondary structure transformation, implies its potential role in microRNA (miRNA) maturation. Conclusions To our knowledge, the results provide the first global view of RNA editing in plant nuclear transcripts. PMID:21143795

  9. Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition.

    PubMed

    Madina, Bhaskara R; Kumar, Vikas; Metz, Richard; Mooers, Blaine H M; Bundschuh, Ralf; Cruz-Reyes, Jorge

    2014-07-01

    Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.

  10. Mediated plastid RNA editing in plant immunity.

    PubMed

    García-Andrade, Javier; Ramírez, Vicente; López, Ana; Vera, Pablo

    2013-10-01

    Plant regulatory circuits coordinating nuclear and plastid gene expression have evolved in response to external stimuli. RNA editing is one of such control mechanisms. We determined the Arabidopsis nuclear-encoded homeodomain-containing protein OCP3 is incorporated into the chloroplast, and contributes to control over the extent of ndhB transcript editing. ndhB encodes the B subunit of the chloroplast NADH dehydrogenase-like complex (NDH) involved in cyclic electron flow (CEF) around photosystem I. In ocp3 mutant strains, ndhB editing efficiency decays, CEF is impaired and disease resistance to fungal pathogens substantially enhanced, a process recapitulated in plants defective in editing plastid RNAs encoding NDH complex subunits due to mutations in previously described nuclear-encoded pentatricopeptide-related proteins (i.e. CRR21, CRR2). Furthermore, we observed that following a pathogenic challenge, wild type plants respond with editing inhibition of ndhB transcript. In parallel, rapid destabilization of the plastidial NDH complex is also observed in the plant following perception of a pathogenic cue. Therefore, NDH complex activity and plant immunity appear as interlinked processes.

  11. Immunology. RNA editing AIDs antibody diversification?

    PubMed

    Neuberger, M S; Scott, J

    2000-09-01

    How do B cells generate the enormous diversity of antibodies that are able to recognize and bind to whichever antigen a B cell might happen to encounter in the body? Several genetic mechanisms that manipulate different combinations of immunoglobulin genes are known. In their Perspective, Neuberger and Scott, highlight another genetic mechanism called RNA editing now shown to be involved in the production of antibody diversity.

  12. REDIdb: an upgraded bioinformatics resource for organellar RNA editing sites.

    PubMed

    Picardi, Ernesto; Regina, Teresa M R; Verbitskiy, Daniil; Brennicke, Axel; Quagliariello, Carla

    2011-03-01

    RNA editing is a post-transcriptional molecular process whereby the information in a genetic message is modified from that in the corresponding DNA template by means of nucleotide substitutions, insertions and/or deletions. It occurs mostly in organelles by clade-specific diverse and unrelated biochemical mechanisms. RNA editing events have been annotated in primary databases as GenBank and at more sophisticated level in the specialized databases REDIdb, dbRES and EdRNA. At present, REDIdb is the only freely available database that focuses on the organellar RNA editing process and annotates each editing modification in its biological context. Here we present an updated and upgraded release of REDIdb with a web-interface refurbished with graphical and computational facilities that improve RNA editing investigations. Details of the REDIdb features and novelties are illustrated and compared to other RNA editing databases. REDIdb is freely queried at http://biologia.unical.it/py_script/REDIdb/.

  13. Alu element-containing RNAs maintain nucleolar structure and function.

    PubMed

    Caudron-Herger, Maïwen; Pankert, Teresa; Seiler, Jeanette; Németh, Attila; Voit, Renate; Grummt, Ingrid; Rippe, Karsten

    2015-11-12

    Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production.

  14. 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.

  15. RNA editing in plants: Machinery and flexibility of site recognition.

    PubMed

    Shikanai, Toshiharu

    2015-09-01

    In plants, RNA editing is a process that deaminates specific cytidines (C) to uridines (U). PLS subfamily members of PPR proteins function in site recognition of the target C. In silico analysis has predicted the code used for PPR motif-nucleotide interaction, and the crystal structure of a protein-RNA complex supports this model. Despite progress in understanding the RNA-binding mechanism of PPR proteins, some of the flexibility of RNA recognition observed in trans-factors of RNA editing has not been fully explained. It is probably necessary to consider another unknown mechanism, and this consideration is related to the question of how PPR proteins have managed the creation of RNA editing sites during evolution. This question may be related to the mystery of the biological function of RNA editing in plants. MORF/RIP family members are required for RNA editing at multiple editing sites and are components of the RNA editosome in plants. The DYW domain has been a strong candidate for the C deaminase activity required for C-to-U conversion in RNA editing. So far, the activity of this enzyme has not been detected in recombinant DYW proteins, and several puzzling experimental results need to be explained to support the model. It is still difficult to resolve the entire image of the editosome in RNA editing in plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

  16. Genome sequence-independent identification of RNA editing sites.

    PubMed

    Zhang, Qing; Xiao, Xinshu

    2015-04-01

    RNA editing generates post-transcriptional sequence changes that can be deduced from RNA-seq data, but detection typically requires matched genomic sequence or multiple related expression data sets. We developed the GIREMI tool (genome-independent identification of RNA editing by mutual information; https://www.ibp.ucla.edu/research/xiao/GIREMI.html) to predict adenosine-to-inosine editing accurately and sensitively from a single RNA-seq data set of modest sequencing depth. Using GIREMI on existing data, we observed tissue-specific and evolutionary patterns in editing sites in the human population.

  17. 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

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

    PubMed

    Cui, Yalei; Huang, Tianzhi; Zhang, Xiaobo

    2015-12-01

    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.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. Hepatitis delta virus mutant: effect on RNA editing.

    PubMed

    Wu, T T; Bichko, V V; Ryu, W S; Lemon, S M; Taylor, J M

    1995-11-01

    During the replication cycle of hepatitis delta virus (HDV), RNA editing occurs at position 1012 on the 1679-nucleotide RNA genome. This changes an A to G in the amber termination codon, UAG, of the small form of the delta antigen (delta Ag). The resultant UGG codon, tryptophan, allows the translation of a larger form of the delta Ag with a 19-amino-acid C-terminal extension. Using HDV cDNA-transfected cells, we examined the editing potential of HDV RNA mutated from G to A at 1011 on the antigenome, adjacent to normal editing site at 1012. Four procedures were used to study not only the editing of the A at 1012, but also that of the new A at 1011: (i) nucleotide sequencing, (ii) a PCR-based RNA-editing assay, (iii) immunoblot assays, and (iv) immunofluorescence. Five findings are reported. (i) Even after the mutation at 1011, editing still occurred at 1012. (ii) Site 1011 itself now acted as a novel RNA-editing site. (iii) Sites 1011 and 1012 were edited independently. (iv) At later times, both sites became edited, thereby allowing the synthesis of the large form of the delta Ag (delta Ag-L). (v) Via immunofluorescence, such double editing became apparent as a stochastic event, in that groups of cells arose in which the changes had taken place. Evaluation of these findings and of those from previous studies of the stability of the HDV genomic sequence (H.J. Netter et al., J. Virol. 69:1687-1692, 1995) supports both the recent reevaluation of HDV RNA editing as occurring on antigenomic RNA (Casey and Gerin, personal communication) and the interpretation that editing occurs via the RNA-modifying enzyme known as DRADA.

  4. Putative impact of RNA editing on drug discovery.

    PubMed

    Decher, Niels; Netter, Michael F; Streit, Anne K

    2013-01-01

    Virtually all organisms use RNA editing as a powerful post-transcriptional mechanism to recode genomic information and to increase functional protein diversity. The enzymatic editing of pre-mRNA by ADARs and CDARs is known to change the functional properties of neuronal receptors and ion channels regulating cellular excitability. However, RNA editing is also an important mechanism for genes expressed outside the brain. The fact that RNA editing breaks the 'one gene encodes one protein' hypothesis is daunting for scientists and a probable drawback for drug development, as scientists might search for drugs targeting the 'wrong' protein. This possible difficulty for drug discovery and development became more evident from recent publications, describing that RNA editing events have profound impact on the pharmacology of some common drug targets. These recent studies highlight that RNA editing can cause massive discrepancies between the in vitro and in vivo pharmacology. Here, we review the putative impact of RNA editing on drug discovery, as RNA editing has to be considered before using high-throughput screens, rational drug design or choosing the right model organism for target validation.

  5. DNA and RNA editing of retrotransposons accelerate mammalian genome evolution.

    PubMed

    Knisbacher, Binyamin A; Levanon, Erez Y

    2015-04-01

    Genome evolution is commonly viewed as a gradual process that is driven by random mutations that accumulate over time. However, DNA- and RNA-editing enzymes have been identified that can accelerate evolution by actively modifying the genomically encoded information. The apolipoprotein B mRNA editing enzymes, catalytic polypeptide-like (APOBECs) are potent restriction factors that can inhibit retroelements by cytosine-to-uridine editing of retroelement DNA after reverse transcription. In some cases, a retroelement may successfully integrate into the genome despite being hypermutated. Such events introduce unique sequences into the genome and are thus a source of genomic innovation. adenosine deaminases that act on RNA (ADARs) catalyze adenosine-to-inosine editing in double-stranded RNA, commonly formed by oppositely oriented retroelements. The RNA editing confers plasticity to the transcriptome by generating many transcript variants from a single genomic locus. If the editing produces a beneficial variant, the genome may maintain the locus that produces the RNA-edited transcript for its novel function. Here, we discuss how these two powerful editing mechanisms, which both target inserted retroelements, facilitate expedited genome evolution.

  6. RNA editing in the free-living bodonid Bodo saltans.

    PubMed

    Blom, D; de Haan, A; van den Berg, M; Sloof, P; Jirku, M; Lukes, J; Benne, R

    1998-03-01

    In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids. In contrast, B.saltans cytochrome c oxidase (cox) subunit 2 and maxicircle unidentified reading frame (MURF) 2 RNAs display limited editing in the same regions as their trypanosomatid counterparts. A putative intramolecular cox2 gRNA and the gene for gMURF2-I directing the insertion of only one U in the 5' editing domain of MURF2 RNA, are conserved in B.saltans. This lends (further) evolutionary support to the proposed role of these sequences as gRNAs. Phylogenetic analysis showed that B.saltans is more closely related to trypanosomatids than the cryptobiids Trypanoplasma borreli and Cryptobia helicis, in line with the trypanosomatid-like cox2 and MURF2 RNA editing patterns. Nevertheless, other features like the apparent absence of a catenated mtDNA network, are shared with bodonid and cryptobiid species. ND5 RNA editing may represent yet another example of editing 'on the way out' during kinetoplastid evolution, but in view of the fact that cox2 RNA is unedited in T. borreli and C.helicis, we infer that the editing of this RNA may have arisen relatively recently. Our results provide the first examples of RNA editing in a free-living kinetoplastid, indicating that there is no direct link between U-insertion/deletion editing and a parasitic lifestyle.

  7. RNA editing in the free-living bodonid Bodo saltans.

    PubMed Central

    Blom, D; de Haan, A; van den Berg, M; Sloof, P; Jirku, M; Lukes, J; Benne, R

    1998-01-01

    In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids. In contrast, B.saltans cytochrome c oxidase (cox) subunit 2 and maxicircle unidentified reading frame (MURF) 2 RNAs display limited editing in the same regions as their trypanosomatid counterparts. A putative intramolecular cox2 gRNA and the gene for gMURF2-I directing the insertion of only one U in the 5' editing domain of MURF2 RNA, are conserved in B.saltans. This lends (further) evolutionary support to the proposed role of these sequences as gRNAs. Phylogenetic analysis showed that B.saltans is more closely related to trypanosomatids than the cryptobiids Trypanoplasma borreli and Cryptobia helicis, in line with the trypanosomatid-like cox2 and MURF2 RNA editing patterns. Nevertheless, other features like the apparent absence of a catenated mtDNA network, are shared with bodonid and cryptobiid species. ND5 RNA editing may represent yet another example of editing 'on the way out' during kinetoplastid evolution, but in view of the fact that cox2 RNA is unedited in T. borreli and C.helicis, we infer that the editing of this RNA may have arisen relatively recently. Our results provide the first examples of RNA editing in a free-living kinetoplastid, indicating that there is no direct link between U-insertion/deletion editing and a parasitic lifestyle. PMID:9469817

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

    PubMed

    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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. Activation of RNA polymerase III transcription of human Alu repetitive elements by adenovirus type 5: requirement for the E1b 58-kilodalton protein and the products of E4 open reading frames 3 and 6.

    PubMed Central

    Panning, B; Smiley, J R

    1993-01-01

    We found that transcription of endogenous human Alu elements by RNA polymerase III was strongly stimulated following infection of HeLa cells with adenovirus type 5, leading to the accumulation of high levels of Alu transcripts initiated from Alu polymerase III promoters. In contrast to previously reported cases of adenovirus-induced activation of polymerase III transcription, induction required the E1b 58-kDa protein and the products of E4 open reading frames 3 and 6 in addition to the 289-residue E1a protein. In addition, E1a function was not required at high multiplicities of infection, suggesting that E1a plays an indirect role in Alu activation. These results suggest previously unsuspected regulatory properties of the adenovirus E1b and E4 gene products and provide a novel approach to the study of the biology of the most abundant class of dispersed repetitive DNA in the human genome. Images PMID:7684492

  14. Molecular Evolution: The Perplexing Diversity of Mitochondrial RNA Editing Systems.

    PubMed

    Sloan, Daniel B; Wu, Zhiqiang

    2016-01-11

    New analysis of rapidly evolving mitochondrial genomes in calcaronean sponges has demonstrated that accurate gene expression requires systematic nucleotide insertion throughout RNA transcripts, altering previous views that RNA editing systems are difficult to maintain in genomes with high mutation rates. PMID:26766226

  15. 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

  16. 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

  17. 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

    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.

  18. Editing independent effects of ADARs on the miRNA/siRNA pathways

    PubMed Central

    Heale, Bret S E; Keegan, Liam P; McGurk, Leeanne; Michlewski, Gracjan; Brindle, James; Stanton, Chloe M; Caceres, Javier F; O'Connell, Mary A

    2009-01-01

    Adenosine deaminases acting on RNA (ADARs) are best known for altering the coding sequences of mRNA through RNA editing, as in the GluR-B Q/R site. ADARs have also been shown to affect RNA interference (RNAi) and microRNA processing by deamination of specific adenosines to inosine. Here, we show that ADAR proteins can affect RNA processing independently of their enzymatic activity. We show that ADAR2 can modulate the processing of mir-376a2 independently of catalytic RNA editing activity. In addition, in a Drosophila assay for RNAi deaminase-inactive ADAR1 inhibits RNAi through the siRNA pathway. These results imply that ADAR1 and ADAR2 have biological functions as RNA-binding proteins that extend beyond editing per se and that even genomically encoded ADARs that are catalytically inactive may have such functions. PMID:19713932

  19. 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

  20. 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

  1. Using REDItools to Detect RNA Editing Events in NGS Datasets.

    PubMed

    Picardi, Ernesto; D'Erchia, Anna Maria; Montalvo, Antonio; Pesole, Graziano

    2015-03-09

    RNA editing is a post-transcriptional/co-transcriptional molecular phenomenon whereby a genetic message is modified from the corresponding DNA template by means of substitutions, insertions, and/or deletions. It occurs in a variety of organisms and different cellular locations through evolutionally and biochemically unrelated proteins. RNA editing has a plethora of biological effects including the modulation of alternative splicing and fine-tuning of gene expression. RNA editing events by base substitutions can be detected on a genomic scale by NGS technologies through the REDItools package, an ad hoc suite of Python scripts to study RNA editing using RNA-Seq and DNA-Seq data or RNA-Seq data alone. REDItools implement effective filters to minimize biases due to sequencing errors, mapping errors, and SNPs. The package is freely available at Google Code repository (http://code.google.com/p/reditools/) and released under the MIT license. In the present unit we show three basic protocols corresponding to three main REDItools scripts.

  2. 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

  3. Trans and cis factors affecting A-to-I RNA editing efficiency of a noncoding editing target in C. elegans.

    PubMed

    Washburn, Michael C; Hundley, Heather A

    2016-05-01

    Adenosine-to-inosine RNA editing by ADARs affects thousands of adenosines in an organism's transcriptome. However, adenosines are not edited at equal levels nor do these editing levels correlate well with ADAR expression levels. Therefore, additional mechanisms are utilized by the cell to dictate the editing efficiency at a given adenosine. To examine cis-and trans-acting factors that regulate A-to-I editing levels specifically in neural cells, we utilized the model organism Caenorhabditis elegans We demonstrate that a double-stranded RNA (dsRNA) binding protein, ADR-1, inhibits editing in neurons, which is largely masked when examining editing levels from whole animals. Furthermore, expression of ADR-1 and mRNA expression of the editing target can act synergistically to regulate editing efficiency. In addition, we identify a dsRNA region within the Y75B8A.83' UTR that acts as acis-regulatory element by enhancing ADR-2 editing efficiency. Together, this work identifies mechanisms that regulate editing efficiency of noncoding A-to-I editing sites, which comprise the largest class of ADAR targets.

  4. Cross-reaction of snRNA and an Alu I-like sequence from rat with DNAs from different eucaryotic species.

    PubMed Central

    Blin, N; Weber, T; Alonso, A

    1983-01-01

    Sequence homologies to rat U1-snRNA and U2-snRNA were investigated in DNA from 23 eucaryotic species (3 lower eucaryotes, 4 plants, and 16 animals) using dot hybridization at various stringency conditions. Cross-hybridization among very distantly related species in e.g. plants-insects or mold-vertebrates is not the rule; there are, however, examples for stronger homologies like Rattus-Dictyostelium. Furthermore, DNA from all 23 species was analysed for sequence homologies with the repetitive DNA sequence B1 (an Alu I family equivalent) from rat. We observed a wide range of homologies covering some plants and insects, up to vertebrates. Hybridization at increasing stringency conditions revealed species with higher degree of homology to the rat B1 sequence: maize, chicken, and hamster. Images PMID:6186992

  5. Complete cycles of bloodstream trypanosome RNA editing in vitro.

    PubMed

    Halbig, Kari; De Nova-Ocampo, Monica; Cruz-Reyes, Jorge

    2004-06-01

    RNA editing in kinetoplastid protists is required for the maturation of mitochondrial pre-mRNAs and occurs by protein-catalyzed cycles of uridylate insertion and deletion. During the complex life cycle of Trypanosoma brucei this process is differentially regulated in the mammalian bloodstream and insect procyclic stages. Complementary guide RNAs (gRNAs) direct editing, but the abundance of these transcripts is not developmentally controlled. The establishment of in vitro systems that recreate efficient RNA editing in bloodstream T. brucei would be valuable for mechanistic studies of regulation. Here we describe a robust in vitro system that reconstitutes full cycles of both U insertion and U deletion in bloodstream trypanosomes, and the first direct comparisons of the in vitro systems for strains of mammalian and insect stages.

  6. 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

  7. 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

  8. Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications.

    PubMed

    Licht, Konstantin; Jantsch, Michael F

    2016-04-11

    Advances in next-generation sequencing and mass spectrometry have revealed widespread messenger RNA modifications and RNA editing, with dramatic effects on mammalian transcriptomes. Factors introducing, deleting, or interpreting specific modifications have been identified, and analogous with epigenetic terminology, have been designated "writers," "erasers," and "readers." Such modifications in the transcriptome are referred to as epitranscriptomic changes and represent a fascinating new layer of gene expression regulation that has only recently been appreciated. Here, we outline how RNA editing and RNA modification can rapidly affect gene expression, making both processes as well suited to respond to cellular stress and to regulate the transcriptome during development or circadian periods.

  9. Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications.

    PubMed

    Licht, Konstantin; Jantsch, Michael F

    2016-04-11

    Advances in next-generation sequencing and mass spectrometry have revealed widespread messenger RNA modifications and RNA editing, with dramatic effects on mammalian transcriptomes. Factors introducing, deleting, or interpreting specific modifications have been identified, and analogous with epigenetic terminology, have been designated "writers," "erasers," and "readers." Such modifications in the transcriptome are referred to as epitranscriptomic changes and represent a fascinating new layer of gene expression regulation that has only recently been appreciated. Here, we outline how RNA editing and RNA modification can rapidly affect gene expression, making both processes as well suited to respond to cellular stress and to regulate the transcriptome during development or circadian periods. PMID:27044895

  10. Comprehensive high-resolution analysis of the role of an Arabidopsis gene family in RNA editing.

    PubMed

    Bentolila, Stéphane; Oh, Julyun; Hanson, Maureen R; Bukowski, Robert

    2013-06-01

    In flowering plants, mitochondrial and chloroplast mRNAs are edited by C-to-U base modification. In plant organelles, RNA editing appears to be generally a correcting mechanism that restores the proper function of the encoded product. Members of the Arabidopsis RNA editing-Interacting Protein (RIP) family have been recently shown to be essential components of the plant editing machinery. We report the use of a strand- and transcript-specific RNA-seq method (STS-PCRseq) to explore the effect of mutation or silencing of every RIP gene on plant organelle editing. We confirm RIP1 to be a major editing factor that controls the editing extent of 75% of the mitochondrial sites and 20% of the plastid C targets of editing. The quantitative nature of RNA sequencing allows the precise determination of overlapping effects of RIP factors on RNA editing. Over 85% of the sites under the influence of RIP3 and RIP8, two moderately important mitochondrial factors, are also controlled by RIP1. Previously uncharacterized RIP family members were found to have only a slight effect on RNA editing. The preferential location of editing sites controlled by RIP7 on some transcripts suggests an RNA metabolism function for this factor other than editing. In addition to a complete characterization of the RIP factors for their effect on RNA editing, our study highlights the potential of RNA-seq for studying plant organelle editing. Unlike previous attempts to use RNA-seq to analyze RNA editing extent, our methodology focuses on sequencing of organelle cDNAs corresponding to known transcripts. As a result, the depth of coverage of each editing site reaches unprecedented values, assuring a reliable measurement of editing extent and the detection of numerous new sites. This strategy can be applied to the study of RNA editing in any organism.

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

    PubMed

    Nishikura, Kazuko

    2016-02-01

    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

  12. 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

  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. Variable frequency of plastid RNA editing among ferns and repeated loss of uridine-to-cytidine editing from vascular plants.

    PubMed

    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.

  15. The organization, structure, and in vitro transcription of Alu family RNA polymerase III transcription units in the human alpha-like globin gene cluster: precipitation of in vitro transcripts by lupus anti-La antibodies.

    PubMed

    Shen, C K; Maniatis, T

    1982-01-01

    We have studied the location, structure, and in vitro transcription of repetitive DNA sequences within the human alpha-like globin gene cluster. At least eight different Alu family repeats were identified, each of which is transcribed in vitro to produce discrete RNA transcripts. The nucleotide sequence of one Alu repeat sequence, located on the 3' side of the alpha l globin gene (3'-alpha l), was determined and compared to published Alu repeat sequences. In vitro transcription of this repeat sequence generates RNA fragments of approximately 410, 260, 160, and 86 nucleotides. To determine whether these transcripts associate with specific proteins in vitro, we carried out immunoprecipitation experiments using an antiserum from systemic lupus erythematosus (SLE) patients. We find that the antiserum anti-La, which was shown to precipitate ribonucleoproteins (RNPs) containing the adenovirus VAI RNA from virus infected cells, preferentially precipitates the smallest two in vitro transcripts of the 3'-alpha l Alu repeat. These results suggest that the RNAs interact with specific factors in the in vitro transcription reaction mix to form RNP.

  16. Cell type-specific loss of atp6 RNA editing in cytoplasmic male sterile Sorghum bicolor.

    PubMed

    Howad, W; Kempken, F

    1997-09-30

    RNA editing and cytoplasmic male sterility are two important phenomena in higher plant mitochondria. To determine whether correlations might exist between the two, RNA editing in different tissues of Sorghum bicolor was compared employing reverse transcription-PCR and subsequent sequence analysis. In etiolated shoots, RNA editing of transcripts of plant mitochondrial atp6, atp9, nad3, nad4, and rps12 genes was identical among fertile or cytoplasmic male sterile plants. We then established a protocol for mitochondrial RNA isolation from plant anthers and pollen to include in these studies. Whereas RNA editing of atp9, nad3, nad4, and rps12 transcripts in anthers was similar to etiolated shoots, mitochondrial atp6 RNA editing was strongly reduced in anthers of the A3Tx398 male sterile line of S. bicolor. atp6 transcripts of wheat and selected plastid transcripts in S. bicolor showed normal RNA editing, indicating that loss of atp6 RNA editing is specific for cytoplasmic male sterility S. bicolor mitochondria. Restoration of fertility in F1 and F2 lines correlated with an increase in RNA editing of atp6 transcripts. Our data suggest that loss of atp6 RNA editing contributes to or causes cytoplasmic male sterility in S. bicolor. Further analysis of the mechanism of cell type-specific loss of atp6 RNA editing activity may advance our understanding of the mechanism of RNA editing.

  17. Identification of a pentatricopeptide repeat RNA editing factor in Physcomitrella patens chloroplasts.

    PubMed

    Ichinose, Mizuho; Uchida, Masato; Sugita, Mamoru

    2014-11-01

    The moss Physcomitrella patens has two RNA editing sites in the chloroplasts. Here we identified a novel DYW-subclass pentatricopeptide repeat (PPR) protein, PpPPR_45, as a chloroplast RNA editing factor in P. patens. Knockdown of the PpPPR_45 gene reduced the extent of RNA editing at the chloroplast rps14-C2 site, whereas over-expression of PpPPR_45 increased the levels of RNA editing at both the rps14-C2 site and its neighboring C site. This indicates that the expression level of PpPPR_45 affects the extent of RNA editing at the two neighboring sites.

  18. 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

  19. The domain structure and distribution of Alu elements in long noncoding RNAs and mRNAs.

    PubMed

    Kim, Eugene Z; Wespiser, Adam R; Caffrey, Daniel R

    2016-02-01

    Approximately 75% of the human genome is transcribed and many of these spliced transcripts contain primate-specific Alu elements, the most abundant mobile element in the human genome. The majority of exonized Alu elements are located in long noncoding RNAs (lncRNAs) and the untranslated regions of mRNA, with some performing molecular functions. To further assess the potential for Alu elements to be repurposed as functional RNA domains, we investigated the distribution and evolution of Alu elements in spliced transcripts. Our analysis revealed that Alu elements are underrepresented in mRNAs and lncRNAs, suggesting that most exonized Alu elements arising in the population are rare or deleterious to RNA function. When mRNAs and lncRNAs retain exonized Alu elements, they have a clear preference for Alu dimers, left monomers, and right monomers. mRNAs often acquire Alu elements when their genes are duplicated within Alu-rich regions. In lncRNAs, reverse-oriented Alu elements are significantly enriched and are not restricted to the 3' and 5' ends. Both lncRNAs and mRNAs primarily contain the Alu J and S subfamilies that were amplified relatively early in primate evolution. Alu J subfamilies are typically overrepresented in lncRNAs, whereas the Alu S dimer is overrepresented in mRNAs. The sequences of Alu dimers tend to be constrained in both lncRNAs and mRNAs, whereas the left and right monomers are constrained within particular Alu subfamilies and classes of RNA. Collectively, these findings suggest that Alu-containing RNAs are capable of forming stable structures and that some of these Alu domains might have novel biological functions.

  20. 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

  1. A-to-I editing of protein coding and noncoding RNAs.

    PubMed

    Mallela, Arka; Nishikura, Kazuko

    2012-01-01

    Adenosine deaminase acting on RNA (ADAR) catalyzes the hydrolytic deamination of adenosine to inosine in double-stranded RNA (dsRNA) substrates. Inosine pairs preferentially with cytidine, as opposed to uridine; therefore, ADAR editing alters the sequence and base pairing properties of both protein-coding and non-coding RNA. Editing can directly alter the sequence of protein-coding transcripts and modify splicing, or affect a variety of non-coding targets, including microRNA, small interfering RNA, viral transcripts, and repeat elements such as Alu and LINE. Such editing has a wide range of physiological effects, including modification of targets in the brain and in disease states.

  2. Trypanosome RNA editing: the complexity of getting U in and taking U out.

    PubMed

    Read, Laurie K; Lukeš, Julius; Hashimi, Hassan

    2016-01-01

    RNA editing, which adds sequence information to RNAs post-transcriptionally, is a widespread phenomenon throughout eukaryotes. The most complex form of this process is the uridine (U) insertion/deletion editing that occurs in the mitochondria of kinetoplastid protists. RNA editing in these flagellates is specified by trans-acting guide RNAs and entails the insertion of hundreds and deletion of dozens of U residues from mitochondrial RNAs to produce mature, translatable mRNAs. An emerging model indicates that the machinery required for trypanosome RNA editing is much more complicated than previously appreciated. A family of RNA editing core complexes (RECCs), which contain the required enzymes and several structural proteins, catalyze cycles of U insertion and deletion. A second, dynamic multiprotein complex, the Mitochondrial RNA Binding 1 (MRB1) complex, has recently come to light as another essential component of the trypanosome RNA editing machinery. MRB1 likely serves as the platform for kinetoplastid RNA editing, and plays critical roles in RNA utilization and editing processivity. MRB1 also appears to act as a hub for coordination of RNA editing with additional mitochondrial RNA processing events. This review highlights the current knowledge regarding the complex molecular machinery involved in trypanosome RNA editing. WIREs RNA 2016, 7:33-51. doi: 10.1002/wrna.1313 For further resources related to this article, please visit the WIREs website.

  3. Widespread establishment and regulatory impact of Alu exons in human genes.

    PubMed

    Shen, Shihao; Lin, Lan; Cai, James J; Jiang, Peng; Kenkel, Elizabeth J; Stroik, Mallory R; Sato, Seiko; Davidson, Beverly L; Xing, Yi

    2011-02-15

    The Alu element has been a major source of new exons during primate evolution. Thousands of human genes contain spliced exons derived from Alu elements. However, identifying Alu exons that have acquired genuine biological functions remains a major challenge. We investigated the creation and establishment of Alu exons in human genes, using transcriptome profiles of human tissues generated by high-throughput RNA sequencing (RNA-Seq) combined with extensive RT-PCR analysis. More than 25% of Alu exons analyzed by RNA-Seq have estimated transcript inclusion levels of at least 50% in the human cerebellum, indicating widespread establishment of Alu exons in human genes. Genes encoding zinc finger transcription factors have significantly higher levels of Alu exonization. Importantly, Alu exons with high splicing activities are strongly enriched in the 5'-UTR, and two-thirds (10/15) of 5'-UTR Alu exons tested by luciferase reporter assays significantly alter mRNA translational efficiency. Mutational analysis reveals the specific molecular mechanisms by which newly created 5'-UTR Alu exons modulate translational efficiency, such as the creation or elongation of upstream ORFs that repress the translation of the primary ORFs. This study presents genomic evidence that a major functional consequence of Alu exonization is the lineage-specific evolution of translational regulation. Moreover, the preferential creation and establishment of Alu exons in zinc finger genes suggest that Alu exonization may have globally affected the evolution of primate and human transcriptomes by regulating the protein production of master transcriptional regulators in specific lineages.

  4. Conserved microRNA editing in mammalian evolution, development and disease

    PubMed Central

    2014-01-01

    Background Mammalian microRNAs (miRNAs) are sometimes subject to adenosine-to-inosine RNA editing, which can lead to dramatic changes in miRNA target specificity or expression levels. However, although a few miRNAs are known to be edited at identical positions in human and mouse, the evolution of miRNA editing has not been investigated in detail. In this study, we identify conserved miRNA editing events in a range of mammalian and non-mammalian species. Results We demonstrate deep conservation of several site-specific miRNA editing events, including two that date back to the common ancestor of mammals and bony fishes some 450 million years ago. We also find evidence of a recent expansion of an edited miRNA family in placental mammals and show that editing of these miRNAs is associated with changes in target mRNA expression during primate development and aging. While global patterns of miRNA editing tend to be conserved across species, we observe substantial variation in editing frequencies depending on tissue, age and disease state: editing is more frequent in neural tissues compared to heart, kidney and testis; in older compared to younger individuals; and in samples from healthy tissues compared to tumors, which together suggests that miRNA editing might be associated with a reduced rate of cell proliferation. Conclusions Our results show that site-specific miRNA editing is an evolutionarily conserved mechanism, which increases the functional diversity of mammalian miRNA transcriptomes. Furthermore, we find that although miRNA editing is rare compared to editing of long RNAs, miRNAs are greatly overrepresented among conserved editing targets. PMID:24964909

  5. 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

  6. 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

  7. RNA editing in plant mitochondria—connecting RNA target sequences and acting proteins.

    PubMed

    Takenaka, Mizuki; Verbitskiy, Daniil; Zehrmann, Anja; Härtel, Barbara; Bayer-Császár, Eszter; Glass, Franziska; Brennicke, Axel

    2014-11-01

    RNA editing changes several hundred cytidines to uridines in the mRNAs of mitochondria in flowering plants. The target cytidines are identified by a subtype of PPR proteins characterized by tandem modules which each binds with a specific upstream nucleotide. Recent progress in correlating repeat structures with nucleotide identities allows to predict and identify target sites in mitochondrial RNAs. Additional proteins have been found to play a role in RNA editing; their precise function still needs to be elucidated. The enzymatic activity performing the C to U reaction may reside in the C-terminal DYW extensions of the PPR proteins; however, this still needs to be proven. Here we update recent progress in understanding RNA editing in flowering plant mitochondria.

  8. In vivo dissection of cis-acting determinants for plastid RNA editing.

    PubMed

    Bock, R; Hermann, M; Kössel, H

    1996-09-16

    Substitutional RNA editing changes single C nucleotides in higher plant chloroplast transcripts into U residues. To determine the cis-acting sequence elements involved in plastid RNA editing, we constructed a series of chloroplast transformation vectors harboring selected editing sites of the tobacco ndhB transcript in a chimeric context. The constructs were inserted into the tobacco plastid genome by biolistic transformation leading to the production of stable chimeric RNAs. Analysis of RNA editing revealed unexpected differences in the size of the essential cis elements or in their distance from the editing site. Flanking sequences of identical size direct virtually complete editing for one pair of editing sites, partial editing for a second and no editing at all for a third pair of sites. Serial 5' and 3' deletions allowed us to define the cis-acting elements more precisely and to identify a sequence element essential for editing site recognition. In addition, a single nucleotide substitution immediately upstream of an editing position was introduced. This mutation was found drastically and selectively to reduce the editing efficiency of the downstream editing site, demonstrating that position -1 is important for either site recognition or catalysis. Our results indicate that the editing of adjacent sites is likely to be mechanistically coupled. In no case did the presence in the plastome of the additional editing sites have any effect on the editing efficiency of the endogenous ndhB sites, indicating that the availability of site-specific trans-acting factors is not rate limiting.

  9. RNA Editing of Cytochrome Oxidase Subunit III in Sunflower Mitochondria 1

    PubMed Central

    Saiardi, Adolfo; Quagliariello, Carla

    1992-01-01

    Direct sequencing of cytochrome oxidase subunit III (coxIII) mRNA with a specific primer confirms RNA editing in sunflower (Helianthus annus) mitochondria. Six instances of mRNA editing could be verified, one of these specific to this species. All the editing events involve C to U transitions in the coxIII mRNA causing codon changes that lead to amino acids better conserved in evolution than those encoded in the genomic DNA. This observation confirms RNA editing to be widespread in higher plant mitochondria. ImagesFigure 1 PMID:16668785

  10. Ku antigen binds to Alu family DNA.

    PubMed

    Tsuchiya, T; Saëgusa, Y; Taira, T; Mimori, T; Iguchi-Ariga, S M; Ariga, H

    1998-01-01

    The GC-rich segment containing GGAGGC (Alu core) is conserved within the RNA polymerase III (pol III) promoters of Alu family sequences. We have shown that the GGAGGC motif functions as a modulator of DNA replication as well as of transcription, and identified the proteins binding to the motif in human HeLa cells. In this study, the Alu core binding proteins were partially purified from human Raji cells by using an Alu core DNA affinity column. Both the proteins thus purified were implied to be subunits of Ku antigen based on the following criteria: The molecular weights of the proteins estimated on gel electrophoreses were 70 and 85 kDa, respectively, under denaturing conditions, while under non-denaturing conditions only one band was observed for the same sample at 150 kDa, probably representing hetero-dimer formed between the 70 and 85 kDa proteins. The sizes and the hetero-dimer formation are reminiscent of the 70 and 80 kDa subunits of Ku antigen (Ku-p70 and Ku-p80). Moreover, the purified proteins were immunoreactive with anti-Ku antibodies, and the specific DNA-protein complex on the Alu core element was cancelled by the anti-Ku antibodies. The nucleoprotein complex showed the same clipping patterns as those of the complex between the Alu core element and an authentically purified Ku antigen after proteolytic cleavage with trypsin and chymotrypsin.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. 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

  16. 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

  17. A new Ebola virus nonstructural glycoprotein expressed through RNA editing.

    PubMed

    Mehedi, Masfique; Falzarano, Darryl; Seebach, Jochen; Hu, Xiaojie; Carpenter, Michael S; Schnittler, Hans-Joachim; Feldmann, Heinz

    2011-06-01

    Ebola virus (EBOV), an enveloped, single-stranded, negative-sense RNA virus, causes severe hemorrhagic fever in humans and nonhuman primates. The EBOV glycoprotein (GP) gene encodes the nonstructural soluble glycoprotein (sGP) but also produces the transmembrane glycoprotein (GP₁,₂) through transcriptional editing. A third GP gene product, a small soluble glycoprotein (ssGP), has long been postulated to be produced also as a result of transcriptional editing. To identify and characterize the expression of this new EBOV protein, we first analyzed the relative ratio of GP gene-derived transcripts produced during infection in vitro (in Vero E6 cells or Huh7 cells) and in vivo (in mice). The average percentages of transcripts encoding sGP, GP₁,₂, and ssGP were approximately 70, 25, and 5%, respectively, indicating that ssGP transcripts are indeed produced via transcriptional editing. N-terminal sequence similarity with sGP, the absence of distinguishing antibodies, and the abundance of sGP made it difficult to identify ssGP through conventional methodology. Optimized 2-dimensional (2D) gel electrophoresis analyses finally verified the expression and secretion of ssGP in tissue culture during EBOV infection. Biochemical analysis of recombinant ssGP characterized this protein as a disulfide-linked homodimer that was exclusively N glycosylated. In conclusion, we have identified and characterized a new EBOV nonstructural glycoprotein, which is expressed as a result of transcriptional editing of the GP gene. While ssGP appears to share similar structural properties with sGP, it does not appear to have the same anti-inflammatory function on endothelial cells as sGP.

  18. 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.

  19. Incorporating alternative splicing and mRNA editing into the genetic analysis of complex traits

    PubMed Central

    Hassan, Musa A.; Saeij, Jeroen P.J.

    2014-01-01

    The nomination of candidate genes underlying complex traits is often focused on genetic variations that alter mRNA abundance or result in non-conservative changes in amino acids. Although inconspicuous in complex trait analysis, genetic variants that affect splicing or RNA editing can also generate proteomic diversity and impact genetic traits. Indeed it is known that splicing and RNA editing modulate several traits in humans and model organisms. Using high-throughput RNA sequencing (RNA-seq) analysis, it is now possible to integrate the genetics of transcript abundance, alternative splicing and editing with the analysis of complex traits. We recently demonstrated that both alternative splicing and mRNA editing are modulated by genetic and environmental factors, and potentially engender phenotypic diversity in a genetically segregating mouse population. Therefore, the analysis of splicing and RNA editing will expand not only the regulatory landscape of transcriptome and proteome complexity, but also the repertoire of candidate genes for complex traits. PMID:25171292

  20. 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

  1. Mitochondrial tRNA 5′-Editing in Dictyostelium discoideum and Polysphondylium pallidum*

    PubMed Central

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

    2014-01-01

    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

  2. In vitro RNA-binding assay for studying trans-factors for RNA editing in chloroplasts.

    PubMed

    Shikanai, Toshiharu; Okuda, Kenji

    2011-01-01

    In plant organelles, specific C residues are modified to U by RNA editing. Short RNA sequences surrounding the target site (i.e., cis-elements) are recognized by trans-factors, which were recently shown to be pentatricopeptide repeat (PPR) proteins. PPR proteins consist of tandem arrays of a highly degenerate unit of 35 (pentatrico) amino acids, and PPR motifs are believed to recognize specific RNA sequences. In Arabidopsis thaliana, more than 450 sites are edited in mitochondria and plastids, and a similar number of PPR proteins are encoded in the nuclear genome. To study how the tandem array of a PPR motif facilitates the recognition of RNA sequences, an efficient biochemical strategy is an in vitro binding assay of recombinant PPR proteins with target RNA. This analysis is especially powerful with a combination of in vivo analyses based on the phenotypes of mutants and transgenic plants. In this chapter, we describe methods for the expression of recombinant PPR proteins in Escherichia coli, preparation of probe RNAs, and RNA gel shift assays. These methods can also be utilized for other RNA-binding proteins.

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

    PubMed

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

    2016-02-01

    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.

  4. 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

  5. Two RNA recognition motif-containing proteins are plant mitochondrial editing factors

    PubMed Central

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

    2015-01-01

    Post-transcriptional C-to-U RNA editing occurs in plant plastid and mitochondrial transcripts. Members of the Arabidopsis RNA-editing factor interacting protein (RIP) family and ORRM1 (Organelle RNA Recognition Motif-containing protein 1) have been recently characterized as essential components of the chloroplast RNA editing apparatus. ORRM1 belongs to a distinct clade of RNA Recognition Motif (RRM)-containing proteins, most of which are predicted to be organelle-targeted. Here we report the identification of two proteins, ORRM2 (organelle RRM protein 2) and ORRM3 (organelle RRM protein 3), as the first members of the ORRM clade to be identified as mitochondrial editing factors. Transient silencing of ORRM2 and ORRM3 resulted in reduced editing efficiency at ∼6% of the mitochondrial C targets. In addition to an RRM domain at the N terminus, ORRM3 carries a glycine-rich domain at the C terminus. The N-terminal RRM domain by itself provides the editing activity of ORRM3. In yeast-two hybrid assays, ORRM3 interacts with RIP1, ORRM2 and with itself. Transient silencing of ORRM2 in the orrm3 mutant further impairs the editing activity at sites controlled by both ORRM2 and ORRM3. Identification of the effect of ORRM2 and ORRM3 on RNA editing reveals a previously undescribed role of RRM-containing proteins as mitochondrial RNA editing factors. PMID:25800738

  6. 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.

  7. Rewriting the information in DNA: RNA editing in kinetoplastids and myxomycetes.

    PubMed

    Horton, Tamara L; Landweber, Laura F

    2002-12-01

    RNA editing has a major impact on the genes and genomes that it modifies. Editing by insertion, deletion and base conversion exists in nuclear, mitochondrial and viral genomes throughout the eukaryotic lineage. Editing was first discovered in kinetoplastids, and recent work has resulted in the characterization of some components of the editing machinery. Two proteins with ligase activity have been identified in Trypanosoma brucei, and other proteins in the editosome complex are yielding to the probe of research. A second group of protists, myxomycetes, are unique in their use of four different types of editing within a single transcript. Phylogenetic analysis of editing in representative myxomycetes revealed a different history of the four types of editing in this lineage. Development of a soluble in vitro editing system has provided further support for the co-transcriptional nature of editing in Physarum polycephalum, and will certainly provide future opportunities for understanding this mysterious process.

  8. Discriminative Prediction of A-To-I RNA Editing Events from DNA Sequence

    PubMed Central

    Sun, Jiangming; Singh, Pratibha; Bagge, Annika; Valtat, Bérengère; Vikman, Petter; Spégel, Peter; Mulder, Hindrik

    2016-01-01

    RNA editing is a post-transcriptional alteration of RNA sequences that, via insertions, deletions or base substitutions, can affect protein structure as well as RNA and protein expression. Recently, it has been suggested that RNA editing may be more frequent than previously thought. A great impediment, however, to a deeper understanding of this process is the paramount sequencing effort that needs to be undertaken to identify RNA editing events. Here, we describe an in silico approach, based on machine learning, that ameliorates this problem. Using 41 nucleotide long DNA sequences, we show that novel A-to-I RNA editing events can be predicted from known A-to-I RNA editing events intra- and interspecies. The validity of the proposed method was verified in an independent experimental dataset. Using our approach, 203 202 putative A-to-I RNA editing events were predicted in the whole human genome. Out of these, 9% were previously reported. The remaining sites require further validation, e.g., by targeted deep sequencing. In conclusion, the approach described here is a useful tool to identify potential A-to-I RNA editing events without the requirement of extensive RNA sequencing. PMID:27764195

  9. 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

  10. Association of guide RNA binding protein gBP21 with active RNA editing complexes in Trypanosoma brucei.

    PubMed

    Allen, T E; Heidmann, S; Reed, R; Myler, P J; Göringer, H U; Stuart, K D

    1998-10-01

    RNA editing in Trypanosoma brucei mitochondria produces mature mRNAs by a series of enzyme-catalyzed reactions that specifically insert or delete uridylates in association with a macromolecular complex. Using a mitochondrial fraction enriched for in vitro RNA editing activity, we produced several monoclonal antibodies that are specific for a 21-kDa guide RNA (gRNA) binding protein initially identified by UV cross-linking. Immunofluorescence studies localize the protein to the mitochondrion, with a preference for the kinetoplast. The antibodies cause a supershift of previously identified gRNA-specific ribonucleoprotein complexes and immunoprecipitate in vitro RNA editing activities that insert and delete uridylates. The immunoprecipitated material also contains gRNA-specific endoribonuclease, terminal uridylyltransferase, and RNA ligase activities as well as gRNA and both edited and unedited mRNA. The immunoprecipitate contains numerous proteins, of which the 21-kDa protein, a 90-kDa protein, and novel 55- and 16-kDa proteins can be UV cross-linked to gRNA. These studies indicate that the 21-kDa protein associates with the ribonucleoprotein complex (or complexes) that catalyze RNA editing.

  11. Determinants for tRNA-Dependent Pretransfer Editing in the Synthetic Site of Isoleucyl-tRNA Synthetase

    PubMed Central

    2015-01-01

    The accurate expression of genetic information relies on the fidelity of amino acid–tRNA coupling by aminoacyl-tRNA synthetases (aaRS). When the specificity against structurally similar noncognate amino acids in the synthetic reaction does not support a threshold fidelity level for translation, the aaRS employ intrinsic hydrolytic editing to correct errors in aminoacylation. Escherichia coli isoleucyl-tRNA synthetase (EcIleRS) is a class I aaRS that is notable for its use of tRNA-dependent pretransfer editing to hydrolyze noncognate valyl-adenylate prior to aminoacyl-tRNA formation. On the basis of the finding that IleRS possessing an inactivated post-transfer editing domain is still capable of robust tRNA-dependent editing, we have recently proposed that the pretransfer editing activity resides within the synthetic site. Here we apply an improved methodology that allows quantitation of the AMP fraction that arises particularly from tRNA-dependent aa-AMP hydrolysis. By this approach, we demonstrate that tRNA-dependent pretransfer editing accounts for nearly one-third of the total proofreading by EcIleRS and that a highly conserved tyrosine within the synthetic site modulates both editing and aminoacylation. Therefore, synthesis of aminoacyl-tRNA and hydrolysis of aminoacyl-adenylates employ overlapping amino acid determinants. We suggest that this overlap hindered the evolution of synthetic site-based pretransfer editing as the predominant proofreading pathway, because that activity is difficult to accommodate in the context of efficient aminoacyl-tRNA synthesis. Instead, the acquisition of a spatially separate domain dedicated to post-transfer editing alone allowed for the development of a powerful deacylation machinery that effectively competes with dissociation of misacylated tRNAs. PMID:25207837

  12. When you can't trust the DNA: RNA editing changes transcript sequences.

    PubMed

    Knoop, Volker

    2011-02-01

    RNA editing describes targeted sequence alterations in RNAs so that the transcript sequences differ from their DNA template. Since the original discovery of RNA editing in trypanosomes nearly 25 years ago more than a dozen such processes of nucleotide insertions, deletions, and exchanges have been identified in evolutionarily widely separated groups of the living world including plants, animals, fungi, protists, bacteria, and viruses. In many cases gene expression in mitochondria is affected, but RNA editing also takes place in chloroplasts and in nucleocytosolic genetic environments. While some RNA editing systems largely seem to repair defect genes (cryptogenes), others have obvious functions in modulating gene activities. The present review aims for an overview on the current states of research in the different systems of RNA editing by following a historic timeline along the respective original discoveries.

  13. The RNA-Editing Enzyme ADAR1 Controls Innate Immune Responses to RNA

    PubMed Central

    Mannion, Niamh M.; Greenwood, Sam M.; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P.; McLaughlin, Paul J.; Jantsch, Michael F.; Dorin, Julia; Adams, Ian R.; Scadden, A.D.J.; Öhman, Marie; Keegan, Liam P.; O’Connell, Mary A.

    2014-01-01

    Summary The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. PMID:25456137

  14. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA.

    PubMed

    Mannion, Niamh M; Greenwood, Sam M; Young, Robert; Cox, Sarah; Brindle, James; Read, David; Nellåker, Christoffer; Vesely, Cornelia; Ponting, Chris P; McLaughlin, Paul J; Jantsch, Michael F; Dorin, Julia; Adams, Ian R; Scadden, A D J; Ohman, Marie; Keegan, Liam P; O'Connell, Mary A

    2014-11-20

    The ADAR RNA-editing enzymes deaminate adenosine bases to inosines in cellular RNAs. Aberrant interferon expression occurs in patients in whom ADAR1 mutations cause Aicardi-Goutières syndrome (AGS) or dystonia arising from striatal neurodegeneration. Adar1 mutant mouse embryos show aberrant interferon induction and die by embryonic day E12.5. We demonstrate that Adar1 embryonic lethality is rescued to live birth in Adar1; Mavs double mutants in which the antiviral interferon induction response to cytoplasmic double-stranded RNA (dsRNA) is prevented. Aberrant immune responses in Adar1 mutant mouse embryo fibroblasts are dramatically reduced by restoring the expression of editing-active cytoplasmic ADARs. We propose that inosine in cellular RNA inhibits antiviral inflammatory and interferon responses by altering RLR interactions. Transfecting dsRNA oligonucleotides containing inosine-uracil base pairs into Adar1 mutant mouse embryo fibroblasts reduces the aberrant innate immune response. ADAR1 mutations causing AGS affect the activity of the interferon-inducible cytoplasmic isoform more severely than the nuclear isoform. PMID:25456137

  15. Uridine insertion/deletion RNA editing in trypanosome mitochondria: a complex business.

    PubMed

    Simpson, Larry; Sbicego, Sandro; Aphasizhev, Ruslan

    2003-03-01

    The basic mechanism of uridine insertion/deletion RNA editing in mitochondria of kinetoplastid protists has been established for some time but the molecular details remained largely unknown. Recently, there has been significant progress in defining the molecular components of the editing reaction. A number of factors have been isolated from trypanosome mitochondria, some of which have been definitely implicated in the uridine insertion/deletion RNA editing reaction and others of which have been circumstantially implicated. Several protein complexes have been isolated which exhibit some editing activities, and the macromolecular organization of these complexes is being analyzed. In addition, there have been several important technical advances in the in vitro analysis of editing. In this review we critically examine the various factors and complexes proposed to be involved in RNA editing.

  16. 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

  17. 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

  18. Molecular Evidence of RNA Editing in Bombyx Chemosensory Protein Family

    PubMed Central

    Xuan, Ning; Bu, Xun; Liu, Yan Yan; Yang, Xue; Liu, Guo Xia; Fan, Zhong Xue; Bi, Yu Ping; Yang, Lian Qun; Lou, Qi Nian; Rajashekar, Balaji; Leppik, Getter; Kasvandik, Sergo; Picimbon, Jean-François

    2014-01-01

    Chemosensory proteins (CSPs) are small scavenger proteins that are mainly known as transporters of pheromone/odor molecules at the periphery of sensory neurons in the insect antennae and in the producing cells from the moth female pheromone gland. Sequencing cDNAs of RNA encoding CSPs in the antennae, legs, head, pheromone gland and wings from five single individual adult females of the silkworm moth Bombyx mori showed that they differed from genomic sequences by subtle nucleotide replacement (RDD). Both intronless and intronic CSP genes expressed RDDs, although in different rates. Most interestingly, in our study the degree of RDDs in CSP genes were found to be tissue-specific. The proportion of CSP-RDDs was found to be significantly much higher in the pheromone gland. In addition, Western blot analysis of proteins in different tissues showed existence of multiple CSP protein variant chains particularly found in the pheromone gland. Peptide sequencing demonstrated the occurrence of a pleiad of protein variants for most of all BmorCSPs from the pheromone gland. Our findings show that RNA editing is an important feature in the expression of CSPs and that a high variety of RDDs is found to expand drastically thus altering the repertoire of CSP proteins in a tissue-specific manner. PMID:24551045

  19. Unraveling pleiotropic functions of A-to-I RNA editing in Drosophila.

    PubMed

    Jepson, James E; Reenan, Robert A

    2010-01-01

    In metazoan cells, transcripts that fold into double-strand RNA structures are endowed with the capacity to undergo A-to-I RNA editing, during which adenosines are catalytically deaminated to inosines by a class of enzymes known as ADARs (adenosine deaminases acting on RNA). In Drosophila, a wide range of coding mRNAs associated with signaling in the nervous system undergo A-to-I editing, and loss of editing results in extreme behavioral defects. Furthermore, there are indications that the precursors of endogenous small interfering RNAs also undergo editing. However, the mechanism by which A-to-I editing is related to ethology in Drosophila is unclear, as are the precise cell-types and developmental stages in which editing is most crucial. We have investigated these issues by altering small RNA production in flies lacking ADAR, and modulating editing levels in both time and space through a variety of transgenic techniques. Our results indicate that genetic re-coding in the nervous system is likely to be the primary pathway through which editing affects behavioral outputs, and further suggest that editing is required to 'fine-tune' neuro-transmission in the adult brain.

  20. 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

  1. PopAlu: population-scale detection of Alu polymorphisms

    PubMed Central

    Qian, Yu; Kehr, Birte

    2015-01-01

    Alu elements are sequences of approximately 300 basepairs that together comprise more than 10% of the human genome. Due to their recent origin in primate evolution some Alu elements are polymorphic in humans, present in some individuals while absent in others. We present PopAlu, a tool to detect polymorphic Alu elements on a population scale from paired-end sequencing data. PopAlu uses read pair distance and orientation as well as split reads to identify the location and precise breakpoints of polymorphic Alus. Genotype calling enables us to differentiate between homozygous and heterozygous carriers, making the output of PopAlu suitable for use in downstream analyses such as genome-wide association studies (GWAS). We show on a simulated dataset that PopAlu calls Alu elements inserted and deleted with respect to a reference genome with high accuracy and high precision. Our analysis of real data of a human trio from the 1000 Genomes Project confirms that PopAlu is able to produce highly accurate genotype calls. To our knowledge, PopAlu is the first tool that identifies polymorphic Alu elements from multiple individuals simultaneously, pinpoints the precise breakpoints and calls genotypes with high accuracy. PMID:26417547

  2. TRIBE: Hijacking an RNA-Editing Enzyme to Identify Cell-Specific Targets of RNA-Binding Proteins.

    PubMed

    McMahon, Aoife C; Rahman, Reazur; Jin, Hua; Shen, James L; Fieldsend, Allegra; Luo, Weifei; Rosbash, Michael

    2016-04-21

    RNA transcripts are bound and regulated by RNA-binding proteins (RBPs). Current methods for identifying in vivo targets of an RBP are imperfect and not amenable to examining small numbers of cells. To address these issues, we developed TRIBE (targets of RNA-binding proteins identified by editing), a technique that couples an RBP to the catalytic domain of the Drosophila RNA-editing enzyme ADAR and expresses the fusion protein in vivo. RBP targets are marked with novel RNA editing events and identified by sequencing RNA. We have used TRIBE to identify the targets of three RBPs (Hrp48, dFMR1, and NonA). TRIBE compares favorably to other methods, including CLIP, and we have identified RBP targets from as little as 150 specific fly neurons. TRIBE can be performed without an antibody and in small numbers of specific cells.

  3. 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

  4. U-Insertion/Deletion mRNA-Editing Holoenzyme: Definition in Sight.

    PubMed

    Aphasizheva, Inna; Aphasizhev, Ruslan

    2016-02-01

    RNA editing is a process that alters DNA-encoded sequences and is distinct from splicing, 5' capping, and 3' additions. In 30 years since editing was discovered in mitochondria of trypanosomes, several functionally and evolutionarily unrelated mechanisms have been described in eukaryotes, archaea, and viruses. Editing events are predominantly post-transcriptional and include nucleoside insertions and deletions, and base substitutions and modifications. Here, we review the mechanism of uridine insertion/deletion mRNA editing in kinetoplastid protists typified by Trypanosoma brucei. This type of editing corrects frameshifts, introduces translation punctuation signals, and often adds hundreds of uridines to create protein-coding sequences. We focus on protein complexes responsible for editing reactions and their interactions with other elements of the mitochondrial gene expression pathway.

  5. The 3′-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA

    PubMed Central

    GOLDEN, DANIEL E.; HAJDUK, STEPHEN L.

    2005-01-01

    RNA editing in trypanosomes is a post-transcriptional process responsible for correcting the coding sequences of many mitochondrial mRNAs. Uridines are specifically added or deleted from mRNA by an enzymatic cascade in which a pre-edited mRNA is specifically cleaved, uridines are added or removed, and the corrected mRNA is ligated. The process is directed by RNA molecules, termed guide RNAs (gRNA). The ability of this class of small, noncoding RNA to function in RNA editing is essential for these organisms. Typically, gRNAs are transcribed independent of the their cognate mRNA and anneal to form a binary RNA complex . An exception for this process may be cytochrome oxidase subunit II (COII) mRNA since a gene encoding a trans acting gRNA has not been identified. Using an in vitro editing assay we find that the 3′ UTR of COII, indeed, functions as a guide for both the site and number of uridines added to the coding region of the COII mRNA. We further show that the guiding sequence within the COII 3′ UTR can only function in COII editing when contiguous with the editing substrate, indicating that the 3′ UTR of COII lacks sequence or structure information necessary to function as a trans-acting gRNA. While other RNAs have been shown to “guide” RNA processing reactions, our discovery that the COII 3′ UTR directs editing of its cognate mRNA in cis, is a unique function for a 3′ UTR. The findings described here have led us to propose a new model for the evolution of gRNAs in kinetoplastids. PMID:15574518

  6. RNA-interacting proteins act as site-specific repressors of ADAR2-mediated RNA editing and fluctuate upon neuronal stimulation

    PubMed Central

    Tariq, Aamira; Garncarz, Wojciech; Handl, Cornelia; Balik, Ales; Pusch, Oliver; Jantsch, Michael F.

    2013-01-01

    RNA editing by adenosine deaminases that act on RNA (ADARs) diversifies the transcriptome by changing adenosines to inosines. In mammals, editing levels vary in different tissues, during development, and also in pathogenic conditions. From a screen for repressors of editing we have isolated three proteins that repress ADAR2-mediated RNA editing. The three proteins RPS14, SFRS9 and DDX15 interact with RNA. Overexpression or depletion of these proteins can decrease or increase editing levels by 15%, thus allowing a modulation of RNA editing up to 30%. Interestingly, the three proteins alter RNA editing in a substrate-specific manner that correlates with their RNA binding preferences. In mammalian cells, SFRS9 significantly affects editing of the two substrates CFLAR and cyFIP2, while the ribosomal protein RPS14 mostly inhibits editing of cyFIP2 messenger RNA. The helicase DDX15, in turn, has a strong effect on editing in Caenorhabditis elegans. Expression of the three factors decreases during mouse brain development. Moreover, expression levels of SFRS9 and DDX15 respond strongly to neuronal stimulation or repression, showing an inverse correlation with editing levels. Colocalization and immunoprecipitation studies demonstrate a direct interaction of SFRS9 and RPS14 with ADAR2, while DDX15 associates with other helicases and splicing factors. Our data show that different editing sites can be specifically altered in their editing pattern by changing the local RNP landscape. PMID:23275536

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

    PubMed

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

    2014-10-01

    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.

  8. RNA editing and mitochondrial genomic organization in the cryptobiid kinetoplastid protozoan Trypanoplasma borreli.

    PubMed Central

    Maslov, D A; Simpson, L

    1994-01-01

    The bodonids and cryptobiids represent an early diverged sister group to the trypanosomatids among the kinetoplastid protozoa. The trypanosome type of uridine insertion-deletion RNA editing was found to occur in the cryptobiid fish parasite Trypanoplasma borreli. A pan-edited ribosomal protein, S12, and a novel 3'- and 5'-edited cytochrome b, in addition to an unedited cytochrome oxidase III gene and an apparently unedited 12S rRNA gene, were found in a 6-kb fragment of the 80- to 90-kb mitochondrial genome. The gene order differs from that in trypanosomatids, as does the organization of putative guide RNA genes; guide RNA-like molecules are transcribed from tandemly repeated 1-kb sequences organized in 200- and 170-kb molecules instead of minicircles. The presence of pan-editing in this lineage is consistent with an ancient evolutionary origin of this process. Images PMID:7969154

  9. PPR proteins network as site-specific RNA editing factors in plant organelles.

    PubMed

    Zehrmann, Anja; Verbitskiy, Daniil; Härtel, Barbara; Brennicke, Axel; Takenaka, Mizuki

    2011-01-01

    RNA editing in flowering plant mitochondria targets several hundred C nucleotides mostly in mRNAs to be altered to U. Several nuclear encoded genes have been recently identified predominantly in Arabidopsis thaliana which code for proteins involved in specific RNA editing events in plastids or mitochondria. These nuclear genes code for proteins characterized by a stretch of 4-20 repeats of 34-36 amino acids each, accordingly classified as pentatricopeptide repeat (PPR) proteins. These repeats most likely participate in recognizing and binding the specific nucleotide motifs around editing sites which have been defined as essential cis-elements. All of the RNA editing PPR proteins contain at their C-termini an extension of as yet unclear function, the E domain, and some of these are further extended by another domain which terminates with the triplet DYW. While the E domain seems to be always required for their function in RNA editing, the DYW domain can sometimes be removed. At some editing sites a given PPR protein seems to be required, while at others their function can at least partially be compensated by presumably other PPR proteins. These observations suggest that the PPR proteins may act in a complex network to define and to target RNA editing sites.

  10. 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

  11. 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

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. Loss of editing activity during the evolution of mitochondrial phenylalanyl-tRNA synthetase.

    PubMed

    Roy, Hervé; Ling, Jiqiang; Alfonzo, Juan; Ibba, Michael

    2005-11-18

    Accurate selection of amino acids is essential for faithful translation of the genetic code. Errors during amino acid selection are usually corrected by the editing activity of aminoacyl-tRNA synthetases such as phenylalanyl-tRNA synthetases (PheRS), which edit misactivated tyrosine. Comparison of cytosolic and mitochondrial PheRS from the yeast Saccharomyces cerevisiae suggested that the organellar protein might lack the editing activity. Yeast cytosolic PheRS was found to contain an editing site, which upon disruption abolished both cis and trans editing of Tyr-tRNA(Phe). Wild-type mitochondrial PheRS lacked cis and trans editing and could synthesize Tyr-tRNA(Phe), an activity enhanced in active site variants with improved tyrosine recognition. Possible trans editing was investigated in isolated mitochondrial extracts, but no such activity was detected. These data indicate that the mitochondrial protein synthesis machinery lacks the tyrosine proofreading activity characteristic of cytosolic translation. This difference between the mitochondria and the cytosol suggests that either organellar protein synthesis quality control is focused on another step or that translation in this compartment is inherently less accurate than in the cytosol. PMID:16162501

  18. 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

  19. 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

  20. Extensive Mitochondrial mRNA Editing and Unusual Mitochondrial Genome Organization in Calcaronean Sponges.

    PubMed

    Lavrov, Dennis V; Adamski, Marcin; Chevaldonné, Pierre; Adamska, Maja

    2016-01-11

    One of the unusual features of DNA-containing organelles in general and mitochondria in particular is the frequent occurrence of RNA editing [1]. The term "RNA editing" refers to a variety of mechanistically unrelated biochemical processes that alter RNA sequence during or after transcription [2]. The editing can be insertional, deletional, or substitutional and has been found in all major types of RNAs [3, 4]. Although mitochondrial mRNA editing is widespread in some eukaryotic lineages [5-7], it is rare in animals, with reported cases limited both in their scope and in phylogenetic distribution [8-11] (see also [12]). While analyzing genomic data from calcaronean sponges Sycon ciliatum and Leucosolenia complicata, we were perplexed by the lack of recognizable mitochondrial coding sequences. Comparison of genomic and transcriptomic data from these species revealed the presence of mitochondrial cryptogenes whose transcripts undergo extensive editing. This editing consisted of single or double uridylate (U) insertions in pre-existing short poly(U) tracts. Subsequent analysis revealed the presence of similar editing in Sycon coactum and the loss of editing in Petrobiona massiliana, a hypercalcified calcaronean sponge. In addition, mitochondrial genomes of at least some calcaronean sponges were found to have a highly unusual architecture, with nearly all genes located on individual and likely linear chromosomes. Phylogenetic analysis of mitochondrial coding sequences revealed accelerated rates of sequence evolution in this group. The latter observation presents a challenge for the mutational-hazard hypothesis [13], which posits that mRNA editing should not occur in lineages with an elevated mutation rate.

  1. Translation of the edited mRNA for cytochrome b in trypanosome mitochondria

    SciTech Connect

    Horvath, Anton; Berry, Edward A.; Maslov, Dmitri A.

    2000-02-01

    The type of RNA editing found in the kinetoplast-mitochondria of trypanosomes and related protozoa, involving uridylate insertions and deletions, creates translatable messenger RNAs (mRNAs) out of nonsense pre-edited RNAs by correcting encoded defects that vary from simple frameshifts to large ''cryptic'' regions. However, any evidence for translation of these mRNAs in the kinetoplast has been missing for decades. We identified a kinetoplast-encoded protein, apocytochrome b, whose mRNA is edited in the 5' region. The determined amino-terminal sequence of the protein coincides with the predicted sequence derived from the edited region, demonstrating that the cognate apocytochrome b mRNA is translated into a functional protein. This finding represents the first direct evidence for a functional translation system in the kinetoplasts.

  2. 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.

  3. 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

  4. Large-scale detection and analysis of adenosine-to-inosine RNA editing during development in Plutella xylostella.

    PubMed

    He, Tao; Lei, Wenjie; Ge, Chang; Du, Peng; Wang, Li; Li, Fei

    2015-06-01

    Adenosine-to-inosine (A-to-I) RNA editing is site-specific modification of RNAs that increases the diversity of the transcriptome and proteome. Most insects undergo complete metamorphosis, including four life cycle stages: egg, larva, pupa and adult. Many previous studies have confirmed that RNA-editing events occur in a development-specific manner; in other words, RNA-editing levels change during metamorphosis. Here, we describe an effort to identify the developmental specificity of RNA-editing events using a large-scale computational analysis of RNA-seq data derived from four developmental stages of the diamondback moth, Plutella xylostella. One thousand one hundred and eighty-seven A-to-I RNA-editing sites were predicted to be developmental stage specific (false-discovery rate <0.01) and 1,094 of these sites were located in protein-coding regions. Editing of 152 sites resulted in an altered amino acid residue. A putative adult-specific A-to-I RNA-editing site was verified by comparing cDNA sequences with its corresponding genomic locus at different stages of the P. xylostella life cycle. Our findings will help elucidate the role of A-to-I RNA editing in the regulation of metamorphosis. Further studies detailing changes in the extent of editing are needed to establish how as yet unknown regulatory factors are involved in the editing mechanism and what biological functions' editing serves. PMID:25492222

  5. The fraction of RNA that folds into the correct branched secondary structure determines hepatitis delta virus type 3 RNA editing levels

    PubMed Central

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

    2009-01-01

    RNA editing by the host RNA adenosine deaminase ADAR1 at the amber/W site of hepatitis delta virus RNA plays a central role in the viral replication cycle by affecting the balance between viral RNA synthesis and packaging. Previously, we found that HDV genotype III (HDV-3) RNA can form two secondary structures following transcription: an unbranched rod structure, which is characteristic of HDV, and a metastable branched structure that serves as the substrate for editing. The unstable nature of the branched editing substrate structure raised the possibility that structural dynamics of the RNA following transcription could determine the rate at which editing occurs. Here, editing and its control are examined in two HDV-3 isolates, from Peru and Ecuador. Analysis of editing in vitro by ADAR1 indicated that the branched structure formed by RNA derived from the Peruvian isolate is edited more efficiently than that from the Ecuadorian isolate. In contrast, in the context of replication, Peruvian RNA is edited less efficiently than RNA containing Ecuadorian sequences. Computational analyses of RNA folding using the massively parallel genetic algorithm (MPGAfold) indicated that the Peruvian RNA is less likely to form the branched structure required for editing than the Ecuadorian isolate. This difference was confirmed by in vitro transcription of these RNAs. Overall, our data indicate that HDV-3 controls RNA editing levels via (1) the fraction of the RNA that folds, during transcription, into the metastable branched structure required for editing and (2) the efficiency with which ADAR1 edits this branched substrate RNA. PMID:19383766

  6. Reprogramming of microRNAs by adenosine-to-inosine editing and the selective elimination of edited microRNA precursors in mouse oocytes and preimplantation embryos.

    PubMed

    García-López, Jesús; Hourcade, Juan de Dios; Del Mazo, Jesús

    2013-05-01

    Adenosine deaminases-acting-on-RNA (ADAR) proteins induce adenosine-to-inosine editing in double-stranded RNA molecules. This editing generates RNA diversity at the post-transcriptional level, and it has been implicated in the control of cell differentiation and development. The editing of microRNA (miRNA) precursors, along with Tudor-SN (Snd1) activity, could lead to the elimination of selected miRNAs and reprogram miRNA activity. Here, we report the dynamics of adenosine-to-inosine editing in miRNA precursors and their selected elimination during mouse preimplantation development. Adar1p110 and Snd1 were found to be strongly but differentially expressed in oocytes and zygotes with respect to later pre-implantation stages. When the biogenesis of miR-151 was assessed, the majority of miR-151 precursors was edited and subsequently eliminated during early development. Deep sequencing of this and other miRNAs confirmed that, in general, edited precursors were selectively eliminated at early post-zygotic stages. Moreover, in oocytes and throughout the zygote-to-blastocyst stages, Tudor-SN accumulated in newly discovered aggregates termed 'T bodies'. These results provide new insight into how editing and Tudor-SN-mediated elimination of miRNA precursors is regulated during early development.

  7. Structure of the core editing complex (L-complex) involved in uridine insertion/deletion RNA editing in trypanosomatid mitochondria.

    PubMed

    Li, Feng; Ge, Peng; Hui, Wong H; Atanasov, Ivo; Rogers, Kestrel; Guo, Qiang; Osato, Daren; Falick, Arnold M; Zhou, Z Hong; Simpson, Larry

    2009-07-28

    Uridine insertion/deletion RNA editing is a unique form of posttranscriptional RNA processing that occurs in mitochondria of kinetoplastid protists. We have carried out 3D structural analyses of the core editing complex or "L (ligase)-complex" from Leishmania tarentolae mitochondria isolated by the tandem affinity purification procedure (TAP). The purified material, sedimented at 20-25S, migrated in a blue native gel at 1 MDa and exhibited both precleaved and full-cycle gRNA-mediated U-insertion and U-deletion in vitro activities. The purified L-complex was analyzed by electron tomography to determine the extent of heterogeneity. Three-dimensional structural comparisons of individual particles in the tomograms revealed that a majority of the complexes have a similar shape of a slender triangle. An independent single-particle reconstruction, using a featureless Gaussian ball as the initial model, converged to a similar triangular structure. Another single-particle reconstruction, using the averaged tomography structure as the initial model, yielded a similar structure. The REL1 ligase was localized on the model to the base of the apex by decoration with REL1-specific IgG. This structure should prove useful for a detailed analysis of the editing reaction.

  8. Transcriptome-wide identification of A > I RNA editing sites by inosine specific cleavage.

    PubMed

    Cattenoz, Pierre B; Taft, Ryan J; Westhof, Eric; Mattick, John S

    2013-02-01

    Adenosine to inosine (A > I) RNA editing, which is catalyzed by the ADAR family of proteins, is one of the fundamental mechanisms by which transcriptomic diversity is generated. Indeed, a number of genome-wide analyses have shown that A > I editing is not limited to a few mRNAs, as originally thought, but occurs widely across the transcriptome, especially in the brain. Importantly, there is increasing evidence that A > I editing is essential for animal development and nervous system function. To more efficiently characterize the complete catalog of ADAR events in the mammalian transcriptome we developed a high-throughput protocol to identify A > I editing sites, which exploits the capacity of glyoxal to protect guanosine, but not inosine, from RNAse T1 treatment, thus facilitating extraction of RNA fragments with inosine bases at their termini for high-throughput sequencing. Using this method we identified 665 editing sites in mouse brain RNA, including most known sites and suite of novel sites that include nonsynonymous changes to protein-coding genes, hyperediting of genes known to regulate p53, and alterations to non-protein-coding RNAs. This method is applicable to any biological system for the de novo discovery of A > I editing sites, and avoids the complicated informatic and practical issues associated with editing site identification using traditional RNA sequencing data. This approach has the potential to substantially increase our understanding of the extent and function of RNA editing, and thereby to shed light on the role of transcriptional plasticity in evolution, development, and cognition.

  9. Discovery and characterization of Alu repeat sequences via precise local read assembly

    PubMed Central

    Wildschutte, Julia H.; Baron, Alayna; Diroff, Nicolette M.; Kidd, Jeffrey M.

    2015-01-01

    Alu insertions have contributed to >11% of the human genome and ∼30–35 Alu subfamilies remain actively mobile, yet the characterization of polymorphic Alu insertions from short-read data remains a challenge. We build on existing computational methods to combine Alu detection and de novo assembly of WGS data as a means to reconstruct the full sequence of insertion events from Illumina paired end reads. Comparison with published calls obtained using PacBio long-reads indicates a false discovery rate below 5%, at the cost of reduced sensitivity due to the colocation of reference and non-reference repeats. We generate a highly accurate call set of 1614 completely assembled Alu variants from 53 samples from the Human Genome Diversity Project (HGDP) panel. We utilize the reconstructed alternative insertion haplotypes to genotype 1010 fully assembled insertions, obtaining >99% agreement with genotypes obtained by PCR. In our assembled sequences, we find evidence of premature insertion mechanisms and observe 5′ truncation in 16% of AluYa5 and AluYb8 insertions. The sites of truncation coincide with stem-loop structures and SRP9/14 binding sites in the Alu RNA, implicating L1 ORF2p pausing in the generation of 5′ truncations. Additionally, we identified variable AluJ and AluS elements that likely arose due to non-retrotransposition mechanisms. PMID:26503250

  10. 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

  11. Editing of the grapevine mitochondrial cytochrome b mRNA and molecular modeling of the protein.

    PubMed

    Islas-Osuna, María A; Silva-Moreno, Begonia; Caceres-Carrizosa, Nidia; García-Robles, Jesús M; Sotelo-Mundo, Rogerio R; Yepiz-Plascencia, Gloria M

    2006-05-01

    Cytochrome b (COB), the central catalytic subunit of ubiquinol cytochrome c reductase, is a component of the transmembrane electron transfer chain that generates proton motive force. Some plant COB mRNAs are processed by RNA editing, which changes the gene coding sequence. This report presents the sequences of the grapevine (Vitis vinifera L.) mitochondrial gene for apocytochrome b (cob), the edited mRNA and the deduced protein. Grapevine COB is 393 amino acids long and is 98% identical to homologs in rapeseed, Arabidopsis thaliana and Oenothera sp. Twenty-one C-U editing sites were identified in the grapevine cob mRNA, resulting in 20 amino acid changes. These changes increase the overall hydrophobicity of the protein and result in a more conserved protein. Molecular modeling of grapevine COB shows that residues changed by RNA editing fit the secondary structure characteristic of an integral membrane protein. This is the first complete mitochondrial gene reported for grapevine. Novel RNA editing sites were identified in grapevine cob, which have not been previously reported for other plants.

  12. Fmrp Interacts with Adar and Regulates RNA Editing, Synaptic Density and Locomotor Activity in Zebrafish

    PubMed Central

    Porath, Hagit T.; Barak, Michal; Pinto, Yishay; Wachtel, Chaim; Zilberberg, Alona; Lerer-Goldshtein, Tali; Efroni, Sol; Levanon, Erez Y.; Appelbaum, Lior

    2015-01-01

    Fragile X syndrome (FXS) is the most frequent inherited form of mental retardation. The cause for this X-linked disorder is the silencing of the fragile X mental retardation 1 (fmr1) gene and the absence of the fragile X mental retardation protein (Fmrp). The RNA-binding protein Fmrp represses protein translation, particularly in synapses. In Drosophila, Fmrp interacts with the adenosine deaminase acting on RNA (Adar) enzymes. Adar enzymes convert adenosine to inosine (A-to-I) and modify the sequence of RNA transcripts. Utilizing the fmr1 zebrafish mutant (fmr1-/-), we studied Fmrp-dependent neuronal circuit formation, behavior, and Adar-mediated RNA editing. By combining behavior analyses and live imaging of single axons and synapses, we showed hyperlocomotor activity, as well as increased axonal branching and synaptic density, in fmr1-/- larvae. We identified thousands of clustered RNA editing sites in the zebrafish transcriptome and showed that Fmrp biochemically interacts with the Adar2a protein. The expression levels of the adar genes and Adar2 protein increased in fmr1-/- zebrafish. Microfluidic-based multiplex PCR coupled with deep sequencing showed a mild increase in A-to-I RNA editing levels in evolutionarily conserved neuronal and synaptic Adar-targets in fmr1-/- larvae. These findings suggest that loss of Fmrp results in increased Adar-mediated RNA editing activity on target-specific RNAs, which, in turn, might alter neuronal circuit formation and behavior in FXS. PMID:26637167

  13. 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

  14. Hypoxia-inducible C-to-U coding RNA editing downregulates SDHB in monocytes.

    PubMed

    Baysal, Bora E; De Jong, Kitty; Liu, Biao; Wang, Jianmin; Patnaik, Santosh K; Wallace, Paul K; Taggart, Robert T

    2013-01-01

    Background. RNA editing is a post-transcriptional regulatory mechanism that can alter the coding sequences of certain genes in response to physiological demands. We previously identified C-to-U RNA editing (C136U, R46X) which inactivates a small fraction of succinate dehydrogenase (SDH; mitochondrial complex II) subunit B gene (SDHB) mRNAs in normal steady-state peripheral blood mononuclear cells (PBMCs). SDH is a heterotetrameric tumor suppressor complex which when mutated causes paraganglioma tumors that are characterized by constitutive activation of hypoxia inducible pathways. Here, we studied regulation, extent and cell type origin of SDHB RNA editing. Methods. We used short-term cultured PBMCs obtained from random healthy platelet donors, performed monocyte enrichment by cold aggregation, employed a novel allele-specific quantitative PCR method, flow cytometry, immunologic cell separation, gene expression microarray, database analysis and high-throughput RNA sequencing. Results. While the editing rate is low in uncultured monocyte-enriched PBMCs (average rate 2.0%, range 0.4%-6.3%, n = 42), it is markedly upregulated upon exposure to 1% oxygen tension (average rate 18.2%, range 2.8%-49.4%, n = 14) and during normoxic macrophage differentiation in the presence of serum (average rate 10.1%, range 2.7%-18.8%, n = 17). The normoxic induction of SDHB RNA editing was associated with the development of dense adherent aggregates of monocytes in culture. CD14-positive monocyte isolation increased the percentages of C136U transcripts by 1.25-fold in normoxic cultures (n = 5) and 1.68-fold in hypoxic cultures (n = 4). CD14-negative lymphocytes showed no evidence of SDHB editing. The SDHB genomic DNA remained wild-type during increased RNA editing. Microarray analysis showed expression changes in wound healing and immune response pathway genes as the editing rate increased in normoxic cultures. High-throughput sequencing of SDHB and SDHD transcripts confirmed the

  15. Principles Governing A-to-I RNA Editing in the Breast Cancer Transcriptome.

    PubMed

    Fumagalli, Debora; Gacquer, David; Rothé, Françoise; Lefort, Anne; Libert, Frederick; Brown, David; Kheddoumi, Naima; Shlien, Adam; Konopka, Tomasz; Salgado, Roberto; Larsimont, Denis; Polyak, Kornelia; Willard-Gallo, Karen; Desmedt, Christine; Piccart, Martine; Abramowicz, Marc; Campbell, Peter J; Sotiriou, Christos; Detours, Vincent

    2015-10-13

    Little is known about how RNA editing operates in cancer. Transcriptome analysis of 68 normal and cancerous breast tissues revealed that the editing enzyme ADAR acts uniformly, on the same loci, across tissues. In controlled ADAR expression experiments, the editing frequency increased at all loci with ADAR expression levels according to the logistic model. Loci-specific "editabilities," i.e., propensities to be edited by ADAR, were quantifiable by fitting the logistic function to dose-response data. The editing frequency was increased in tumor cells in comparison to normal controls. Type I interferon response and ADAR DNA copy number together explained 53% of ADAR expression variance in breast cancers. ADAR silencing using small hairpin RNA lentivirus transduction in breast cancer cell lines led to less cell proliferation and more apoptosis. A-to-I editing is a pervasive, yet reproducible, source of variation that is globally controlled by 1q amplification and inflammation, both of which are highly prevalent among human cancers.

  16. Maintenance of plastid RNA editing activities independently of their target sites

    PubMed Central

    Tillich, Michael; Poltnigg, Peter; Kushnir, Sergei; Schmitz-Linneweber, Christian

    2006-01-01

    RNA editing in plant organelles is mediated by site-specific, nuclear-encoded factors. Previous data suggested that the maintenance of these factors depends on the presence of their rapidly evolving cognate sites. The surprising ability of allotetraploid Nicotiana tabacum (tobacco) to edit a foreign site in the chloroplast ndhA messenger RNA was thought to be inherited from its diploid male ancestor, Nicotiana tomentosiformis. Here, we show that the same ndhA editing activity is also present in Nicotiana sylvestris, which is the female diploid progenitor of tobacco and which lacks the ndhA site. Hence, heterologous editing is not simply a result of tobacco's allopolyploid genome organization. Analyses of other editing sites after sexual or somatic transfer between land plants showed that heterologous editing occurs at a surprisingly high frequency. This suggests that the corresponding editing activities are conserved despite the absence of their target sites, potentially because they serve other functions in the plant cell. PMID:16415790

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

    PubMed

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

    2015-12-22

    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.

  18. LINE-1 ORF1 protein enhances Alu SINE retrotransposition.

    PubMed

    Wallace, Nicholas; Wagstaff, Bradley J; Deininger, Prescott L; Roy-Engel, Astrid M

    2008-08-01

    Retroelements have contributed over one third of the human genome mass. The currently active LINE-1 (L1) codes for two proteins (ORF1p and ORF2p), both strictly required for retrotransposition. In contrast, the non-coding parasitic SINE (Alu) only appears to need the L1 ORF2p for its own amplification. This requirement was previously determined using a tissue culture assay system in human cells (HeLa). Because HeLa are likely to express functional L1 proteins, it is possible that low levels of endogenous ORF1p are necessary for the observed tagged Alu mobilization. By individually expressing ORF1 and ORF2 proteins from both human (L1RP and LRE3) and rodent (L1A102 and L1spa) L1 sources, we demonstrate that increasing amounts of ORF1 expressing vector enhances tagged Alu mobilization in HeLa cells. In addition, using chicken fibroblast cells as an alternate cell culture source, we confirmed that ORF1p is not strictly required for Alu mobilization in our assay. Supporting our observations in HeLa cells, we find that tagged Alu retrotransposition is improved by supplementation of ORF1p in the cultured chicken cells. We postulate that L1 ORF1p plays either a direct or indirect role in enhancing the interaction between the Alu RNA and the required factors needed for its retrotransposition.

  19. 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

  20. 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

  1. Targeted gene disruption identifies three PPR-DYW proteins involved in RNA editing for five editing sites of the moss mitochondrial transcripts.

    PubMed

    Ohtani, Shotaro; Ichinose, Mizuho; Tasaki, Eiji; Aoki, Yoshiaki; Komura, Yoshihiro; Sugita, Mamoru

    2010-11-01

    In plant organelles, RNA editing frequently occurs in many transcripts, but little is known about its molecular mechanism. Eleven RNA editing sites are present in the moss Physcomitrella patens mitochondria. Recently PpPPR_71, one member of 10 DYW-subclass pentatricopeptide repeat (PPR-DYW) proteins, has been identified as a site-specific recognition factor for RNA editing in the mitochondrial transcript. In this study, we disrupted three genes encoding a PPR-DYW protein-PpPPR_56, PpPPR_77, and PpPPR_91-to investigate whether they are involved in RNA editing. Transient expression of an N-terminal amino acid sequence fused to the green fluorescent protein (GFP) suggests that the three PPR-DYW proteins are targeted to mitochondria. Disruption of each gene by homologous recombination revealed that PpPPR_56 was involved in RNA editing at the nad3 and nad4 sites, PpPPR_77 at the cox2 and cox3 sites, and PpPPR_91 at the nad5-2 site in the mitochondrial transcripts. The nucleotide sequences surrounding the two editing sites targeted by a single PPR-DYW protein share 42 to 56% of their identities. Thus, moss PPR-DYW proteins seem to be site-specific factors for RNA editing in mitochondrial transcripts.

  2. The involvement of a PPR protein of the P subfamily in partial RNA editing of an Arabidopsis mitochondrial transcript.

    PubMed

    Doniwa, Yoko; Ueda, Minoru; Ueta, Masami; Wada, Akira; Kadowaki, Koh-ichi; Tsutsumi, Nobuhiro

    2010-04-01

    C-to-U RNA editing (i.e., alteration of a C in the genomic sequence to U in the transcript) has been confirmed widely in angiosperm organellar genomes. During the C-to-U RNA editing event, incomplete edited transcripts have been observed at many sites in the steady-state mRNA population (partial editing). Here, by using coexpression analysis and the surveillance of whole editing status on the mitochondrial genome, we have revealed that a pentatricopeptide repeat (PPR) protein classified into the P subfamily (PPR596) has site-specific influence on the efficiency of C-to-U RNA editing events at partial editing sites on the Arabidopsis thaliana mitochondrial genome. Previous works have revealed that PPR proteins classified into the PLS subfamily containing the E or E and DYW motif are involved in RNA editing as trans-factors; they are believed to recruit deaminase at editing sites. In contrast with the mutant analyses of PLS-subfamily PPR proteins, the editing efficiency at rps3eU1344SS was revealed to be significantly increased in ppr596 mutants. Our study implies P-subfamily PPR protein is involved in the control of the degree of partial editing.

  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

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

    2005-09-01

    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.

  5. The genetic basis for individual differences in mRNA splicing and APOBEC1 editing activity in murine macrophages

    PubMed Central

    Hassan, Musa A.; Butty, Vincent; Jensen, Kirk D.C.; Saeij, Jeroen P.J.

    2014-01-01

    Alternative splicing and mRNA editing are known to contribute to transcriptome diversity. Although alternative splicing is pervasive and contributes to a variety of pathologies, including cancer, the genetic context for individual differences in isoform usage is still evolving. Similarly, although mRNA editing is ubiquitous and associated with important biological processes such as intracellular viral replication and cancer development, individual variations in mRNA editing and the genetic transmissibility of mRNA editing are equivocal. Here, we have used linkage analysis to show that both mRNA editing and alternative splicing are regulated by the macrophage genetic background and environmental cues. We show that distinct loci, potentially harboring variable splice factors, regulate the splicing of multiple transcripts. Additionally, we show that individual genetic variability at the Apobec1 locus results in differential rates of C-to-U(T) editing in murine macrophages; with mouse strains expressing mostly a truncated alternative transcript isoform of Apobec1 exhibiting lower rates of editing. As a proof of concept, we have used linkage analysis to identify 36 high-confidence novel edited sites. These results provide a novel and complementary method that can be used to identify C-to-U editing sites in individuals segregating at specific loci and show that, beyond DNA sequence and structural changes, differential isoform usage and mRNA editing can contribute to intra-species genomic and phenotypic diversity. PMID:24249727

  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. Integrative analyses of RNA editing, alternative splicing, and expression of young genes in human brain transcriptome by deep RNA sequencing.

    PubMed

    Wu, Dong-Dong; Ye, Ling-Qun; Li, Yan; Sun, Yan-Bo; Shao, Yi; Chen, Chunyan; Zhu, Zhu; Zhong, Li; Wang, Lu; Irwin, David M; Zhang, Yong E; Zhang, Ya-Ping

    2015-08-01

    Next-generation RNA sequencing has been successfully used for identification of transcript assembly, evaluation of gene expression levels, and detection of post-transcriptional modifications. Despite these large-scale studies, additional comprehensive RNA-seq data from different subregions of the human brain are required to fully evaluate the evolutionary patterns experienced by the human brain transcriptome. Here, we provide a total of 6.5 billion RNA-seq reads from different subregions of the human brain. A significant correlation was observed between the levels of alternative splicing and RNA editing, which might be explained by a competition between the molecular machineries responsible for the splicing and editing of RNA. Young human protein-coding genes demonstrate biased expression to the neocortical and non-neocortical regions during evolution on the lineage leading to humans. We also found that a significantly greater number of young human protein-coding genes are expressed in the putamen, a tissue that was also observed to have the highest level of RNA-editing activity. The putamen, which previously received little attention, plays an important role in cognitive ability, and our data suggest a potential contribution of the putamen to human evolution.

  8. 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

  9. RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling.

    PubMed

    Shimokawa, Takashi; Rahman, Mohammed Ferdous-Ur; Tostar, Ulrica; Sonkoly, Enikö; Ståhle, Mona; Pivarcsi, Andor; Palaniswamy, Ramesh; Zaphiropoulos, Peter G

    2013-02-01

    The Hedgehog (HH) signaling pathway has important roles in tumorigenesis and in embryonal patterning. The Glioma-associated oncogene 1 (GLI1) is a key molecule in HH signaling, acting as a transcriptional effector and, moreover, is considered to be a potential therapeutic target for several types of cancer. To extend our previous focus on the implications of alternative splicing for HH signal transduction, we now report on an additional post-transcriptional mechanism with an impact on GLI1 activity, namely RNA editing. The GLI1 mRNA is highly edited at nucleotide 2179 by adenosine deamination in normal cerebellum, but the extent of this modification is reduced in cell lines from the cerebellar tumor medulloblastoma. Additionally, basal cell carcinoma tumor samples exhibit decreased GLI1 editing compared with normal skin. Interestingly, knocking down of either ADAR1 or ADAR2 reduces RNA editing of GLI1. This adenosine to inosine substitution leads to a change from Arginine to Glycine at position 701 that influences not only GLI1 transcriptional activity, but also GLI1-dependent cellular proliferation. Specifically, the edited GLI1, GLI1-701G, has a higher capacity to activate most of the transcriptional targets tested and is less susceptible to inhibition by the negative regulator of HH signaling suppressor of fused. However, the Dyrk1a kinase, implicated in cellular proliferation, is more effective in increasing the transcriptional activity of the non-edited GLI1. Finally, introduction of GLI1-701G into medulloblastoma cells confers a smaller increase in cellular growth relative to GLI1. In conclusion, our findings indicate that RNA editing of GLI1 is a regulatory mechanism that modulates the output of the HH signaling pathway. PMID:23324600

  10. Perturbing A-to-I RNA editing using genetics and homologous recombination.

    PubMed

    Staber, Cynthia J; Gell, Selena; Jepson, James E C; Reenan, Robert A

    2011-01-01

    Evidence for the chemical conversion of adenosine-to-inosine (A-to-I) in messenger RNA (mRNA) has been detected in numerous metazoans, especially those "most successful" phyla: Arthropoda, Mollusca, and Chordata. The requisite enzymes for A-to-I editing, ADARs (adenosine deaminases acting on RNA) are highly conserved and are present in every higher metazoan genome sequenced to date. The fruit fly, Drosophila melanogaster, represents an ideal model organism for studying A-to-I editing, both in terms of fundamental biochemistry and in relation to determining adaptive downstream effects on physiology and behavior. The Drosophila genome contains a single structural gene for ADAR (dAdar), yet the fruit fly transcriptome has the widest range of conserved and validated ADAR targets in coding mRNAs of any known organism. In addition, many of the genes targeted by dADAR have been genetically identified as playing a role in nervous system function, providing a rich source of material to investigate the biological relevance of this intriguing process. Here, we discuss how recent advances in the use of ends-out homologous recombination (HR) in Drosophila make possible both the precise control of the editing status for defined adenosine residues and the engineering of flies with globally altered RNA editing of the fly transcriptome. These new approaches promise to significantly improve our understanding of how mRNA modification contributes to insect physiology and ethology.

  11. 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.

  12. RNA editing underlies temperature adaptation in K+ channels from polar octopuses.

    PubMed

    Garrett, Sandra; Rosenthal, Joshua J C

    2012-02-17

    To operate in the extreme cold, ion channels from psychrophiles must have evolved structural changes to compensate for their thermal environment. A reasonable assumption would be that the underlying adaptations lie within the encoding genes. Here, we show that delayed rectifier K(+) channel genes from an Antarctic and a tropical octopus encode channels that differ at only four positions and display very similar behavior when expressed in Xenopus oocytes. However, the transcribed messenger RNAs are extensively edited, creating functional diversity. One editing site, which recodes an isoleucine to a valine in the channel's pore, greatly accelerates gating kinetics by destabilizing the open state. This site is extensively edited in both Antarctic and Arctic species, but mostly unedited in tropical species. Thus adenosine-to-inosine RNA editing can respond to the physical environment.

  13. 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

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

    PubMed

    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. 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

  16. 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

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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

  5. Messenger RNA editing in mammals: new members of the APOBEC family seeking roles in the family business.

    PubMed

    Wedekind, Joseph E; Dance, Geoffrey S C; Sowden, Mark P; Smith, Harold C

    2003-04-01

    Alteration of mRNA sequence through base modification mRNA editing frequently generates protein diversity. Several proteins have been identified as being similar to C-to-U mRNA editing enzymes based on their structural domains and the occurrence of a catalytic domain characteristic of cytidine deaminases. In light of the hypothesis that these proteins might represent novel mRNA editing systems that could affect proteome diversity, we consider their structure, expression and relevance to biomedically significant processes or pathologies. PMID:12683974

  6. The longest mitochondrial RNA editing PPR protein MEF12 in Arabidopsis thaliana requires the full-length E domain.

    PubMed

    Härtel, Barbara; Zehrmann, Anja; Verbitskiy, Daniil; Takenaka, Mizuki

    2013-01-01

    Mitochondrial RNA editing factor 12 (MEF12) was identified in a screen for editing defects of a chemically mutated plant population in Arabidopsis thaliana. The MEF12 editing protein is required for the C to U change of nucleotide nad5-374. The MEF12 polypeptide is characterized by an exceptionally long stretch of 25 pentatricopeptide repeats (PPR) and a C-terminal extension domain. Editing is lost in mutant plants with a stop codon in the extending element. A T-DNA insertion substituting the 10 C-terminal amino acids of the extension domain reduces RNA editing to about 20% at the target site in a mutant plant. These results support the importance of the full-length extension module for functional RNA editing in plant mitochondria.

  7. RNA editing of hepatitis B virus transcripts by activation-induced cytidine deaminase.

    PubMed

    Liang, Guoxin; Kitamura, Kouichi; Wang, Zhe; Liu, Guangyan; Chowdhury, Sajeda; Fu, Weixin; Koura, Miki; Wakae, Kousho; Honjo, Tasuku; Muramatsu, Masamichi

    2013-02-01

    Activation-induced cytidine deaminase (AID) is essential for the somatic hypermutation (SHM) and class-switch recombination (CSR) of Ig genes. The mechanism by which AID triggers SHM and CSR has been explained by two distinct models. In the DNA deamination model, AID converts cytidine bases in DNA into uridine. The uridine is recognized by the DNA repair system, which produces DNA strand breakages and point mutations. In the alternative model, RNA edited by AID is responsible for triggering CSR and SHM. However, RNA deamination by AID has not been demonstrated. Here we found that C-to-T and G-to-A mutations accumulated in hepatitis B virus (HBV) nucleocapsid DNA when AID was expressed in HBV-replicating hepatic cell lines. AID expression caused C-to-T mutations in the nucleocapsid DNA of RNase H-defective HBV, which does not produce plus-strand viral DNA. Furthermore, the RT-PCR products of nucleocapsid viral RNA from AID-expressing cells exhibited significant C-to-T mutations, whereas viral RNAs outside the nucleocapsid did not accumulate C-to-U mutations. Moreover, AID was packaged within the nucleocapsid by forming a ribonucleoprotein complex with HBV RNA and the HBV polymerase protein. The encapsidation of the AID protein with viral RNA and DNA provides an efficient environment for evaluating AID's RNA and DNA deamination activities. A bona fide RNA-editing enzyme, apolipoprotein B mRNA editing catalytic polypeptide 1, induced a similar level of C-to-U mutations in nucleocapsid RNA as AID. Taken together, the results indicate that AID can deaminate the nucleocapsid RNA of HBV.

  8. 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

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

    PubMed

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

    2014-03-01

    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.

  10. Pentatricopeptide Repeat Proteins with the DYW Motif Have Distinct Molecular Functions in RNA Editing and RNA Cleavage in Arabidopsis Chloroplasts[W

    PubMed Central

    Okuda, Kenji; Chateigner-Boutin, Anne-Laure; Nakamura, Takahiro; Delannoy, Etienne; Sugita, Mamoru; Myouga, Fumiyoshi; Motohashi, Reiko; Shinozaki, Kazuo; Small, Ian; Shikanai, Toshiharu

    2009-01-01

    The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing. PMID:19182104

  11. The SLO1 PPR protein is required for RNA editing at multiple sites with similar upstream sequences in Arabidopsis mitochondria.

    PubMed

    Sung, Tzu-Ying; Tseng, Ching-Chih; Hsieh, Ming-Hsiun

    2010-08-01

    In Arabidopsis, RNA editing changes more than 500 cytidines to uridines in mitochondrial transcripts. The editing enzyme and co-factors involved in these processes are largely unknown. We have identified a nuclear gene SLOW GROWTH1 (SLO1) encoding an E motif-containing pentatricopeptide repeat protein that is required for RNA editing of nad4 and nad9 in Arabidopsis mitochondria. The SLO1 protein is localized to the mitochondrion, and its absence gives rise to small plants with slow growth and delayed development. A survey of approximately 500 mitochondrial RNA editing sites in Arabidopsis reveals that the editing of two sites, nad4-449 and nad9-328, is abolished in the slo1 mutants. Sequence comparison in the upstream (from -1 to -15 bp) of nad4-449 and nad9-328 editing sites shows that nine of the 15 nucleotides are identical. In addition to RNA editing, we used RNA gel blot analysis to compare the abundance and banding patterns of mitochondrial transcripts between the wild type and slo1 mutants. Of the 79 genes and open reading frames examined, steady-state levels of 56 mitochondrial transcripts are increased in the slo1 mutants. These results suggest that the SLO1 protein may indirectly regulate plant growth and development via affecting mitochondrial RNA editing and gene expression.

  12. Nuclear DYW-type PPR gene families diversify with increasing RNA editing frequencies in liverwort and moss mitochondria.

    PubMed

    Rüdinger, Mareike; Volkmar, Ute; Lenz, Henning; Groth-Malonek, Milena; Knoop, Volker

    2012-02-01

    RNA editing in mitochondria and chloroplasts of land plants alters transcript sequences by site-specific conversions of cytidines into uridines. RNA editing frequencies vary extremely between land plant clades, ranging from zero in some liverworts to more than 2,000 sites in lycophytes. Unique pentatricopeptide repeat (PPR) proteins with variable domain extension (E/E+/DYW) have recently been identified as specific editing site recognition factors in model plants. The distinctive functions of these PPR protein domain additions have remained unclear, although deaminase function has been proposed for the DYW domain. To shed light on diversity of RNA editing and DYW proteins at the origin of land plant evolution, we investigated editing patterns of the mitochondrial nad5, nad4, and nad2 genes in a wide sampling of more than 100 liverworts and mosses using the recently developed PREPACT program (www.prepact.de) and exemplarily confirmed predicted RNA editing sites in selected taxa. Extreme variability in RNA editing frequency is seen both in liverworts and mosses. Only few editings exist in the liverwort Lejeunea cavifolia or the moss Pogonatum urnigerum whereas up to 20% of cytidines are edited in the liverwort Haplomitrium mnioides or the moss Takakia lepidozioides. Interestingly, the latter are taxa that branch very early within their respective clades. Amplicons targeting the E/E+/DYW domains and subsequent random clone sequencing show DYW domains among bryophytes to be highly conserved in comparison with their angiosperm counterparts and to correlate well with RNA editing frequencies regarding their diversities. We propose that DYW proteins are the key players of RNA editing at the origin of land plants.

  13. 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

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

    PubMed Central

    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

    2016-01-01

    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

  15. 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

  16. 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

  17. 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

  18. 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

  19. RNA editing and regulation of Drosophila 4f-rnp expression by sas-10 antisense readthrough mRNA transcripts.

    PubMed

    Peters, Nick T; Rohrbach, Justin A; Zalewski, Brian A; Byrkett, Colleen M; Vaughn, Jack C

    2003-06-01

    We have previously described an example of extensively A-to-G edited cDNA derived from adult heads of the fruitfly Drosophila melanogaster. In that study, the source of the predicted antisense RNA pairing strand for template recognition by dADAR editase was not identified, and the biological significance of the observed hyperediting was not known. Here, we address each of these questions. 4f-rnp and sas-10 are closely adjacent X-linked genes located on opposite DNA strands that produce convergent transcripts. We show that developmentally regulated antisense sas-10 readthrough mRNA arises by activation of an upstream promoter P2 during the late embryo stage of fly development. The sas-10 readthrough transcripts pair with 4f-rnp mRNA to form double-stranded molecules, as indicated by A-to-G editing observed in both RNA strands. It would be predicted that perfect RNA duplexes would be targeted for modification/degradation by enzyme pathways that recognize double-stranded RNAs, leading to decline in 4f-rnp mRNA levels, and this is what we observe. The observation using quantitative RT-PCR that sas-10 readthrough and 4f-rnp transcript levels are inversely related suggests a role for the antisense RNA in posttranscriptional regulation of 4f-rnp gene expression during development. Potential molecular mechanisms that could lead to this result are discussed, one of which is targeted transcript degradation via the RNAi pathway. Insofar as the dADAR editase and RNAi pathways are known to be constitutive in this system, it is likely that control of antisense RNA transcription is the rate-limiting factor. The results provide insight into roles of naturally occurring antisense RNAs in regulation of eukaryotic gene expression.

  20. Reverse genetic screening identifies five E-class PPR proteins involved in RNA editing in mitochondria of Arabidopsis thaliana.

    PubMed

    Takenaka, Mizuki; Verbitskiy, Daniil; Zehrmann, Anja; Brennicke, Axel

    2010-08-27

    RNA editing in flowering plant mitochondria post-transcriptionally alters several hundred nucleotides from C to U, mostly in mRNAs. Several factors required for specific RNA-editing events in plant mitochondria and plastids have been identified, all of them PPR proteins of the PLS subclass with a C-terminal E-domain and about half also with an additional DYW domain. Based on this information, we here probe the connection between E-PPR proteins and RNA editing in plant mitochondria. We initiated a reverse genetics screen of T-DNA insertion lines in Arabidopsis thaliana and investigated 58 of the 150 E-PPR-coding genes for a function in RNA editing. Six genes were identified to be involved in mitochondrial RNA editing at specific sites. Homozygous mutants of the five genes MEF18-MEF22 display no gross disturbance in their growth or development patterns, suggesting that the editing sites affected are not crucial at least in the greenhouse. These results show that a considerable percentage of the E-PPR proteins are involved in the functional processing of site-specific RNA editing in plant mitochondria.

  1. Crystal structure of a signal recognition particle Alu domain in the elongation arrest conformation.

    PubMed

    Bousset, Luc; Mary, Camille; Brooks, Mark A; Scherrer, Anne; Strub, Katharina; Cusack, Stephen

    2014-12-01

    The signal recognition particle (SRP) is a conserved ribonucleoprotein particle that targets membrane and secreted proteins to translocation channels in membranes. In eukaryotes, the Alu domain, which comprises the 5' and 3' extremities of the SRP RNA bound to the SRP9/14 heterodimer, is thought to interact with the ribosome to pause translation elongation during membrane docking. We present the 3.2 Å resolution crystal structure of a chimeric Alu domain, comprising Alu RNA from the archaeon Pyrococcus horikoshii bound to the human Alu binding proteins SRP9/14. The structure reveals how intricate tertiary interactions stabilize the RNA 5' domain structure and how an extra, archaeal-specific, terminal stem helps constrain the Alu RNA into the active closed conformation. In this conformation, highly conserved noncanonical base pairs allow unusually tight side-by-side packing of 5' and 3' RNA stems within the SRP9/14 RNA binding surface. The biological relevance of this structure is confirmed by showing that a reconstituted full-length chimeric archaeal-human SRP is competent to elicit elongation arrest in vitro. The structure will be useful in refining our understanding of how the SRP Alu domain interacts with the ribosome.

  2. 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

  3. 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.

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

    PubMed

    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.

  5. 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

  6. Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader-Willi Syndrome

    PubMed Central

    Morabito, Michael V.; Abbas, Atheir I.; Hood, Jennifer L.; Kesterson, Robert A.; Jacobs, Michelle M.; Kump, David S.; Hachey, David L.; Roth, Bryan L.; Emeson, Ronald B.

    2010-01-01

    RNA transcripts encoding the 2C-subtype of serotonin (5HT2C) receptor undergo up to five adenosine-to-inosine editing events to encode twenty-four protein isoforms. To examine the effects of altered 5HT2C editing in vivo, we generated mutant mice solely expressing the fully-edited (VGV) isoform of the receptor. Mutant animals present phenotypic characteristics of Prader-Willi Syndrome (PWS) including a failure to thrive, decreased somatic growth, neonatal muscular hypotonia, and reduced food consumption followed by post-weaning hyperphagia. Though previous studies have identified alterations in both 5HT2C receptor expression and 5HT2C-mediated behaviors in both PWS patients and mouse models of this disorder, to our knowledge the 5HT2C gene is the first locus outside the PWS imprinted region in which mutations can phenocopy numerous aspects of this syndrome. These results not only strengthen the link between the molecular etiology of PWS and altered 5HT2C expression, but also demonstrate the importance of normal patterns of 5HT2C RNA editing in vivo. PMID:20394819

  7. Requirement of the RNA-editing Enzyme ADAR2 for Normal Physiology in Mice*

    PubMed Central

    Horsch, Marion; Seeburg, Peter H.; Adler, Thure; Aguilar-Pimentel, Juan Antonio; Becker, Lore; Calzada-Wack, Julia; Garrett, Lilian; Götz, Alexander; Hans, Wolfgang; Higuchi, Miyoko; Hölter, Sabine M.; Naton, Beatrix; Prehn, Cornelia; Puk, Oliver; Rácz, Ildikó; Rathkolb, Birgit; Rozman, Jan; Schrewe, Anja; Adamski, Jerzy; Busch, Dirk H.; Esposito, Irene; Graw, Jochen; Ivandic, Boris; Klingenspor, Martin; Klopstock, Thomas; Mempel, Martin; Ollert, Markus; Schulz, Holger; Wolf, Eckhard; Wurst, Wolfgang; Zimmer, Andreas; Gailus-Durner, Valérie; Fuchs, Helmut; de Angelis, Martin Hrabě; Beckers, Johannes

    2011-01-01

    ADAR2, an RNA editing enzyme that converts specific adenosines to inosines in certain pre-mRNAs, often leading to amino acid substitutions in the encoded proteins, is mainly expressed in brain. Of all ADAR2-mediated edits, a single one in the pre-mRNA of the AMPA receptor subunit GluA2 is essential for survival. Hence, early postnatal death of mice lacking ADAR2 is averted when the critical edit is engineered into both GluA2 encoding Gria2 alleles. Adar2−/−/Gria2R/R mice display normal appearance and life span, but the general phenotypic effects of global lack of ADAR2 have remained unexplored. Here we have employed the Adar2−/−/Gria2R/R mouse line, and Gria2R/R mice as controls, to study the phenotypic consequences of loss of all ADAR2-mediated edits except the critical one in GluA2. Our extended phenotypic analysis covering ∼320 parameters identified significant changes related to absence of ADAR2 in behavior, hearing ability, allergy parameters and transcript profiles of brain. PMID:21467037

  8. Uridylate-specific 3' 5'-exoribonucleases involved in uridylate-deletion RNA editing in trypanosomatid mitochondria.

    PubMed

    Rogers, Kestrel; Gao, Guanghan; Simpson, Larry

    2007-10-01

    In kinetoplastid protists, maturation of mitochondrial pre-mRNAs involves the insertion and deletion of uridylates (Us) within coding regions, as specified by mitochondrial DNA-encoded guide RNAs. U-deletion editing involves endonucleolytic cleavage of the pre-mRNA at the editing site followed by U-specific 3'-5'-exonucleolytic removal of nonbase-paired Us prior to ligation of the two mRNA cleavage fragments. We showed previously that an exonuclease/endonuclease/phosphatase (EEP) motif protein from Leishmania major, designated RNA editing exonuclease 1 (REX1) (Kang, X., Rogers, K., Gao, G., Falick, A. M., Zhou, S.-L., and Simpson, L. (2005) Proc. Natl. Acad. Sci. U. S. A. 102, 1017-1022), exhibits 3'-5'-exonuclease activity. Two EEP motif proteins have also been identified in the Trypanosoma brucei editing complex. TbREX1 is a homologue of LmREX1, and TbREX2 shows homology to another editing protein in L. major, which lacks the EEP motif (LmREX2*). Here we have expressed the T. brucei EEP motif proteins in insect cells and purified them to homogeneity. We showed that these are U-specific 3'-5'-exonucleases that are inhibited by base pairing of 3' Us. The recombinant EEP motif alone also showed 3'-5' U-specific exonuclease activity, and mutations of the REX EEP motifs greatly reduced exonuclease activity. The absence of enzymatic activity in LmREX2* was confirmed with a purified recombinant protein. We showed that pre-cleaved U-deletion editing could be reconstituted with either TbREX1 or TbREX2 in combination with either RNA ligase, LmREL1, or LmREL2. Down-regulation of TbREX2 expression by conditional RNA interference had little effect on parasite viability or sedimentation of the L-complex, suggesting either that TbREX2 is inactive in vivo or that TbREX1 can compensate for the loss of TbREX2 function in down-regulated cells.

  9. Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing

    PubMed Central

    Esvelt, Kevin M.; Mali, Prashant; Braff, Jonathan L.; Moosburner, Mark; Yaung, Stephanie J.; Church, George M.

    2013-01-01

    The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences and identify a highly targetable protein from Neisseria meningitidis. Our results provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins and adapting them to eukaryotic cells. PMID:24076762

  10. 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.

  11. 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.

  12. From End to End: tRNA Editing at 5'- and 3'-Terminal Positions

    PubMed Central

    Betat, Heike; Long, Yicheng; Jackman, Jane E.; Mörl, Mario

    2014-01-01

    During maturation, tRNA molecules undergo a series of individual processing steps, ranging from exo- and endonucleolytic trimming reactions at their 5'- and 3'-ends, specific base modifications and intron removal to the addition of the conserved 3'-terminal CCA sequence. Especially in mitochondria, this plethora of processing steps is completed by various editing events, where base identities at internal positions are changed and/or nucleotides at 5'- and 3'-ends are replaced or incorporated. In this review, we will focus predominantly on the latter reactions, where a growing number of cases indicate that these editing events represent a rather frequent and widespread phenomenon. While the mechanistic basis for 5'- and 3'-end editing differs dramatically, both reactions represent an absolute requirement for generating a functional tRNA. Current in vivo and in vitro model systems support a scenario in which these highly specific maturation reactions might have evolved out of ancient promiscuous RNA polymerization or quality control systems. PMID:25535083

  13. 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

  14. Targeted genome editing of sweet orange using Cas9/sgRNA.

    PubMed

    Jia, Hongge; Wang, Nian

    2014-01-01

    Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system-a system that holds significant promise for the study of citrus gene function and for targeted genetic modification.

  15. Targeted Genome Editing of Sweet Orange Using Cas9/sgRNA

    PubMed Central

    Jia, Hongge; Wang, Nian

    2014-01-01

    Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance. Targeted genome engineering is expected to contribute significantly to future varietal improvement, and genome editing technologies using zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9/single guide RNA (sgRNA) have already been successfully used to genetically modify plants. However, to date, there has been no reported use of any of the current genome editing approaches in sweet orange, an important fruit crop. In this study, we first developed a novel tool, Xcc-facilitated agroinfiltration, for enhancing transient protein expression in sweet orange leaves. We then successfully employed Xcc-facilitated agroinfiltration to deliver Cas9, along with a synthetic sgRNA targeting the CsPDS gene, into sweet orange. DNA sequencing confirmed that the CsPDS gene was mutated at the target site in treated sweet orange leaves. The mutation rate using the Cas9/sgRNA system was approximately 3.2 to 3.9%. Off-target mutagenesis was not detected for CsPDS-related DNA sequences in our study. This is the first report of targeted genome modification in citrus using the Cas9/sgRNA system—a system that holds significant promise for the study of citrus gene function and for targeted genetic modification. PMID:24710347

  16. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

    PubMed

    Gasiunas, Giedrius; Siksnys, Virginijus

    2013-11-01

    Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects.

  17. 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.

  18. Impact of RNA Editing on Functions of the Serotonin 2C Receptor in vivo

    PubMed Central

    Olaghere da Silva, Uade B.; Morabito, Michael V.; Canal, Clinton E.; Airey, David C.; Emeson, Ronald B.; Sanders-Bush, Elaine

    2009-01-01

    Transcripts encoding 5-HT2C receptors are modified posttranscriptionally by RNA editing, generating up to 24 protein isoforms. In recombinant cells, the fully edited isoform, 5-HT2C-VGV, exhibits blunted G-protein coupling and reduced constitutive activity. The present studies examine the signal transduction properties of 5-HT2C-VGV receptors in brain to determine the in vivo consequences of altered editing. Using mice solely expressing the 5-HT2C-VGV receptor (VGV/Y), we demonstrate reduced G-protein coupling efficiency and high-affinity agonist binding of brain 5-HT2C-VGV receptors. However, enhanced behavioral sensitivity to a 5-HT2C receptor agonist was also seen in mice expressing 5-HT2C-VGV receptors, an unexpected finding given the blunted G-protein coupling. In addition, mice expressing 5-HT2C-VGV receptors had greater sensitivity to a 5-HT2C inverse agonist/antagonist enhancement of dopamine turnover relative to wild-type mice. These behavioral and biochemical results are most likely explained by increases in 5-HT2C receptor binding sites in the brains of mice solely expressing 5-HT2C-VGV receptors. We conclude that 5-HT2C-VGV receptor signaling in brain is blunted, but this deficiency is masked by a marked increase in 5-HT2C receptor binding site density in mice solely expressing the VGV isoform. These findings suggest that RNA editing may regulate the density of 5-HT2C receptor binding sites in brain. We further caution that the pattern of 5-HT2C receptor RNA isoforms may not reflect the pattern of protein isoforms, and hence the inferred overall function of the receptor. PMID:20582266

  19. 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

  20. 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

  1. 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

  2. Assigning DYW-type PPR proteins to RNA editing sites in the funariid mosses Physcomitrella patens and Funaria hygrometrica.

    PubMed

    Rüdinger, Mareike; Szövényi, Péter; Rensing, Stefan A; Knoop, Volker

    2011-07-01

    The plant-specific pentatricopeptide repeat (PPR) proteins with variable PPR repeat lengths (PLS-type) and protein extensions up to the carboxyterminal DYW domain have received attention as specific recognition factors for the C-to-U type of RNA editing events in plant organelles. Here, we report a DYW-protein knockout in the model plant Physcomitrella patens specifically affecting mitochondrial RNA editing positions cox1eU755SL and rps14eU137SL. Assignment of DYW proteins and RNA editing sites might best be corroborated by data from a taxon with a slightly different, yet similarly manageable low number of editing sites and DYW proteins. To this end we investigated the mitochondrial editing status of the related funariid moss Funaria hygrometrica. We find that: (i) Funaria lacks three mitochondrial RNA editing positions present in Physcomitrella, (ii) that F. hygrometrica cDNA sequence data identify nine DYW proteins as clear orthologues of their P. patens counterparts, and (iii) that the 'missing' 10th DYW protein in F. hygrometrica is responsible for two mitochondrial editing sites in P. patens lacking in F. hygrometrica (nad3eU230SL, nad4eU272SL). Interestingly, the third site of RNA editing missing in F. hygrometrica (rps14eU137SL) is addressed by the DYW protein characterized here and the presence of its orthologue in F. hygrometrica is explained through its simultaneous action on site cox1eU755SL conserved in both mosses.

  3. 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

  4. 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

  5. The PPR-DYW proteins are required for RNA editing of rps14, cox1 and nad5 transcripts in Physcomitrella patens mitochondria.

    PubMed

    Uchida, Masato; Ohtani, Shotaro; Ichinose, Mizuho; Sugita, Chieko; Sugita, Mamoru

    2011-07-21

    We identified two DYW subclass pentatricopeptide repeat (PPR) proteins, PpPPR_78 and PpPPR_79, as RNA editing factors in the moss Physcomitrella patens. Disruption of each gene by homologous recombination revealed that PpPPR_78 was involved in RNA editing at the rps14 (rps14-C137) and cox1 (cox1-C755) sites and PpPPR_79 at the nad5-1 (nad5-C598) site in the mitochondrial transcripts. RNA editing defects did not affect transcript patterns of the target genes. Thus, DYW subclass PPR proteins seem to be site-specific trans-acting factors for RNA editing.

  6. Nuclear genes from Tx CMS maintainer lines are unable to maintain atp6 RNA editing in any anther cell-type in the sorghum bicolor A3 cytoplasm.

    PubMed

    Howad, W; Tang, H V; Pring, D R; Kempken, F

    1999-08-01

    RNA editing and cytoplasmic male sterility are two important phenomena associated with higher plant mitochondria. We recently have shown a potential function of RNA editing in CMS development. The frequency of atp6 RNA editing was specifically reduced in anthers of male-sterile Sorghum bicolor, which increased in frequency in partially restored progeny. Here we present data that show that the loss of RNA editing capability also occurs in a second nuclear background that allows the expression of male sterility. Loss of RNA editing thus appears to be associated with unique combinations of male-sterile cytoplasm and non-restoring nuclear backgrounds. In addition, the reduction of RNA editing affects both gametophytic and sporophytic anther cell-types but not other floral tissues. An analysis of F(2) plants exhibiting different levels of fertility indicates a co-segregation of fertility restoration and atp6 RNA editing. The atp6 transcript abundance is similar in seedlings and anthers of male-sterile, partially restored, and male-fertile lines and thus is not associated with loss of atp6 RNA editing in anthers. A model for RNA editing and male sterility based on the data available is presented. Functional correlations with other CMS systems are also discussed.

  7. 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

  8. [CRISPR/Cas: a novel way of RNA-guided genome editing].

    PubMed

    Li, Jun; Zhang, Yi; Chen, Kun-Ling; Shan, Qi-Wei; Wang, Yan-Peng; Liang, Zhen; Gao, Cai-Xia

    2013-11-01

    Bacteria and archaea have evolved an adaptive immune system, known as type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which uses short RNA to direct the degradation of target sequences present in invading viral and plasmid DNAs. Recent advances in CRISPR/Cas system provide an improved method for genome editing, showing robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci. It is the latest technology to modify genome DNA specifically and effectively following zinc finger nucleases (ZFNs) and TALE nucleases (TALENs). Compared with ZFNs and TALENs, CRISPR/Cas is much simpler and easier to engineer. This review summarizes recent progress, and discusses the prospects of CRISPR/Cas system, with an emphasis on its structure, principle, applications and potential challenges.

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

    PubMed

    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.

  10. 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

  11. RNA editing of the GABA(A) receptor alpha3 subunit alters the functional properties of recombinant receptors.

    PubMed

    Nimmich, Mitchell L; Heidelberg, Laura S; Fisher, Janet L

    2009-04-01

    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the alpha3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors containing either the edited or unedited forms of the alpha3 subunit along with beta3 and gamma2L. Editing substantially altered the GABA sensitivity and deactivation rate of the receptors, with the unedited form showing a lower GABA EC50 and slower decay. Comparable effects were observed with a mutation at the homologous location in the alpha1 subunit, suggesting a common role for this site in regulation of channel gating. Except for the response to GABA, the pharmacological properties of the receptor were unaffected by editing, with similar enhancement by a variety of modulators. Since RNA editing of the alpha3 subunit increases through development, our findings suggest that GABAergic neurotransmission may be more effective early in development, with greater GABA sensitivity and slower decay rates conferred by the unedited alpha3 subunit.

  12. Empty pericarp5 encodes a pentatricopeptide repeat protein that is required for mitochondrial RNA editing and seed development in maize.

    PubMed

    Liu, Yu-Jun; Xiu, Zhi-Hui; Meeley, Robert; Tan, Bao-Cai

    2013-03-01

    In flowering plants, RNA editing is a posttranscriptional mechanism that converts specific cytidines to uridines in both mitochondrial and plastidial transcripts, altering the information encoded by these genes. Here, we report the molecular characterization of the empty pericarp5 (emp5) mutants in maize (Zea mays). Null mutation of Emp5 results in abortion of embryo and endosperm development at early stages. Emp5 encodes a mitochondrion-targeted DYW subgroup pentatricopeptide repeat (PPR) protein. Analysis of the mitochondrial transcripts revealed that loss of the EMP5 function abolishes the C-to-U editing of ribosomal protein L16 at the rpl16-458 site (100% edited in the wild type), decreases the editing at nine sites in NADH dehydrogenase9 (nad9), cytochrome c oxidase3 (cox3), and ribosomal protein S12 (rps12), and surprisingly increases the editing at five sites of ATP synthase F0 subunit a (atp6), apocytochrome b (cob), nad1, and rpl16. Mutant EMP5-4 lacking the E+ and DYW domains still retains the substrate specificity and editing function, only at reduced efficiency. This suggests that the E+ and DYW domains of EMP5 are not essential to the EMP5 editing function but are necessary for efficiency. Analysis of the ortholog in rice (Oryza sativa) indicates that rice EMP5 has a conserved function in C-to-U editing of the rice mitochondrial rpl16-458 site. EMP5 knockdown expression in transgenics resulted in slow growth and defective seeds. These results demonstrate that Emp5 encodes a PPR-DYW protein that is required for the editing of multiple transcripts in mitochondria, and the editing events, particularly the C-to-U editing at the rpl16-458 site, are critical to the mitochondrial functions and, hence, to seed development in maize. PMID:23463776

  13. RNA editing of the transcript coding for subunit 4 of NADH dehydrogenase in wheat mitochondria: uneven distribution of the editing sites among the four exons.

    PubMed Central

    Lamattina, L; Grienenberger, J M

    1991-01-01

    The wheat mitochondrial (mt) NADH dehydrogenase subunit 4 gene (nad4) has been localized and sequenced. This gene, about 8 kb long, is composed of four exons separated by three class II introns. The nad4 gene exists as a single copy in the wheat mitochondrial genome and it is transcribed into one abundant mRNA of 1.8 kb, whose extremities have been mapped. The complete cDNA sequence corresponding to the nad4 transcript has been determined by combining the direct sequencing of uncloned cDNA and a method involving cDNA synthesis and PCR amplification using specific oligonucleotides as primers, followed by cloning and sequencing of the amplification product. Comparison of the genomic sequence with that of the cDNA shows that all nad4 transcripts are fully edited at 23 positions, with an uneven distribution of the editing sites between the different exons: While exon 1 and exon 4 are extensively edited (with a change of 11% of the amino acid sequence), exon 2 is not edited at all and exon 3 is 0.5% edited. This uneven distribution is discussed. Images PMID:1712098

  14. Two DYW subclass PPR proteins are involved in RNA editing of ccmFc and atp9 transcripts in the moss Physcomitrella patens: first complete set of PPR editing factors in plant mitochondria.

    PubMed

    Ichinose, Mizuho; Sugita, Chieko; Yagi, Yusuke; Nakamura, Takahiro; Sugita, Mamoru

    2013-11-01

    The moss Physcomitrella patens has 11 RNA editing sites in mitochondrial transcripts. We previously identified six DYW subclass pentatricopeptide repeat (PPR) proteins as RNA editing factors for nine out of 11 sites. In this study, we identified two novel DYW subclass PPR proteins, PpPPR_65 and PpPPR_98, as RNA editing factors. Disruption of the PpPPR_65 gene resulted in a complete loss of RNA editing at two neighboring sites, ccmFc-C103 and ccmFc-C122, in the mitochondrial ccmFc transcript. To confirm this result, we further generated PpPPR_65 knockdown (KD) mutants by an inducible RNA interference (RNAi) system. The generated RNAi lines displayed reduced levels of RNA editing at both ccmFc-C103 and ccmFc-C122 sites. Next, we characterized the function of PpPPR_98 by constructing a KD mutant of PpPPR_98 expression. The KD mutant showed a 30% reduction in the level of atp9-C92 editing. When PpPPR_98 cDNA was introduced into the KD mutant, RNA editing levels were restored to the wild-type level. This indicates that PpPPR_98 is an editing factor for the atp9-C92 site. The recombinant PpPPR_98 protein bound to the upstream sequence of the editing site that was created by splicing of atp9 transcript. This suggests that atp9 RNA editing occurs after splicing of atp9 transcript. Our present and previous data provide the first evidence that all 11 known editing events require at least eight DYW subclass PPR proteins in the moss mitochondria.

  15. 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

  16. The eukaryotic way to defend and edit genomes by sRNA-targeted DNA deletion.

    PubMed

    Swart, Estienne C; Nowacki, Mariusz

    2015-04-01

    While there is currently burgeoning interest in the application of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) to genome editing, it is perhaps not widely appreciated that this is the second discovery of a small RNA (sRNA)-targeted DNA-deletion system. The first sRNA-targeted DNA-deletion system to be discovered, which we call IES/Ias (internal eliminated sequence/IES-associated genes) to contrast with CRISPR/Cas, is found in ciliates, and, like CRISPR/Cas, is thought to serve as a form of immune defense against invasive DNAs. The manner in which the ciliate IES/Ias system functions is distinct from that of the CRISPR/Cas system in archaea and bacteria, and arose independently through a synthesis of RNA interference-derived and DNA-specific molecular components. Despite the major differences between CRISPR/Cas and IES/Ias, both systems face similar conceptual challenges in targeting invasive DNAs. In this review, we focus on the discovery, effects, function, and evolutionary consequences of the IES/Ias system.

  17. 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

  18. 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

  19. 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

  20. Retrotransposition and Crystal Structure of an Alu RNP in the Ribosome-Stalling Conformation.

    PubMed

    Ahl, Valentina; Keller, Heiko; Schmidt, Steffen; Weichenrieder, Oliver

    2015-12-01

    The Alu element is the most successful human genomic parasite affecting development and causing disease. It originated as a retrotransposon during early primate evolution of the gene encoding the signal recognition particle (SRP) RNA. We defined a minimal Alu RNA sufficient for effective retrotransposition and determined a high-resolution structure of its complex with the SRP9/14 proteins. The RNA adopts a compact, closed conformation that matches the envelope of the SRP Alu domain in the ribosomal translation elongation factor-binding site. Conserved structural elements in SRP RNAs support an ancient function of the closed conformation that predates SRP9/14. Structure-based mutagenesis shows that retrotransposition requires the closed conformation of the Alu ribonucleoprotein particle and is consistent with the recognition of stalled ribosomes. We propose that ribosome stalling is a common cause for the cis-preference of the mammalian L1 retrotransposon and for the efficiency of the Alu RNA in hijacking nascent L1 reverse transcriptase. PMID:26585389

  1. 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

  2. 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.

  3. 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

  4. 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

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

    PubMed

    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.

  6. Alu retrotransposons promote differentiation of human carcinoma cells through the aryl hydrocarbon receptor

    PubMed Central

    Morales-Hernández, Antonio; González-Rico, Francisco J.; Román, Angel C.; Rico-Leo, Eva; Alvarez-Barrientos, Alberto; Sánchez, Laura; Macia, Ángela; Heras, Sara R.; García-Pérez, José L.; Merino, Jaime M.; Fernández-Salguero, Pedro M.

    2016-01-01

    Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions. PMID:26883630

  7. 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

  8. Growing Slowly 1 locus encodes a PLS-type PPR protein required for RNA editing and plant development in Arabidopsis

    PubMed Central

    Xie, Tingting; Chen, Dan; Wu, Jian; Huang, Xiaorong; Wang, Yifan; Tang, Keli; Li, Jiayang; Sun, Mengxiang; Peng, Xiongbo

    2016-01-01

    Most pentatricopeptide repeat (PPR) proteins are involved in organelle post-transcriptional processes, including RNA editing. The PPR proteins include the PLS subfamily, containing characteristic triplets of P, L, and S motifs; however, their editing mechanisms and roles in developmental processes are not fully understood. In this study, we isolated the Arabidopsis thaliana Growing slowly 1 (AtGRS1) gene and showed that it functions in RNA editing and plant development. Arabidopsis null mutants of grs1 exhibit slow growth and sterility. Further analysis showed that cell division activity was reduced dramatically in the roots of grs1 plants. We determined that GRS1 is a nuclear-encoded mitochondria-localized PPR protein, and is a member of the PLS subfamily. GRS1 is responsible for the RNA editing at four specific sites of four mitochondrial mRNAs: nad1-265, nad4L-55, nad6-103, and rps4-377. The first three of these mRNAs encode for the subunits of complex I of the electron transport chain in mitochondria. Thus, the activity of complex I is strongly reduced in grs1. Changes in RPS4 editing in grs1 plants affect mitochondrial ribosome biogenesis. Expression of the alternative respiratory pathway and the abscisic acid response gene ABI5 were up-regulated in grs1 mutant plants. Genetic analysis revealed that ABI5 is involved in the short root phenotype of grs1. Taken together, our results indicate that AtGRS1 regulates plant development by controlling RNA editing in Arabidopsis. PMID:27670716

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

    PubMed

    Arakawa, Hiroshi

    2016-08-01

    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.

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

    PubMed

    Arakawa, Hiroshi

    2016-08-01

    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

  11. 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

  12. Extra double-stranded RNA binding domain (dsRBD) in a squid RNA editing enzyme confers resistance to high salt environment.

    PubMed

    Palavicini, Juan Pablo; Correa-Rojas, Rodrigo A; Rosenthal, Joshua J C

    2012-05-18

    A-to-I RNA editing is particularly common in coding regions of squid mRNAs. Previously, we isolated a squid editing enzyme (sqADAR2) that shows a unique structural feature when compared with other ADAR2 family members: an additional double-stranded RNA (dsRNA) binding domain (dsRBD). Alternative splicing includes or excludes this motif, generating a novel or a conventional variant termed sqADAR2a and sqADAR2b, respectively. The extra dsRBD of sqADAR2a increases its editing activity in vitro. We hypothesized that the high activity is due to an increase in the affinity of the enzyme for dsRNA. This may be important because protein-RNA interactions can be influenced by physical factors. We became particularly interested in analyzing the effects of salt on interactions between sqADAR2 and RNA because squid cells have a ∼3-fold higher ionic strength and proportionally more Cl(-) than vertebrate cells. To date, in vitro biochemical analyses of adenosine deamination have been conducted using vertebrate-like ionic strength buffers containing chloride as the major anion, although the vast majority of cellular anions are known to be organic. We found that squid-like salt conditions severely impair the binding affinity of conventional ADAR2s for dsRNA, leading to a decrease in nonspecific and site-specific editing activity. Inhibition of editing was mostly due to high Cl(-) levels and not to the high concentrations of K(+), Na(+), and organic anions like glutamate. Interestingly, the extra dsRBD in sqADAR2a conferred resistance to the high Cl(-) levels found in squid neurons. It does so by increasing the affinity of sqADAR2 for dsRNA by 30- or 100-fold in vertebrate-like or squid-like conditions, respectively. Site-directed mutagenesis of squid ADAR2a showed that its increased affinity and editing activity are directly attributable to the RNA binding activity of the extra dsRBD.

  13. 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

  14. 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.

  15. 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

  16. 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

  17. PPR protein PDM1/SEL1 is involved in RNA editing and splicing of plastid genes in Arabidopsis thaliana.

    PubMed

    Zhang, Hong-Dao; Cui, Yong-Lan; Huang, Chao; Yin, Qian-Qian; Qin, Xue-Mei; Xu, Te; He, Xiao-Fang; Zhang, Yi; Li, Zi-Ran; Yang, Zhong-Nan

    2015-12-01

    After transcription, most chloroplast precursor RNAs undergo further post-transcriptional processing including cleavage, editing, and splicing. Previous investigation has shown that the cleavage of the rpoA transcript and most editing sites, including accD-1, are defective in the knockout mutant of PDM1/SEL1, a PLS-type PPR protein, and that PDM1 is associated with the rpoA transcript. In this work, we found that the splicing of group II introns in trnK and ndhA is also affected in pdm1. Co-immunoprecipitation mass spectrometry experiments were performed to identify proteins that are associated with PDM1. We obtained 126 non-redundant proteins, of which MORF9 was reported to be involved in RNA editing in chloroplast. Yeast two-hybrid assays showed that PDM1 interacts directly with MORF9, MORF2, and MORF8. RNA immunoprecipitation showed that PDM1 associates with the transcripts of trnK and ndhA, as well as accD-1, suggesting that PDM1 is involved in RNA editing and splicing. Therefore, PDM1 is an important protein for post-transcriptional regulation in chloroplast.

  18. Uneven spread of cis- and trans-editing aminoacyl-tRNA synthetase domains within translational compartments of P. falciparum.

    PubMed

    Khan, Sameena; Sharma, Arvind; Jamwal, Abhishek; Sharma, Vinay; Pole, Anil Kumar; Thakur, Kamal Kishor; Sharma, Amit

    2011-01-01

    Accuracy of aminoacylation is dependent on maintaining fidelity during attachment of amino acids to cognate tRNAs. Cis- and trans-editing protein factors impose quality control during protein translation, and 8 of 36 Plasmodium falciparum aminoacyl-tRNA synthetase (aaRS) assemblies contain canonical putative editing modules. Based on expression and localization profiles of these 8 aaRSs, we propose an asymmetric distribution between the parasite cytoplasm and its apicoplast of putative editing-domain containing aaRSs. We also show that the single copy alanyl- and threonyl-tRNA synthetases are dually targeted to parasite cytoplasm and apicoplast. This bipolar presence of two unique synthetases presents opportunity for inhibitor targeting their aminoacylation and editing activities in twin parasite compartments. We used this approach to identify specific inhibitors against the alanyl- and threonyl-tRNA synthetases. Further development of such inhibitors may lead to anti-parasitics which simultaneously block protein translation in two key parasite organelles, a strategy of wider applicability for pathogen control.

  19. 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

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

    PubMed Central

    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

  1. A-to-I editing in the miRNA seed region regulates target mRNA selection and silencing efficiency

    PubMed Central

    Kume, Hideaki; Hino, Kimihiro; Galipon, Josephine; Ui-Tei, Kumiko

    2014-01-01

    Hydrolytic deamination of adenosine to inosine (A-to-I) by adenosine deaminases acting on RNA (ADARs) is a post-transcriptional modification which results in a discrepancy between genomic DNA and the transcribed RNA sequence, thus contributing to the diversity of the transcriptome. Inosine preferentially base pairs with cytidine, meaning that A-to-I modifications in the mRNA sequences may be observed as A-to-G substitutions by the protein-coding machinery. Genome-wide studies have revealed that the majority of editing events occur in non-coding RNA sequences, but little is known about their functional meaning. MiRNAs are small non-coding RNAs that regulate the expression of target mRNAs with complementarities to their seed region. Here, we confirm that A-to-I editing in the miRNA seed duplex globally reassigns their target mRNAs in vivo, and reveal that miRNA containing inosine in the seed region exhibits a different degree of silencing efficiency compared to the corresponding miRNA with guanosine at the same position. The difference in base-pairing stability, deduced by melting temperature measurements, between seed-target duplexes containing either C:G or I:C pairs may account for the observed silencing efficiency. These findings unequivocally show that C:G and I:C pairs are biologically different in terms of gene expression regulation by miRNAs. PMID:25056317

  2. 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

  3. Mitochondrial 3' tRNA editing in the jakobid Seculamonas ecuadoriensis: a novel mechanism and implications for tRNA processing.

    PubMed

    Leigh, Jessica; Lang, B Franz

    2004-04-01

    The jakobid flagellates are bacteriovorus protists with mitochondrial genomes that are the most ancestral identified to date, in that they most resemble the genomes of the alpha-proteobacterial ancestors of the mitochondrion. Because of the bacterial character of jakobid mitochondrial genomes, it was expected that mechanisms for gene expression and RNA structures would be bacterial in nature. However, sequencing of the mitochondrial genome of the jakobid Seculamonas ecuadoriensis revealed several apparent mismatches in the acceptor stems of two predicted tRNAs. To investigate this observation, we determined the cDNA sequences of these tRNAs by RT-PCR. Our results show that the last three positions of the 3' extremity, plus the discriminator position of seryl and glutamyl tRNAs, are altered posttranscriptionally, restoring orthodox base-pairing and replacing the discriminator with an adenosine residue, in an editing process that resembles that of the metazoan Lithobius forficatus. However, the most 5' of the edited nucleotides is occasionally left unedited, indicating that the editing mechanism proceeds initially by exonucleolytic degradation, followed by repair of the degraded region. This 3' tRNA editing mechanism is likely distinct from that of L. forficatus, despite the apparent similarities between the two systems.

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. RNA editing of the serotonin 2C receptor and expression of Gαq protein: genetic mouse models do not support a role for regulation or compensation

    PubMed Central

    Canal, Clinton E.; Mahautmr, Kontip C.; Cao, Chike; Sanders-Bush, Elaine; Airey, David C.

    2009-01-01

    The serotonin 2C (5-HT2C) receptor undergoes RNA editing at five bases in a region of the pre-mRNA encoding the second intracellular loop, generating many unique 5-HT2C receptor isoforms. Mechanisms regulating in vivo expression of different edited 5-HT2C receptor isoforms are poorly understood, as are the adaptive consequences of variation in editing profiles. Recent findings suggest a putative relationship between expression levels of Gαq/11 protein and the degree of editing of 5-HT2C receptor transcripts. To elucidate the potential regulatory or adaptive role of Gαq/11 protein levels, we quantified editing of 5-HT2C receptor RNA transcripts in Gαq null mice and protein levels of Gαq and Gα11 in transgenic male mice solely expressing either the non-edited (INI) or the fully edited (VGV) isoforms of the 5-HT2C receptor. Pyrosequencing of RNA isolated from amygdaloid cortex in Gαq null and wild-type mice revealed no significant differences in 5-HT2C receptor mRNA editing profiles. Cortical tissue from INI/y, VGV/y, and wild-type mice was assayed for expression of Gαq and Gα11 subunits by Western blotting. No differences in signal density between wild-type and INI/y or VGV/y groups were found, indicating equivalent levels of Gαq and Gα11 protein. Together, these data do not support a causal or compensatory relationship between 5-HT2C receptor RNA editing and Gq protein levels. PMID:19154337

  12. 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

  13. 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.

  14. The Basques according to polymorphic Alu insertions.

    PubMed

    de Pancorbo, M M; López-Martínez, M; Martínez-Bouzas, C; Castro, A; Fernández-Fernández, I; de Mayolo, G A; de Mayolo, A A; de Mayolo, P A; Rowold, D J; Herrera, R J

    2001-08-01

    Polymorphic Alu insertions provide a set of DNA markers of interest in human population genetics. Approximately 1000-2000 of these insertions have not reached fixation within the human genome. Each one of these polymorphic loci most probably resulted from a unique insertional event, and therefore all individuals possessing the insertion are related by descent not just state. In addition, the direction of mutational change is toward the gain of the Alu element at a particular locus. Therefore, the improved knowledge of both the ancestral state and the direction of mutational change greatly facilitates the analysis of population relationships. As a result, Alu insertion polymorphisms represent a significant tool for population genetic studies. In this study, polymorphic Alu insertions have been employed to ascertain phylogenetic relationships among Basque groups and worldwide populations. The Basques are considered to be a geographic isolate with a unique language and customs. They may be direct descendants of Cro-Magnon enclaves from the upper Paleolithic (38,000 to 10,000 years). The Basques are distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. With the aim of studying this possible effect, we have analyzed six autosomal polymorphic Alu loci from four different sites within the Spanish Basque region in order to ascertain any genetic heterogeneity among the Basques. The results are consistent with a lack of homogeneity among these four autochthonous Basque groups.

  15. The Basques according to polymorphic Alu insertions.

    PubMed

    de Pancorbo, M M; López-Martínez, M; Martínez-Bouzas, C; Castro, A; Fernández-Fernández, I; de Mayolo, G A; de Mayolo, A A; de Mayolo, P A; Rowold, D J; Herrera, R J

    2001-08-01

    Polymorphic Alu insertions provide a set of DNA markers of interest in human population genetics. Approximately 1000-2000 of these insertions have not reached fixation within the human genome. Each one of these polymorphic loci most probably resulted from a unique insertional event, and therefore all individuals possessing the insertion are related by descent not just state. In addition, the direction of mutational change is toward the gain of the Alu element at a particular locus. Therefore, the improved knowledge of both the ancestral state and the direction of mutational change greatly facilitates the analysis of population relationships. As a result, Alu insertion polymorphisms represent a significant tool for population genetic studies. In this study, polymorphic Alu insertions have been employed to ascertain phylogenetic relationships among Basque groups and worldwide populations. The Basques are considered to be a geographic isolate with a unique language and customs. They may be direct descendants of Cro-Magnon enclaves from the upper Paleolithic (38,000 to 10,000 years). The Basques are distributed among narrow valleys in northeastern Spain with little migration between them until recently. This characteristic may have had an effect on allelic frequency distributions. With the aim of studying this possible effect, we have analyzed six autosomal polymorphic Alu loci from four different sites within the Spanish Basque region in order to ascertain any genetic heterogeneity among the Basques. The results are consistent with a lack of homogeneity among these four autochthonous Basque groups. PMID:11511929

  16. U-INSERTION/DELETION RNA EDITING MULTIPROTEIN COMPLEXES AND MITOCHONDRIAL RIBOSOMES IN LEISHMANIA TARENTOLAE ARE LOCATED IN ANTIPODAL NODES ADJACENT TO THE KINETOPLAST DNA

    PubMed Central

    Wong, Richard G; Kazane, Katelynn; Maslov, Dmitri A; Rogers, Kestrel; Aphasizhev, Ruslan; Simpson, Larry

    2015-01-01

    We studied the intramitochondrial localization of several multiprotein complexes involved in U-insertion/deletion RNA editing in trypanosome mitochondria. The editing complexes are located in one or two antipodal nodes adjacent to the kinetoplast DNA (kDNA) disk, which are distinct from but associated with the minicircle catenation nodes. In some cases the proteins are in a bilateral sheet configuration. We also found that mitoribosomes have a nodal configuration. This type of organization is consistent with evidence for protein and RNA interactions of multiple editing complexes to form a ~40S editosome and also an interaction of editosomes with mitochondrial ribosomes. PMID:26462764

  17. U-insertion/deletion RNA editing multiprotein complexes and mitochondrial ribosomes in Leishmania tarentolae are located in antipodal nodes adjacent to the kinetoplast DNA.

    PubMed

    Wong, Richard G; Kazane, Katelynn; Maslov, Dmitri A; Rogers, Kestrel; Aphasizhev, Ruslan; Simpson, Larry

    2015-11-01

    We studied the intramitochondrial localization of several multiprotein complexes involved in U-insertion/deletion RNA editing in trypanosome mitochondria. The editing complexes are located in one or two antipodal nodes adjacent to the kinetoplast DNA (kDNA) disk, which are distinct from but associated with the minicircle catenation nodes. In some cases the proteins are in a bilateral sheet configuration. We also found that mitoribosomes have a nodal configuration. This type of organization is consistent with evidence for protein and RNA interactions of multiple editing complexes to form an ~40S editosome and also an interaction of editosomes with mitochondrial ribosomes.

  18. 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.

  19. 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

  20. Functional conservation in human and Drosophila of Metazoan ADAR2 involved in RNA editing: loss of ADAR1 in insects

    PubMed Central

    Keegan, Liam P.; McGurk, Leeane; Palavicini, Juan Pablo; Brindle, James; Paro, Simona; Li, Xianghua; Rosenthal, Joshua J. C.; O'Connell, Mary A.

    2011-01-01

    Flies with mutations in the single Drosophila Adar gene encoding an RNA editing enzyme involved in editing 4% of all transcripts have severe locomotion defects and develop age-dependent neurodegeneration. Vertebrates have two ADAR-editing enzymes that are catalytically active; ADAR1 and ADAR2. We show that human ADAR2 rescues Drosophila Adar mutant phenotypes. Neither the short nuclear ADAR1p110 isoform nor the longer interferon-inducible cytoplasmic ADAR1p150 isoform rescue walking defects efficiently, nor do they correctly edit specific sites in Drosophila transcripts. Surprisingly, human ADAR1p110 does suppress age-dependent neurodegeneration in Drosophila Adar mutants whereas ADAR1p150 does not. The single Drosophila Adar gene was previously assumed to represent an evolutionary ancestor of the multiple vertebrate ADARs. The strong functional similarity of human ADAR2 and Drosophila Adar suggests rather that these are true orthologs. By a combination of direct cloning and searching new invertebrate genome sequences we show that distinct ADAR1 and ADAR2 genes were present very early in the Metazoan lineage, both occurring before the split between the Bilateria and Cnidarians. The ADAR1 gene has been lost several times, including during the evolution of insects and crustacea. These data complement our rescue results, supporting the idea that ADAR1 and ADAR2 have evolved highly conserved, distinct functions. PMID:21622951

  1. GluA2 AMPA glutamate receptor subunit exhibits codon 607 Q/R RNA editing in the lens.

    PubMed

    Farooq, Mohammed; Kaswala, Rajesh H; Kleiman, Norman J; Kasinathan, Chinnaswamy; Frederikse, Peter H

    2012-02-10

    Regulated GluA2 AMPA receptor subunit expression, RNA editing, and membrane localization are fundamental determinants of neuronal Ca(2+) influx, and underlie basic functions such as memory and the primary brain disorder epilepsy. Consistent with this, AMPARs, and specifically GluA2, are targets of common antiepileptic drugs (AEDs) and antidepressants. Recently, epidemiological associations between epilepsy and increased cataract prevalence were found comparable to cataract links with diabetes and smoking. Similarly, use of AEDs and several antidepressants also showed links with increased cataract. Here, we demonstrated GluA2 in lenses, consistent with REST/NRSF and REST4 we described previously in lenses, as well as GluA1 and ADAR2 in the lens. Surprisingly, we found predominant neuron-like Q/R editing of GluA2 RNAs also occurs in the lens and evidence of lens GluA2 phosphorylation and STEP phosphatases linked with GluA2 membrane localization in neurons. This study is among the first to show GluA2 expression and predominant Q/R RNA editing in a non-neural cell. Our results suggest GluA2 AMPARs have related roles in lens physiology and disease processes, and provide evidence these anticonvulsant and antidepressant drug targets also occur in the lens. PMID:22266371

  2. A bridge between the aminoacylation and editing domains of leucyl-tRNA synthetase is crucial for its synthetic activity

    PubMed Central

    Huang, Qian; Zhou, Xiao-Long; Hu, Qin-Hua; Lei, Hui-Yan; Fang, Zhi-Peng; Yao, Peng; Wang, En-Duo

    2014-01-01

    Leucyl-tRNA synthetases (LeuRSs) catalyze the linkage of leucine with tRNALeu. LeuRS contains a catalysis domain (aminoacylation) and a CP1 domain (editing). CP1 is inserted 35 Å from the aminoacylation domain. Aminoacylation and editing require CP1 to swing to the coordinated conformation. The neck between the CP1 domain and the aminoacylation domain is defined as the CP1 hairpin. The location of the CP1 hairpin suggests a crucial role in the CP1 swing and domain–domain interaction. Here, the CP1 hairpin of Homo sapiens cytoplasmic LeuRS (hcLeuRS) was deleted or substituted by those from other representative species. Lack of a CP1 hairpin led to complete loss of aminoacylation, amino acid activation, and tRNA binding; however, the mutants retained post-transfer editing. Only the CP1 hairpin from Saccharomyces cerevisiae LeuRS (ScLeuRS) could partly rescue the hcLeuRS functions. Further site-directed mutagenesis indicated that the flexibility of small residues and the charge of polar residues in the CP1 hairpin are crucial for the function of LeuRS. PMID:25051973

  3. The mitochondrial PPR protein LOVASTATIN INSENSITIVE 1 plays regulatory roles in cytosolic and plastidial isoprenoid biosynthesis through RNA editing.

    PubMed

    Tang, Jianwei; Kobayashi, Keiko; Suzuki, Masashi; Matsumoto, Shogo; Muranaka, Toshiya

    2010-02-01

    Unlike animals, plants synthesize isoprenoids via two pathways, the cytosolic mevalonate (MVA) pathway and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. Little information is known about the mechanisms that regulate these complex biosynthetic networks over multiple organelles. To understand such regulatory mechanisms of the biosynthesis of isoprenoids in plants, we previously characterized the Arabidopsis mutant, lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, specific inhibitors of the MVA and MEP pathways, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria that is thought to have RNA binding ability and function in post-transcriptional regulation of mitochondrial gene expression. LOI1 belongs to the DYW subclass of PPR proteins, which is hypothesized to be correlated with RNA editing. As a result of analysis of RNA editing of mitochondrial genes in loi1, a defect in RNA editing of three genes, nad4, ccb203 and cox3, was identified in loi1. These genes are related to the respiratory chain. Wild type (WT) treated with some respiration inhibitors mimicked the loi1 phenotype. Interestingly, HMG-CoA reductase activity of WT treated with lovastatin combined with antimycin A, an inhibitor of complex III in the respiratory chain, was higher than that of WT treated with only lovastatin, despite the lack of alteration of transcript or protein levels of HMGR. These results suggest that HMGR enzyme activity is regulated through the respiratory cytochrome pathway. Although various mechanisms exist for isoprenoid biosynthesis, our studies demonstrate the novel possibility that mitochondrial respiration plays potentially regulatory roles in isoprenoid biosynthesis.

  4. Tissue- and stage-specific modulation of RNA editing of the psbF and psbL transcript from spinach plastids--a new regulatory mechanism?

    PubMed

    Bock, R; Hagemann, R; Kössel, H; Kudla, J

    1993-08-01

    The psbE operon of spinach chloroplasts, which includes the genes psbE, psbF, psbL and psbJ, encodes two RNA editing sites. One site corresponds to the initiation codon of the psbL transcript, as has been described earlier for the homologous transcript from tobacco, while at a second editing site, newly reported here, an internal phenylalanine codon of the psbF transcript is restored. Both these sites were investigated with respect to the extent of editing in spinach plastids at various developmental stages. The apparent existence of only completely edited transcripts in etioplasts and chloroplasts, indicates that light-induced processes are not acting as determinants in eliciting the editing process. Reduced editing is, however, observed in the psbF and psbL transcript from seeds and roots. This finding suggests that the RNA editing process is differentially down-regulated in leucoplasts and proplastids and that editing may, therefore, function as a regulatory device in plastid gene expression. PMID:8355656

  5. 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.

  6. The E-class PPR protein MEF3 of Arabidopsis thaliana can also function in mitochondrial RNA editing with an additional DYW domain.

    PubMed

    Verbitskiy, Daniil; Merwe, Johannes A van der; Zehrmann, Anja; Härtel, Barbara; Takenaka, Mizuki

    2012-02-01

    In plants, RNA editing is observed in mitochondria and plastids, changing selected C nucleotides into Us in both organelles. We here identify the PPR (pentatricopeptide repeat) protein MEF3 (mitochondrial editing factor 3) of the E domain PPR subclass by genetic mapping of a variation between ecotypes Columbia (Col) and Landsberg erecta (Ler) in Arabidopsis thaliana to be required for a specific RNA editing event in mitochondria. The Ler variant of MEF3 differs from Col in two amino acids in repeats 9 and 10, which reduce RNA editing levels at site atp4-89 to about 50% in Ler. In a T-DNA insertion line, editing at this site is completely lost. In Vitis vinifera the gene most similar to MEF3 continues into a DYW extension beyond the common E domain. Complementation assays with various combinations of PPR and E domains from the vine and A. thaliana proteins show that the vine E region can substitute for the A. thaliana E region with or without the DYW domain. These findings suggest that the additional DYW domain does not disturb the MEF3 protein function in mitochondrial RNA editing in A. thaliana.

  7. 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.

  8. DYW-type PPR proteins in a heterolobosean protist: plant RNA editing factors involved in an ancient horizontal gene transfer?

    PubMed

    Knoop, Volker; Rüdinger, Mareike

    2010-10-22

    A particular type of pentatricopeptide repeat (PPR) proteins with variable length of the 35 aa PPR motifs and conserved carboxyterminal extensions, named the PLS proteins, was so far exclusively identified in land plants. Several PLS proteins with such domain extensions (E, E+, DYW) were shown to be involved in plant organellar RNA editing but their evolutionary origin had remained enigmatic. We here report the first case of DYW-type PLS proteins outside of the plant kingdom in the protist Naegleria gruberi and hypothesize on horizontal gene transfer in very early land plant evolution.

  9. 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

  10. Structural Variation of Alu Element and Human Disease

    PubMed Central

    Kim, Songmi; Cho, Chun-Sung; Han, Kyudong

    2016-01-01

    Transposable elements are one of major sources to cause genomic instability through various mechanisms including de novo insertion, insertion-mediated genomic deletion, and recombination-associated genomic deletion. Among them is Alu element which is the most abundant element, composing ~10% of the human genome. The element emerged in the primate genome 65 million years ago and has since propagated successfully in the human and non-human primate genomes. Alu element is a non-autonomous retrotransposon and therefore retrotransposed using L1-enzyme machinery. The 'master gene' model has been generally accepted to explain Alu element amplification in primate genomes. According to the model, different subfamilies of Alu elements are created by mutations on the master gene and most Alu elements are amplified from the hyperactive master genes. Alu element is frequently involved in genomic rearrangements in the human genome due to its abundance and sequence identity between them. The genomic rearrangements caused by Alu elements could lead to genetic disorders such as hereditary disease, blood disorder, and neurological disorder. In fact, Alu elements are associated with approximately 0.1% of human genetic disorders. The first part of this review discusses mechanisms of Alu amplification and diversity among different Alu subfamilies. The second part discusses the particular role of Alu elements in generating genomic rearrangements as well as human genetic disorders. PMID:27729835

  11. The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana.

    PubMed

    Brehme, Nadja; Bayer-Császár, Eszter; Glass, Franziska; Takenaka, Mizuki

    2015-01-01

    RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C) to uridine (U) mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35) to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209), for cytochrome b (cob-286) and for subunit 4 of complex I (nad4-1373), respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins.

  12. The DYW Subgroup PPR Protein MEF35 Targets RNA Editing Sites in the Mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana

    PubMed Central

    Glass, Franziska; Takenaka, Mizuki

    2015-01-01

    RNA editing in plant mitochondria and plastids alters specific nucleotides from cytidine (C) to uridine (U) mostly in mRNAs. A number of PLS-class PPR proteins have been characterized as RNA recognition factors for specific RNA editing sites, all containing a C-terminal extension, the E domain, and some an additional DYW domain, named after the characteristic C-terminal amino acid triplet of this domain. Presently the recognition factors for more than 300 mitochondrial editing sites are still unidentified. In order to characterize these missing factors, the recently proposed computational prediction tool could be of use to assign target RNA editing sites to PPR proteins of yet unknown function. Using this target prediction approach we identified the nuclear gene MEF35 (Mitochondrial Editing Factor 35) to be required for RNA editing at three sites in mitochondria of Arabidopsis thaliana. The MEF35 protein contains eleven PPR repeats and E and DYW extensions at the C-terminus. Two T-DNA insertion mutants, one inserted just upstream and the other inside the reading frame encoding the DYW domain, show loss of editing at a site in each of the mRNAs for protein 16 in the large ribosomal subunit (site rpl16-209), for cytochrome b (cob-286) and for subunit 4 of complex I (nad4-1373), respectively. Editing is restored upon introduction of the wild type MEF35 gene in the reading frame mutant. The MEF35 protein interacts in Y2H assays with the mitochondrial MORF1 and MORF8 proteins, mutation of the latter also influences editing at two of the three MEF35 target sites. Homozygous mutant plants develop indistinguishably from wild type plants, although the RPL16 and COB/CYTB proteins are essential and the amino acids encoded after the editing events are conserved in most plant species. These results demonstrate the feasibility of the computational target prediction to screen for target RNA editing sites of E domain containing PLS-class PPR proteins. PMID:26470017

  13. 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

  14. 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

  15. RNA splicing and editing modulation of 5-HT(2C) receptor function: relevance to anxiety and aggression in VGV mice.

    PubMed

    Martin, C B P; Ramond, F; Farrington, D T; Aguiar, A S; Chevarin, C; Berthiau, A-S; Caussanel, S; Lanfumey, L; Herrick-Davis, K; Hamon, M; Madjar, J J; Mongeau, R

    2013-06-01

    Changes in serotonin(2C) receptor (5-HTR2c) editing, splicing and density were found in conditions such as depression and suicide, but mechanisms explaining the changes in 5-HTR2c function are unknown. Thus, mice expressing only the fully edited VGV isoform of 5-HTR2c, in which clinically relevant behavioral changes are associated with alterations in splicing and receptor density, were studied. VGV mice displayed enhanced anxiety-like behavior in response to a preferential 5-HTR2c agonist in the social interaction test. Nearly half of interactions between pairs of VGV congeners consisted of fighting behaviors, whereas no fighting occurred in wild-type (WT) mice. VGV mice also exhibited a striking increase in freezing behaviors in reaction to an innately aversive ultrasonic stimulus. This behavioral phenotype occurred in conjunction with decreased brain 5-HT turnover during stress. These functional data were put in relation with the 5-HTR2c mRNA splicing process generating a truncated protein (5-HTR2c-Tr) in addition to the full-length receptor (5-HTR2c-Fl). 5-HTR2c-Tr mRNA was less abundant in many brain regions of VGV mice, which concomitantly had more 5-HTR2c than WT mice. Fluorescence resonance energy transfer and bioluminescence resonance energy transfer studies in transfected living HEK293T cells showed that 5-HTR2c-Tr interacts with 5-HTR2c-Fl. The 5-HTR2c-Tr was localized in the endoplasmic reticulum where it retained 5-HTR2c-Fl, preventing the latter to reach the plasma membrane. Consequently, 5-HTR2c-Tr decreased (3)H-mesulergine binding to 5-HTR2c-Fl at the plasma membrane in a concentration-dependent manner and more strongly with edited 5-HTR2c-Fl. These results suggest that 5-HTR2c pre-mRNA editing and splicing are entwined processes determining increased 5-HTR2c levels in pathological conditions through a deficit in 5-HTR2c-Tr.

  16. Variations in efficiency of plastidial RNA editing within ndh transcripts of perennial ryegrass (Lolium perenne) are not linked to differences in drought tolerance

    PubMed Central

    Van Den Bekerom, Rob J. M.; Dix, Philip J.; Diekmann, Kerstin; Barth, Susanne

    2013-01-01

    Maintenance of healthy grasslands is essential for efficient livestock production, yet projected climate change is likely to place a heavy drought stress burden on key grassland species, such as perennial ryegrass (Lolium perenne). It is therefore important to gather an in-depth knowledge of the underlying plant response to this stress. The present study is focused on RNA editing (post-transcriptional nucleotide modifications resulting in altered transcripts) within plastidial transcripts of the NADH:ubiquinone oxidoreductase (NDH) complex (NADH dehydrogenase complex) in relation to the drought response of several accessions of perennial ryegrass. Previous studies have shown that the NDH complex is involved in countering oxidative stress during environmental stresses like drought. Owing to the nature of RNA editing within this complex, the RNA editing machinery could play a potential role in regulating the activity of the NDH complex. The investigation revealed dramatic and reproducible differences in RNA editing efficiency between accessions, but efficiency was not influenced by imposition of drought stress, and a direct relationship between editing behaviour and drought response was not detected.

  17. C to U editing of the anticodon of imported mitochondrial tRNA(Trp) allows decoding of the UGA stop codon in Leishmania tarentolae.

    PubMed

    Alfonzo, J D; Blanc, V; Estévez, A M; Rubio, M A; Simpson, L

    1999-12-15

    All mitochondrial tRNAs in kinetoplastid protists are encoded in the nucleus and imported into the organelle. The tRNA(Trp)(CCA) can decode the standard UGG tryptophan codon but can not decode the mitochondrial UGA tryptophan codon. We show that the mitochondrial tRNA(Trp) undergoes a specific C to U nucleotide modification in the first position of the anticodon, which allows decoding of mitochondrial UGA codons as tryptophan. Functional evidence for the absence of a UGA suppressor tRNA in the cytosol, using a reporter gene, was also obtained, which is consistent with a mitochondrial localization of this editing event. Leishmania cells have dealt with the problem of a lack of expression within the organelle of this non-universal tRNA by compartmentalizing an editing activity that modifies the anticodon of the imported tRNA.

  18. 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

  19. Sequence Analysis and Characterization of Active Human Alu Subfamilies Based on the 1000 Genomes Pilot Project.

    PubMed

    Konkel, Miriam K; Walker, Jerilyn A; Hotard, Ashley B; Ranck, Megan C; Fontenot, Catherine C; Storer, Jessica; Stewart, Chip; Marth, Gabor T; Batzer, Mark A

    2015-08-29

    The goal of the 1000 Genomes Consortium is to characterize human genome structural variation (SV), including forms of copy number variations such as deletions, duplications, and insertions. Mobile element insertions, particularly Alu elements, are major contributors to genomic SV among humans. During the pilot phase of the project we experimentally validated 645 (611 intergenic and 34 exon targeted) polymorphic "young" Alu insertion events, absent from the human reference genome. Here, we report high resolution sequencing of 343 (322 unique) recent Alu insertion events, along with their respective target site duplications, precise genomic breakpoint coordinates, subfamily assignment, percent divergence, and estimated A-rich tail lengths. All the sequenced Alu loci were derived from the AluY lineage with no evidence of retrotransposition activity involving older Alu families (e.g., AluJ and AluS). AluYa5 is currently the most active Alu subfamily in the human lineage, followed by AluYb8, and many others including three newly identified subfamilies we have termed AluYb7a3, AluYb8b1, and AluYa4a1. This report provides the structural details of 322 unique Alu variants from individual human genomes collectively adding about 100 kb of genomic variation. Many Alu subfamilies are currently active in human populations, including a surprising level of AluY retrotransposition. Human Alu subfamilies exhibit continuous evolution with potential drivers sprouting new Alu lineages.

  20. Under the genomic radar: The Stealth model of Alu amplification

    PubMed Central

    Han, Kyudong; Xing, Jinchuan; Wang, Hui; Hedges, Dale J.; Garber, Randall K.; Cordaux, Richard; Batzer, Mark A.

    2005-01-01

    Alu elements are the most successful SINEs (Short INterspersed Elements) in primate genomes and have reached more than 1,000,000 copies in the human genome. The amplification of most Alu elements is thought to occur through a limited number of hyperactive “master” genes that produce a high number of copies during long evolutionary periods of time. However, the existence of long-lived, low-activity Alu lineages in the human genome suggests a more complex propagation mechanism. Using both computational and wet-bench approaches, we reconstructed the evolutionary history of the AluYb lineage, one of the most active Alu lineages in the human genome. We show that the major AluYb lineage expansion in humans is a species-specific event, as nonhuman primates possess only a handful of AluYb elements. However, the oldest existing AluYb element resided in an orthologous position in all hominoid primate genomes examined, demonstrating that the AluYb lineage originated 18–25 million years ago. Thus, the history of the AluYb lineage is characterized by ∼20 million years of retrotranspositional quiescence preceding a major expansion in the human genome within the past few million years. We suggest that the evolutionary success of the Alu family may be driven at least in part by “stealth-driver” elements that maintain low retrotranspositional activity over extended periods of time and occasionally produce short-lived hyperactive copies responsible for the formation and remarkable expansion of Alu elements within the genome. PMID:15867427

  1. 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

  2. Hyaloraphidium curvatum: a linear mitochondrial genome, tRNA editing, and an evolutionary link to lower fungi.

    PubMed

    Forget, Lise; Ustinova, Jana; Wang, Zhang; Huss, Volker A R; Lang, B Franz

    2002-03-01

    We have sequenced the mitochondrial DNA (mtDNA) of Hyaloraphidium curvatum, an organism previously classified as a colorless green alga but now recognized as a lower fungus based on molecular data. The 29.97-kbp mitochondrial chromosome is maintained as a monomeric, linear molecule with identical, inverted repeats (1.43 kbp) at both ends, a rare genome architecture in mitochondria. The genome encodes only 14 known mitochondrial proteins, 7 tRNAs, the large subunit rRNA and small subunit rRNA (SSU rRNA), and 3 ORFs. The SSU rRNA is encoded in two gene pieces that are located 8 kbp apart on the mtDNA. Scrambled and fragmented mitochondrial rRNAs are well known from green algae and alveolate protists but are unprecedented in fungi. Protein genes code for apocytochrome b; cytochrome oxidase 1, 2, and 3, NADH dehydrogenase 1, 2, 3, 4, 4L, 5, and 6, and ATP synthase 6, 8, and 9 subunits, and several of these genes are organized in operon-like clusters. The set of seven mitochondrially encoded tRNAs is insufficient to recognize all codons that occur in the mitochondrial protein genes. When taking into account the pronounced codon bias, at least 16 nuclear-encoded tRNAs are assumed to be imported into the mitochondria. Three of the seven predicted mitochondria-encoded tRNA sequences carry mispairings in the first three positions of the acceptor stem. This strongly suggests that these tRNAs are edited by a mechanism similar to the one seen in the fungus Spizellomyces punctatus and the rhizopod amoeba Acanthamoeba castellanii. Our phylogenetic analysis confirms with overwhelming support that H. curvatum is a member of the chytridiomycete fungi, specifically related to the Monoblepharidales.

  3. 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

  4. 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

  5. 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

  6. Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA editing complex.

    PubMed

    Panigrahi, A K; Gygi, S P; Ernst, N L; Igo, R P; Palazzo, S S; Schnaufer, A; Weston, D S; Carmean, N; Salavati, R; Aebersold, R; Stuart, K D

    2001-01-01

    RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3'-terminal uridylyl transferase, 3'-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an approximately 49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.

  7. 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

  8. 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

  9. 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

  10. Effective Alu Repeat Based RT-Qpcr Normalization in Cancer Cell Perturbation Experiments

    PubMed Central

    Rihani, Ali; Van Maerken, Tom; Pattyn, Filip; Van Peer, Gert; Beckers, Anneleen; De Brouwer, Sara; Kumps, Candy; Mets, Evelien; Van der Meulen, Joni; Rondou, Pieter; Leonelli, Carina; Mestdagh, Pieter; Speleman, Frank; Vandesompele, Jo

    2013-01-01

    Background Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. Results The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5). Conclusions We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments. PMID:23977142

  11. Alu repeats as markers for forensic DNA analyses

    SciTech Connect

    Batzer, M.A.; Alegria-Hartman, M.; Kass, D.H.

    1994-01-01

    The Human-Specific (HS) subfamily of Alu sequences is comprised of a group of 500 nearly identical members which are almost exclusively restricted to the human genome. Individual subfamily members share an average of 98.9% nucleotide identity with the HS subfamily consensus sequence, and have an average age of 2.8 million years. We have developed a Polymerase Chain Reaction (PCR) based assay using primers complementary to the 5 inch and 3 inch unique flanking DNA sequences from each HS Alu that allow the locus to be assayed for the presence or absence of the Alu repeat. The dimorphic HS Alu sequences probably inserted in the human genome after the radiation of modem humans (within the last 200,000-one million years) and represent a unique source of information for human population genetics and forensic DNA analyses. These sites can be developed into Dimorphic Alu Sequence Tagged Sites (DASTS) for the Human Genome Project. HS Alu family member insertions differ from other types of polymorphism (e.g. Variable Number of Tandem Repeat [VNTR] or Restriction Fragment Length Polymorphism [RFLP]) in that polymorphisms due to Alu insertions arise as a result of a unique event which has occurred only one time in the human population and spread through the population from that point. Therefore, individuals that share HS Alu repeats inherited these elements from a common ancestor. Most VNTR and RFLP polymorphisms may arise multiple times in parallel within a population.

  12. Comparative Analysis of Alu Repeats in Primate Genomes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Alu repeats are SINEs (Short intersperse repetitive elements) which enjoy a successful application in genome evolution, population biology, phylogenetics and forensics. Human Alu consensus sequences were widely used as surrogates in nonhuman primate studies with an assumption that all p...

  13. RNA-guided genome editing in Drosophila with the purified Cas9 protein.

    PubMed

    Lee, Jeong-Soo; Kwak, Su-Jin; Kim, Jungeun; Kim, Ae-Kyeong; Noh, Hae Min; Kim, Jin-Soo; Yu, Kweon

    2014-07-01

    We report a method for generating Drosophila germline mutants effectively via injection of the complex of the purified Cas9 protein, tracrRNA, and gene-specific crRNAs, which may reduce delayed mutations because of the transient activity of the Cas9 protein, combined with the simple mutation detection in GO founders by the T7E1 assay. PMID:24875628

  14. 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

  15. 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

  16. The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana.

    PubMed

    Handa, Hirokazu

    2003-10-15

    The entire mitochondrial genome of rapeseed (Brassica napus L.) was sequenced and compared with that of Arabidopsis thaliana. The 221 853 bp genome contains 34 protein-coding genes, three rRNA genes and 17 tRNA genes. This gene content is almost identical to that of Arabidopsis: However the rps14 gene, which is a pseudo-gene in Arabidopsis, is intact in rapeseed. On the other hand, five tRNA genes are missing in rapeseed compared to Arabidopsis, although the set of mitochondrially encoded tRNA species is identical in the two Cruciferae. RNA editing events were systematically investigated on the basis of the sequence of the rapeseed mitochondrial genome. A total of 427 C to U conversions were identified in ORFs, which is nearly identical to the number in Arabidopsis (441 sites). The gene sequences and intron structures are mostly conserved (more than 99% similarity for protein-coding regions); however, only 358 editing sites (83% of total editings) are shared by rapeseed and Arabidopsis: Non-coding regions are mostly divergent between the two plants. One-third (about 78.7 kb) and two-thirds (about 223.8 kb) of the rapeseed and Arabidopsis mitochondrial genomes, respectively, cannot be aligned with each other and most of these regions do not show any homology to sequences registered in the DNA databases. The results of the comparative analysis between the rapeseed and Arabidopsis mitochondrial genomes suggest that higher plant mitochondria are extremely conservative with respect to coding sequences and somewhat conservative with respect to RNA editing, but that non-coding parts of plant mitochondrial DNA are extraordinarily dynamic with respect to structural changes, sequence acquisition and/or sequence loss.

  17. Alu repeats as markers for human population genetics

    SciTech Connect

    Batzer, M.A.; Alegria-Hartman, M.; Bazan, H.

    1993-09-01

    The Human-Specific (HS) subfamily of Alu sequences is comprised of a group of 500 nearly identical members which are almost exclusively restricted to the human genome. Individual subfamily members share an average of 97.9% nucleotide identity with each other and an average of 98.9% nucleotide identity with the HS subfamily consensus sequence. HS Alu family members are thought to be derived from a single source ``master`` gene, and have an average age of 2.8 million years. We have developed a Polymerase Chain Reaction (PCR) based assay using primers complementary to the 5 in. and 3 in. unique flanking DNA sequences from each HS Alu that allows the locus to be assayed for the presence or absence of an Alu repeat. Individual HS Alu sequences were found to be either monomorphic or dimorphic for the presence or absence of each repeat. The monomorphic HS Alu family members inserted in the human genome after the human/great ape divergence (which is thought to have occurred 4--6 million years ago), but before the radiation of modem man. The dimorphic HS Alu sequences inserted in the human genome after the radiation of modem man (within the last 200,000-one million years) and represent a unique source of information for human population genetics and forensic DNA analyses. These sites can be developed into Dimorphic Alu Sequence Tagged Sites (DASTS) for the Human Genome Project as well. HS Alu family member insertion dimorphism differs from other types of polymorphism (e.g. Variable Number of Tandem Repeat [VNTR] or Restriction Fragment Length Polymorphism [RFLP]) because individuals share HS Alu family member insertions based upon identity by descent from a common ancestor as a result of a single event which occurred one time within the human population. The VNTR and RFLP polymorphisms may arise multiple times within a population and are identical by state only.

  18. News Editing. Second Edition.

    ERIC Educational Resources Information Center

    Westley, Bruce H.

    A revision of the first edition of "News Editing," this is a textbook for the newspaper editor. The duties of the editor are detailed, as are those of other newspaper employees. Among the basic editing skills the author includes suggestions for sentence structure, word usage, and vocabulary. Examples are given of editing for objectivity, handling…

  19. 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

  20. 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.

  1. Alu Elements as Novel Regulators of Gene Expression in Type 1 Diabetes Susceptibility Genes?

    PubMed Central

    Kaur, Simranjeet; Pociot, Flemming

    2015-01-01

    Despite numerous studies implicating Alu repeat elements in various diseases, there is sparse information available with respect to the potential functional and biological roles of the repeat elements in Type 1 diabetes (T1D). Therefore, we performed a genome-wide sequence analysis of T1D candidate genes to identify embedded Alu elements within these genes. We observed significant enrichment of Alu elements within the T1D genes (p-value < 10e−16), which highlights their importance in T1D. Functional annotation of T1D genes harboring Alus revealed significant enrichment for immune-mediated processes (p-value < 10e−6). We also identified eight T1D genes harboring inverted Alus (IRAlus) within their 3' untranslated regions (UTRs) that are known to regulate the expression of host mRNAs by generating double stranded RNA duplexes. Our in silico analysis predicted the formation of duplex structures by IRAlus within the 3'UTRs of T1D genes. We propose that IRAlus might be involved in regulating the expression levels of the host T1D genes. PMID:26184322

  2. Alu Elements as Novel Regulators of Gene Expression in Type 1 Diabetes Susceptibility Genes?

    PubMed

    Kaur, Simranjeet; Pociot, Flemming

    2015-07-13

    Despite numerous studies implicating Alu repeat elements in various diseases, there is sparse information available with respect to the potential functional and biological roles of the repeat elements in Type 1 diabetes (T1D). Therefore, we performed a genome-wide sequence analysis of T1D candidate genes to identify embedded Alu elements within these genes. We observed significant enrichment of Alu elements within the T1D genes (p-value < 10e-16), which highlights their importance in T1D. Functional annotation of T1D genes harboring Alus revealed significant enrichment for immune-mediated processes (p-value < 10e-6). We also identified eight T1D genes harboring inverted Alus (IRAlus) within their 3' untranslated regions (UTRs) that are known to regulate the expression of host mRNAs by generating double stranded RNA duplexes. Our in silico analysis predicted the formation of duplex structures by IRAlus within the 3'UTRs of T1D genes. We propose that IRAlus might be involved in regulating the expression levels of the host T1D genes.

  3. African origin of human-specific polymorphic Alu insertions

    SciTech Connect

    Batzer, M.A.; Alegria-Hartman, M. ); Stoneking, M. ); Bazan, H.; Kass, D.H.; Shaikh, T.H.; Scheer, W.D. ); Novick, G.E.; Herrera, R.J. ); Ioannou, P.A. )

    1994-12-06

    Alu elements are a family of interspersed repeats that have mobilized throughout primate genomes by retroposition from a few [open quotes]master[close quotes] genes. Among the 500,000 Alu elements in the human genome are members of the human-specific subfamily that are not fixed in the human species. Four such polymorphic human-specific Alu insertions were analyzed by a rapid, PCR-based assay. These four polymorphic Alu insertions were shown to be absent from the genomes of a number of nonhuman primates, consistent with their arising as human genetic polymorphisms sometime after the human/African ape divergence. Analysis of 664 unrelated individuals from 16 population groups from around the world revealed substantial levels of variation within population groups and significant genetic differentiation among groups. No significant associations were found among the four loci, consistent with their location on different chromosomes. A maximum-likelihood tree of population relationships showed four major groupings consisting of Africa, Europe, Asia/Americas, and Australia/New Guinea, which is concordant with similar trees based on other loci. A particularly useful feature of the polymorphic Alu insertions is that the ancestral state is known to be the absence of the Alu element, and the presence of the Alu element at a particular chromosomal site reflects a single, unique event in human evolution. A hypothetical ancestral group can then be included in the tree analysis, with the frequency of each insertion set to zero. The ancestral group connected to the maximum-likelihood tree within the African branch, which suggests an African origin of these polymorphic Alu insertions. These data are concordant with other diverse data sets, which lends further support to the recent African origin hypothesis for modern humans. Polymorphic Alu insertions represent a source of genetic variation for studying human population structure and evolution. 45 refs., 2 figs., 1 tab.

  4. Polymorphic Alu insertions among Mayan populations.

    PubMed

    Herrera, R J; Rojas, D P; Terreros, M C

    2007-01-01

    The Mayan homeland within Mesoamerica spans five countries: Belize, El Salvador, Guatemala, Honduras and Mexico. There are indications that the people we call the Maya migrated from the north to the highlands of Guatemala as early as 4000 B.C. Their existence was village-based and agricultural. The culture of these Preclassic Mayans owes much to the earlier Olmec civilization, which flourished in the southern portion of North America. In this study, four different Mayan groups were examined to assess their genetic variability. Ten polymorphic Alu insertion (PAI) loci were employed to ascertain the genetic affinities among these Mayan groups. North American, African, European and Asian populations were also examined as reference populations. Our results suggest that the Mayan groups examined in this study are not genetically homogeneous. PMID:17151812

  5. The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition.

    PubMed

    Moldovan, John B; Moran, John V

    2015-05-01

    Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements.

  6. The Zinc-Finger Antiviral Protein ZAP Inhibits LINE and Alu Retrotransposition.

    PubMed

    Moldovan, John B; Moran, John V

    2015-05-01

    Long INterspersed Element-1 (LINE-1 or L1) is the only active autonomous retrotransposon in the human genome. To investigate the interplay between the L1 retrotransposition machinery and the host cell, we used co-immunoprecipitation in conjunction with liquid chromatography and tandem mass spectrometry to identify cellular proteins that interact with the L1 first open reading frame-encoded protein, ORF1p. We identified 39 ORF1p-interacting candidate proteins including the zinc-finger antiviral protein (ZAP or ZC3HAV1). Here we show that the interaction between ZAP and ORF1p requires RNA and that ZAP overexpression in HeLa cells inhibits the retrotransposition of engineered human L1 and Alu elements, an engineered mouse L1, and an engineered zebrafish LINE-2 element. Consistently, siRNA-mediated depletion of endogenous ZAP in HeLa cells led to a ~2-fold increase in human L1 retrotransposition. Fluorescence microscopy in cultured human cells demonstrated that ZAP co-localizes with L1 RNA, ORF1p, and stress granule associated proteins in cytoplasmic foci. Finally, molecular genetic and biochemical analyses indicate that ZAP reduces the accumulation of full-length L1 RNA and the L1-encoded proteins, yielding mechanistic insight about how ZAP may inhibit L1 retrotransposition. Together, these data suggest that ZAP inhibits the retrotransposition of LINE and Alu elements. PMID:25951186

  7. 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

  8. 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.

  9. Nonlinear analysis of correlations in Alu repeat sequences in DNA

    NASA Astrophysics Data System (ADS)

    Xiao, Yi; Huang, Yanzhao; Li, Mingfeng; Xu, Ruizhen; Xiao, Saifeng

    2003-12-01

    We report on a nonlinear analysis of deterministic structures in Alu repeats, one of the richest repetitive DNA sequences in the human genome. Alu repeats contain the recognition sites for the restriction endonuclease AluI, which is what gives them their name. Using the nonlinear prediction method developed in chaos theory, we find that all Alu repeats have novel deterministic structures and show strong nonlinear correlations that are absent from exon and intron sequences. Furthermore, the deterministic structures of Alus of younger subfamilies show panlike shapes. As young Alus can be seen as mutation free copies from the “master genes,” it may be suggested that the deterministic structures of the older subfamilies are results of an evolution from a “panlike” structure to a more diffuse correlation pattern due to mutation.

  10. The use of dimorphic Alu insertions in human DNA fingerprinting

    SciTech Connect

    Novick, G.E.; Gonzalez, T.; Garrison, J.; Novick, C.C.; Herrera, R.J.; Batzer, M.A.; Deininger, P.L.

    1992-12-04

    We have characterized certain Human Specific Alu Insertions as either dimorphic (TPA25, PV92, APO), sightly dimorphic (C2N4 and C4N4) or monomorphic (C3N1, C4N6, C4N2, C4N5, C4N8), based on studies of Caucasian, Asian, American Black and African Black populations. Our approach is based upon: (1) PCR amplification using primers directed to the sequences that flank the site of insertion of the different Alu elements studied; (2) gel electrophoresis and scoring according to the presence or absence of an Alu insertion in one or both homologous chromosomes; (3) allelic frequencies calculated and compared according to Hardy-Weinberg equilibrium. Our DNA fingerprinting procedure using PCR amplification of dimorphic Human Specific Alu insertions, is stable enough to be used not only as a tool for genetic mapping but also to characterize populations, study migrational patterns and track the inheritance of human genetic disorders.

  11. Origin and diversification of minisatellites derived from human Alu sequences.

    PubMed

    Jurka, Jerzy; Gentles, Andrew J

    2006-01-01

    We analyze minisatellites derived from Alu fragments corresponding approximately to the first 44 bases of human Alu consensus sequences from different subfamilies. The origin of Alu-derived minisatellites appears to have been mediated by short flanking repeats, as first proposed by Haber and Louis [Haber, J.E., Louis, E.J., 1998. Minisatellite origins in yeast and humans. Genomics 48, 132-135.]. We also present evidence for base substitutions and deletions introduced to minisatellites by gene conversion with partially similar but unrelated flanking regions. Segments flanked by short direct repeats are relatively common in different regions of Alu and other repetitive sequences. Our analysis shows that they can be effectively used in comparative studies of the overall sequence context which may contribute to instability of DNA segments flanked by short direct repeats.

  12. 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.

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

    PubMed Central

    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

  14. 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

  15. Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis.

    PubMed

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

    2015-10-01

    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.

  16. Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis.

    PubMed

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

    2015-10-01

    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

  17. Deficient RNA-editing enzyme ADAR2 in an amyotrophic lateral sclerosis patient with a FUS(P525L) mutation.

    PubMed

    Aizawa, Hitoshi; Hideyama, Takuto; Yamashita, Takenari; Kimura, Takashi; Suzuki, Naoki; Aoki, Masashi; Kwak, Shin

    2016-10-01

    Mutations in the fused in sarcoma (FUS) gene can cause amyotrophic lateral sclerosis (ALS), and FUS gene mutations have been reported in sporadic ALS patients with basophilic cytoplasmic inclusions. Deficiency of adenosine deaminase acting on RNA 2 (ADAR2), an enzyme that specifically catalyzes GluA2 Q/R site-editing, has been reported in considerable proportions of spinal motor neurons of the majority of sporadic ALS patients. We describe the relationship between GluA2 Q/R site-editing efficiency and FUS-positive inclusions in a patient with FUS(P525L). A 24-year-old woman with ALS presented with basophilic cytoplasmic inclusions, significantly reduced GluA2 Q/R site-editing efficiency in the spinal motor neurons, and markedly decreased ADAR2 mRNA levels. Neuropathologic examination showed that not all spinal motor neurons expressed ADAR2 and revealed FUS-positive cytoplasmic inclusions in motor neurons irrespective of ADAR2 immunoreactivity. There were no phosphorylated transactive response (TAR) DNA-binding protein 43 kDa (TDP-43)-positive inclusions, indicating that there was no tight correlation between ADAR2 deficiency and TDP-43 deposition. ADAR2 deficiency can occur in ALS patients with a FUS(P525L) mutation and is unrelated to the presence of FUS-positive inclusions. FUS-associated ALS may share neurodegenerative characteristics with classical sporadic ALS.

  18. LINE-1 and Alu hypomethylation in mucoepidermoid carcinoma

    PubMed Central

    2013-01-01

    Background Mucoepidermoid carcinoma (MEC) can be classified into low-, intermediate-, and high-grade tumors based on its histological features. MEC is mainly composed of three cell types (squamous or epidermoid, mucous and intermediate cells), which correlates with the histological grade and reflects its clinical behavior. Most cancers exhibit reduced methylation of repetitive sequences such as Long INterspersed Element-1 (LINE-1) and Alu elements. However, to date very little information is available on the LINE-1 and Alu methylation status in MEC. The aim of this study was to investigate LINE-1 and Alu element methylation in MEC and compare if key differences in the methylation status exist between the three different cell types, and adjacent normal salivary gland cells, to see if this may reflect the histological grade. Methods LINE-1 and Alu element methylation of 24 MEC, and 14 normal salivary gland tissues were compared using Combine Bisulfite Restriction Analysis (COBRA). Furthermore, the three different cell types from MEC samples were isolated for enrichment by laser capture microdissection (LCM), essentially to see if COBRA was likely to increase the predictive value of LINE-1 and Alu element methylation. Results LINE-1 and Alu element methylation levels were significantly different (p<0.001) between the cell types, and showed a stepwise decrease from the adjacent normal salivary gland to the intermediate, mucous and squamous cells. The reduced methylation levels of LINE-1 were correlated with a poorer histological grade. In addition, MEC tissue showed a significantly lower level of LINE-1 and Alu element methylation overall compared to normal salivary gland tissue (p<0.001). Conclusions Our findings suggest that LINE-1 methylation differed among histological grade mucoepidermoid carcinoma. Hence, this epigenetic event may hold value for MEC diagnosis and prognostic prediction. PMID:23510117

  19. Alu-associated enhancement of single nucleotide polymorphisms in the human genome.

    PubMed

    Ng, Siu-Kin; Xue, Hong

    2006-03-01

    Identifying features shaping the architecture of sequence variations is important for understanding genome evolution and mapping disease loci. In this study, high-resolution scanning of Alu-centered alignments of the human genome sequences has revealed a striking elevation of the frequency of single nucleotide polymorphisms (SNP) in the body and tail of Alu sequences compared to flanking regions. This enhancement in SNP density is evident for all twenty-four chromosomes, and in both the Alu-body and Alu-tail, which together may be referred to as the Alu-SNPs. Reduced levels of Alu-SNPs in the sex chromosomes, especially in the non-recombining NRY region of the Y chromosome, are consistent with recombination events playing an important role in the enhancement. The Alu elements are unstable recombination-mutation hotspots in the human genome, and it is suggested that the Alu-SNPs represent a key manifestation of this instability. Variations in Alu-SNPs among the HapMap populations of northern and western European ancestry (CEU), Han Chinese from Beijing (CHB), Japanese from Tokyo (JPT), and Yoruba from Ibadan, Nigeria (YRI) indicate that the Alu-SNPs provide useful sequence markers, in addition to the Alu-insertion polymorphisms themselves, for the delineation of human genome evolution. That Alu-SNP levels are highest in the youngest Alu-Y, intermediate in the Alu-S of intermediate age, and lowest in the oldest Alu-J is consistent with the occurrence of not only genetic drift but also natural selection on the Alu-SNPs. Such evolutionary selection in turn suggests that Alu-SNPs might include potential sites of disease association, and therefore deserve detailed investigation. PMID:16380220

  20. The Contribution of Alu Elements to Mutagenic DNA Double-Strand Break Repair

    PubMed Central

    Streva, Vincent A.; DeFreece, Cecily B.; Hedges, Dale J.; Deininger, Prescott L.

    2015-01-01

    Alu elements make up the largest family of human mobile elements, numbering 1.1 million copies and comprising 11% of the human genome. As a consequence of evolution and genetic drift, Alu elements of various sequence divergence exist throughout the human genome. Alu/Alu recombination has been shown to cause approximately 0.5% of new human genetic diseases and contribute to extensive genomic structural variation. To begin understanding the molecular mechanisms leading to these rearrangements in mammalian cells, we constructed Alu/Alu recombination reporter cell lines containing Alu elements ranging in sequence divergence from 0%-30% that allow detection of both Alu/Alu recombination and large non-homologous end joining (NHEJ) deletions that range from 1.0 to 1.9 kb in size. Introduction of as little as 0.7% sequence divergence between Alu elements resulted in a significant reduction in recombination, which indicates even small degrees of sequence divergence reduce the efficiency of homology-directed DNA double-strand break (DSB) repair. Further reduction in recombination was observed in a sequence divergence-dependent manner for diverged Alu/Alu recombination constructs with up to 10% sequence divergence. With greater levels of sequence divergence (15%-30%), we observed a significant increase in DSB repair due to a shift from Alu/Alu recombination to variable-length NHEJ which removes sequence between the two Alu elements. This increase in NHEJ deletions depends on the presence of Alu sequence homeology (similar but not identical sequences). Analysis of recombination products revealed that Alu/Alu recombination junctions occur more frequently in the first 100 bp of the Alu element within our reporter assay, just as they do in genomic Alu/Alu recombination events. This is the first extensive study characterizing the influence of Alu element sequence divergence on DNA repair, which will inform predictions regarding the effect of Alu element sequence divergence on both

  1. Germline Chromothripsis Driven by L1-Mediated Retrotransposition and Alu/Alu Homologous Recombination.

    PubMed

    Nazaryan-Petersen, Lusine; Bertelsen, Birgitte; Bak, Mads; Jønson, Lars; Tommerup, Niels; Hancks, Dustin C; Tümer, Zeynep

    2016-04-01

    Chromothripsis (CTH) is a phenomenon where multiple localized double-stranded DNA breaks result in complex genomic rearrangements. Although the DNA-repair mechanisms involved in CTH have been described, the mechanisms driving the localized "shattering" process remain unclear. High-throughput sequence analysis of a familial germline CTH revealed an inserted SVAE retrotransposon associated with a 110-kb deletion displaying hallmarks of L1-mediated retrotransposition. Our analysis suggests that the SVAE insertion did not occur prior to or after, but concurrent with the CTH event. We also observed L1-endonuclease potential target sites in other breakpoints. In addition, we found four Alu elements flanking the 110-kb deletion and associated with an inversion. We suggest that chromatin looping mediated by homologous Alu elements may have brought distal DNA regions into close proximity facilitating DNA cleavage by catalytically active L1-endonuclease. Our data provide the first evidence that active and inactive human retrotransposons can serve as endogenous mutagens driving CTH in the germline.

  2. The effect of the ketogenic diet on hippocampal GluR5 and Glu(6 mRNA expression and Q/R site editing in the kainate-induced epilepsy model.

    PubMed

    Xu, Xiangping; Sun, Ruopeng; Jin, Ruifeng

    2008-10-01

    Treatment with the ketogenic diet has been used to control refractory epilepsy for many years, although its anticonvulsant mechanism is unknown. By modulating synaptic transmission in the hippocampus, kainate receptors and their RNA editing might play a role in the antiseizure action of the treatment. To investigate the potential effect of the ketogenic diet on GluR(5) mRNA and GluR(6) mRNA expression and Q/R site editing, we used the kainate-induced epilepsy model in the present study. Reverse transcription polymerase chain reaction was performed to determine GluR(5) and GluR(6) mRNA expression, and RNA editing was analyzed with the BbvIota restriction enzyme assay. The results demonstrated that expression of GluR(6) mRNA, but not GluR(5) mRNA, was significantly increased after 8 weeks of dietary treatment. Neither the GluR(5) nor the GluR(6) RNA editing rate at the Q/R site was significantly changed by dietary treatment. These data indicate that GluR(6) may be involved in the anticonvulsant mechanism of ketogenic diet treatment.

  3. A knowledgebase of the human Alu repetitive elements.

    PubMed

    Mallona, Izaskun; Jordà, Mireia; Peinado, Miguel A

    2016-04-01

    Alu elements are the most abundant retrotransposons in the human genome with more than one million copies. Alu repeats have been reported to participate in multiple processes related with genome regulation and compartmentalization. Moreover, they have been involved in the facilitation of pathological mutations in many diseases, including cancer. The contribution of Alus and other repeats in genomic regulation is often overlooked because their study poses technical and analytical challenges hardly attainable with conventional strategies. Here we propose the integration of ontology-based semantic methods to query a knowledgebase for the human Alus. The knowledgebase for the human Alus leverages Sequence (SO) and Gene Ontologies (GO) and is devoted to address functional and genetic information in the genomic context of the Alus. For each Alu element, the closest gene and transcript are stored, as well their functional annotation according to GO, the state of the chromatin and the transcription factors binding sites inside the Alu. The model uses Web Ontology Language (OWL) and Semantic Web Rule Language (SWRL). As a case of use and to illustrate the utility of the tool, we have evaluated the epigenetic states of Alu repeats associated with gene promoters according to their transcriptional activity. The ontology is easily extendable, offering a scaffold for the inclusion of new experimental data. The RDF/XML formalization is freely available at http://aluontology.sourceforge.net/.

  4. A knowledgebase of the human Alu repetitive elements.

    PubMed

    Mallona, Izaskun; Jordà, Mireia; Peinado, Miguel A

    2016-04-01

    Alu elements are the most abundant retrotransposons in the human genome with more than one million copies. Alu repeats have been reported to participate in multiple processes related with genome regulation and compartmentalization. Moreover, they have been involved in the facilitation of pathological mutations in many diseases, including cancer. The contribution of Alus and other repeats in genomic regulation is often overlooked because their study poses technical and analytical challenges hardly attainable with conventional strategies. Here we propose the integration of ontology-based semantic methods to query a knowledgebase for the human Alus. The knowledgebase for the human Alus leverages Sequence (SO) and Gene Ontologies (GO) and is devoted to address functional and genetic information in the genomic context of the Alus. For each Alu element, the closest gene and transcript are stored, as well their functional annotation according to GO, the state of the chromatin and the transcription factors binding sites inside the Alu. The model uses Web Ontology Language (OWL) and Semantic Web Rule Language (SWRL). As a case of use and to illustrate the utility of the tool, we have evaluated the epigenetic states of Alu repeats associated with gene promoters according to their transcriptional activity. The ontology is easily extendable, offering a scaffold for the inclusion of new experimental data. The RDF/XML formalization is freely available at http://aluontology.sourceforge.net/. PMID:26827622

  5. Inhibition of LINE-1 and Alu retrotransposition by exosomes encapsidating APOBEC3G and APOBEC3F.

    PubMed

    Khatua, Atanu K; Taylor, Harry E; Hildreth, James E K; Popik, Waldemar

    2010-04-25

    Human cytidine deaminases, including APOBEC3G (A3G) and A3F, are part of a cellular defense system against retroviruses and retroelements including non-LTR retrotransposons LINE-1 (L1) and Alu. Expression of cellular A3 proteins is sufficient for inhibition of L1 and Alu retrotransposition, but the effect of A3 proteins transferred in exosomes on retroelement mobilization is unknown. Here, we demonstrate for the first time that exosomes secreted by CD4(+)H9 T cells and mature monocyte-derived dendritic cells encapsidate A3G and A3F and inhibit L1 and Alu retrotransposition. A3G is the major contributor to the inhibitory activity of exosomes, however, the contribution of A3F in H9 exosomes cannot be excluded. Additionally, we show that exosomes encapsidate mRNAs coding for A3 proteins. A3G mRNA, and less so A3F, was enriched in exosomes secreted by H9 cells. Exosomal A3G mRNA was functional in vitro. Whether exosomes inhibit retrotransposons in vivo requires further investigation.

  6. Cellular inhibitors of long interspersed element 1 and Alu retrotransposition.

    PubMed

    Bogerd, Hal P; Wiegand, Heather L; Hulme, Amy E; Garcia-Perez, José L; O'Shea, K Sue; Moran, John V; Cullen, Bryan R

    2006-06-01

    Long interspersed element (LINE) 1 retrotransposons are major genomic parasites that represent approximately 17% of the human genome. The LINE-1 ORF2 protein is also responsible for the mobility of Alu elements, which constitute a further approximately 11% of genomic DNA. Representative members of each element class remain mobile, and deleterious retrotransposition events can induce spontaneous genetic diseases. Here, we demonstrate that APOBEC3A and APOBEC3B, two members of the APOBEC3 family of human innate antiretroviral resistance factors, can enter the nucleus, where LINE-1 and Alu reverse transcription occurs, and specifically inhibit both LINE-1 and Alu retrotransposition. These data suggest that the APOBEC3 protein family may have evolved, at least in part, to defend the integrity of the human genome against endogenous retrotransposons.

  7. Structure of a putative trans-editing enzyme for prolyl-tRNA synthetase from Aeropyrum pernix K1 at 1.7 Å resolution

    SciTech Connect

    Murayama, Kazutaka; Kato-Murayama, Miyuki; Katsura, Kazushige; Uchikubo-Kamo, Tomomi; Yamaguchi-Hirafuji, Machiko; Kawazoe, Masahito; Akasaka, Ryogo; Hanawa-Suetsugu, Kyoko; Hori-Takemoto, Chie; Terada, Takaho; Shirouzu, Mikako; Yokoyama, Shigeyuki

    2005-01-01

    The three-dimensional structure of the APE2540 protein from A. pernix K1 has been determined by the multiple anomalous dispersion method at 1.7 Å resolution. The structure includes two monomers in the asymmetric unit and shares structural similarity with the YbaK protein or cysteinyl-tRNA{sup Pro} deacylase from H. influenzae. The crystal structure of APE2540, the putative trans-editing enzyme ProX from Aeropyrum pernix K1, was determined in a high-throughput manner. The crystal belongs to the monoclinic space group P2{sub 1}, with unit-cell parameters a = 47.4, b = 58.9, c = 53.6 Å, β = 106.8°. The structure was solved by the multiwavelength anomalous dispersion method at 1.7 Å and refined to an R factor of 16.8% (R{sub free} = 20.5%). The crystal structure includes two protein molecules in the asymmetric unit. Each monomer consists of eight β-strands and seven α-helices. A structure-homology search revealed similarity between the trans-editing enzyme YbaK (or cysteinyl-tRNA{sup Pro} deacylase) from Haemophilus influenzae (HI1434; 22% sequence identity) and putative ProX proteins from Caulobacter crescentus (16%) and Agrobacterium tumefaciens (21%)

  8. Co-occurrence of TDP-43 mislocalization with reduced activity of an RNA editing enzyme, ADAR2, in aged mouse motor neurons.

    PubMed

    Hideyama, Takuto; Teramoto, Sayaka; Hachiga, Kosuke; Yamashita, Takenari; Kwak, Shin

    2012-01-01

    TDP-43 pathology in spinal motor neurons is a neuropathological hallmark of sporadic amyotrophic lateral sclerosis (ALS) and has recently been shown to be closely associated with the downregulation of an RNA editing enzyme called adenosine deaminase acting on RNA 2 (ADAR2) in the motor neurons of sporadic ALS patients. Because TDP-43 pathology is found more frequently in the brains of elderly patients, we investigated the age-related changes in the TDP-43 localization and ADAR2 activity in mouse motor neurons. We found that ADAR2 was developmentally upregulated, and its mRNA expression level was progressively decreased in the spinal cords of aged mice. Motor neurons normally exhibit nuclear ADAR2 and TDP-43 immunoreactivity, whereas fast fatigable motor neurons in aged mice demonstrated a loss of ADAR2 and abnormal TDP-43 localization. Importantly, these motor neurons expressed significant amounts of the Q/R site-unedited AMPA receptor subunit 2 (GluA2) mRNA. Because expression of unedited GluA2 has been demonstrated as a lethality-causing molecular abnormality observed in the motor neurons, these results suggest that age-related decreases in ADAR2 activity play a mechanistic role in aging and serve as one of risk factors for ALS.

  9. Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas

    PubMed Central

    2010-01-01

    Background Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. Methods Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. Results From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A ~ Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). Conclusions Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively. PMID:20163738

  10. 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.

  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. 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.

  13. 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

  14. Alu sequence involvement in transcriptional insulation of the keratin 18 gene in transgenic mice.

    PubMed Central

    Thorey, I S; Ceceña, G; Reynolds, W; Oshima, R G

    1993-01-01

    The human keratin 18 (K18) gene is expressed in a variety of adult simple epithelial tissues, including liver, intestine, lung, and kidney, but is not normally found in skin, muscle, heart, spleen, or most of the brain. Transgenic animals derived from the cloned K18 gene express the transgene in appropriate tissues at levels directly proportional to the copy number and independently of the sites of integration. We have investigated in transgenic mice the dependence of K18 gene expression on the distal 5' and 3' flanking sequences and upon the RNA polymerase III promoter of an Alu repetitive DNA transcription unit immediately upstream of the K18 promoter. Integration site-independent expression of tandemly duplicated K18 transgenes requires the presence of either an 825-bp fragment of the 5' flanking sequence or the 3.5-kb 3' flanking sequence. Mutation of the RNA polymerase III promoter of the Alu element within the 825-bp fragment abolishes copy number-dependent expression in kidney but does not abolish integration site-independent expression when assayed in the absence of the 3' flanking sequence of the K18 gene. The characteristics of integration site-independent expression and copy number-dependent expression are separable. In addition, the formation of the chromatin state of the K18 gene, which likely restricts the tissue-specific expression of this gene, is not dependent upon the distal flanking sequences of the 10-kb K18 gene but rather may depend on internal regulatory regions of the gene. Images PMID:7692231

  15. An alu-based phylogeny of lemurs (infraorder: Lemuriformes).

    PubMed

    McLain, Adam T; Meyer, Thomas J; Faulk, Christopher; Herke, Scott W; Oldenburg, J Michael; Bourgeois, Matthew G; Abshire, Camille F; Roos, Christian; Batzer, Mark A

    2012-01-01

    LEMURS (INFRAORDER: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55-60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus) was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the exclusion of

  16. An Alu-Based Phylogeny of Lemurs (Infraorder: Lemuriformes)

    PubMed Central

    McLain, Adam T.; Meyer, Thomas J.; Faulk, Christopher; Herke, Scott W.; Oldenburg, J. Michael; Bourgeois, Matthew G.; Abshire, Camille F.

    2012-01-01

    Lemurs (infraorder: Lemuriformes) are a radiation of strepsirrhine primates endemic to the island of Madagascar. As of 2012, 101 lemur species, divided among five families, have been described. Genetic and morphological evidence indicates all species are descended from a common ancestor that arrived in Madagascar ∼55–60 million years ago (mya). Phylogenetic relationships in this species-rich infraorder have been the subject of debate. Here we use Alu elements, a family of primate-specific Short INterspersed Elements (SINEs), to construct a phylogeny of infraorder Lemuriformes. Alu elements are particularly useful SINEs for the purpose of phylogeny reconstruction because they are identical by descent and confounding events between loci are easily resolved by sequencing. The genome of the grey mouse lemur (Microcebus murinus) was computationally assayed for synapomorphic Alu elements. Those that were identified as Lemuriformes-specific were analyzed against other available primate genomes for orthologous sequence in which to design primers for PCR (polymerase chain reaction) verification. A primate phylogenetic panel of 24 species, including 22 lemur species from all five families, was examined for the presence/absence of 138 Alu elements via PCR to establish relationships among species. Of these, 111 were phylogenetically informative. A phylogenetic tree was generated based on the results of this analysis. We demonstrate strong support for the monophyly of Lemuriformes to the exclusion of other primates, with Daubentoniidae, the aye-aye, as the basal lineage within the infraorder. Our results also suggest Lepilemuridae as a sister lineage to Cheirogaleidae, and Indriidae as sister to Lemuridae. Among the Cheirogaleidae, we show strong support for Microcebus and Mirza as sister genera, with Cheirogaleus the sister lineage to both. Our results also support the monophyly of the Lemuridae. Within Lemuridae we place Lemur and Hapalemur together to the exclusion of

  17. Detecting Alu insertions from high-throughput sequencing data

    PubMed Central

    David, Matei; Mustafa, Harun; Brudno, Michael

    2013-01-01

    High-throughput sequencing technologies have allowed for the cataloguing of variation in personal human genomes. In this manuscript, we present alu-detect, a tool that combines read-pair and split-read information to detect novel Alus and their precise breakpoints directly from either whole-genome or whole-exome sequencing data while also identifying insertions directly in the vicinity of existing Alus. To set the parameters of our method, we use simulation of a faux reference, which allows us to compute the precision and recall of various parameter settings using real sequencing data. Applying our method to 100 bp paired Illumina data from seven individuals, including two trios, we detected on average 1519 novel Alus per sample. Based on the faux-reference simulation, we estimate that our method has 97% precision and 85% recall. We identify 808 novel Alus not previously described in other studies. We also demonstrate the use of alu-detect to study the local sequence and global location preferences for novel Alu insertions. PMID:23921633

  18. 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

  19. Creative News Editing. Second Edition.

    ERIC Educational Resources Information Center

    Crowell, Alfred A.

    Designed to teach college students how to edit, this book provides an overview of skills required to produce the newspaper, emphasizing the editing of copy, the writing of headlines, and layout and production techniques. The author discusses several theories about how to edit newspapers; the importance of staff organization on metropolitan…

  20. 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.

  1. 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

  2. Alu Insertions and Genetic Diversity: A Preliminary Investigation by an Undergraduate Bioinformatics Class

    ERIC Educational Resources Information Center

    Elwess, Nancy L.; Duprey, Stephen L.; Harney, Lindesay A.; Langman, Jessie E.; Marino, Tara C.; Martinez, Carolina; McKeon, Lauren L.; Moss, Chantel I. E.; Myrie, Sasha S.; Taylor, Luke Ryan

    2008-01-01

    "Alu"-insertion polymorphisms were used by an undergraduate Bioinformatics class to study how these insertion sites could be the basis for an investigation in human population genetics. Based on the students' investigation, both allele and genotype "Alu" frequencies were determined for African-American and Japanese populations as well as a…

  3. Assessment of Azorean ancestry by Alu insertion polymorphisms.

    PubMed

    Branco, Claudia C; Palla, Raquel; Lino, Sílvia; Pacheco, Paula R; Cabral, Rita; De Fez, Laura; Peixoto, Bernardo R; Mota-Vieira, Luisa

    2006-01-01

    Knowledge of population ancestry from genetic markers is essential, for example, to understand the history of human migration and to carry out admixture and association studies. Here we assess the genome ancestry of the Azorean population through analysis of six Alu polymorphic sites (TPA-25, ACE, APO, B65, PV92, and D1) in 65 Azoreans and 30 Portuguese unrelated blood donors and compare data for the Y-chromosome and mtDNA. Allele frequencies were calculated by direct counting. Statistical analysis was performed using Arlequin 2.0. Nei's genetic distance was calculated with DISPAN software, and trees were constructed by neighbor joining (NJ) using PHYLIP 3.63. The results show that all Alu insertions were polymorphic. APO is the closest to fixation. The less frequent insertions are PV92 and D1 in the Azores and Portugal, respectively. ACE and TPA-25 show the highest values of heterozygosity in both populations. Allele frequencies are very similar to those obtained in European populations. These results are validated by the Y-chromosome and mtDNA data, where the majority of the maternal and paternal lineages are European. Overall, these data are reflected in the phylogenetic tree, in which the Azoreans and the Portuguese branch with Catalans, Andalusians, Moroccans, and Algerians. We conclude that the population of the Azores shows no significant genetic differences from that of mainland Portugal and that it is an outbred population. Moreover, the data validate the use of Alu insertion polymorphisms to assess the origin and history of human populations. PMID:16493635

  4. Super-resolution imaging of fluorescently labeled, endogenous RNA Polymerase II in living cells with CRISPR/Cas9-mediated gene editing

    PubMed Central

    Cho, Won-Ki; Jayanth, Namrata; Mullen, Susan; Tan, Tzer Han; Jung, Yoon J.; Cissé, Ibrahim I.

    2016-01-01

    Live cell imaging of mammalian RNA polymerase II (Pol II) has previously relied on random insertions of exogenous, mutant Pol II coupled with the degradation of endogenous Pol II using a toxin, α-amanitin. Therefore, it has been unclear whether over-expression of labeled Pol II under an exogenous promoter may have played a role in reported Pol II dynamics in vivo. Here we label the endogenous Pol II in mouse embryonic fibroblast (MEF) cells using the CRISPR/Cas9 gene editing system. Using single-molecule based super-resolution imaging in the living cells, we captured endogenous Pol II clusters. Consistent with previous studies, we observed that Pol II clusters were short-lived (cluster lifetime ~8 s) in living cells. Moreover, dynamic responses to serum-stimulation, and drug-mediated transcription inhibition were all in agreement with previous observations in the exogenous Pol II MEF cell line. Our findings suggest that previous exogenously tagged Pol II faithfully recapitulated the endogenous polymerase clustering dynamics in living cells, and our approach may in principle be used to directly label transcription factors for live cell imaging. PMID:27782203

  5. Alu methylation serves as a biomarker for non-invasive diagnosis of glioma

    PubMed Central

    Zuo, Hao; Zhao, Longxiang; Huang, Chuanjun; Liu, Xiaojiang; Hou, Shiqiang; Qi, Jing; Shi, Wei

    2016-01-01

    Current techniques for diagnosing glioma are invasive and do not accurately predict prognosis. We developed a novel, non-invasive liquid chip assay to diagnose glioma and predict prognosis. Using this method, we determined the methylation state of the Alu element in cell-free DNA extracted from the serum of 109 glioma patients. Controls included 56 patients with benign intracranial tumors and 50 healthy subjects. Matched tumor tissues were processed for 36 patients. The cfDNA from glioma patients showed lower levels of Alu methylation than the controls (P<0.01). Alu methylation was also lower in high-grade than low-grade gliomas (P<0.01), indicating that Alu methylation correlates negatively with disease severity. Moreover, Alu methylation correlated positively with survival (P<0.01). These findings suggest high-throughput liquid chip could serve as a non-invasive diagnostic assay for glioma. PMID:27028997

  6. 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…

  7. 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

  8. Alu and LINE-1 hypomethylation is associated with HER2 enriched subtype of breast cancer.

    PubMed

    Park, So Yeon; Seo, An Na; Jung, Hae Yoen; Gwak, Jae Moon; Jung, Namhee; Cho, Nam-Yun; Kang, Gyeong Hoon

    2014-01-01

    The changes in DNA methylation status in cancer cells are characterized by hypermethylation of promoter CpG islands and diffuse genomic hypomethylation. Alu and long interspersed nucleotide element-1 (LINE-1) are non-coding genomic repetitive sequences and methylation of these elements can be used as a surrogate marker for genome-wide methylation status. This study was designed to evaluate the changes of Alu and LINE-1 hypomethylation during breast cancer progression from normal to pre-invasive lesions and invasive breast cancer (IBC), and their relationship with characteristics of IBC. We analyzed the methylation status of Alu and LINE-1 in 145 cases of breast samples including normal breast tissue, atypical ductal hyperplasia/flat epithelial atypia (ADH/FEA), ductal carcinoma in situ (DCIS) and IBC, and another set of 129 cases of IBC by pyrosequencing. Alu methylation showed no significant changes during multistep progression of breast cancer, although it tended to decrease during the transition from DCIS to IBC. In contrast, LINE-1 methylation significantly decreased from normal to ADH/FEA, while it was similar in ADH/FEA, DCIS and IBC. In IBC, Alu hypomethylation correlated with negative estrogen receptor (ER) status, and LINE-1 hypomethylation was associated with negative ER status, ERBB2 (HER2) amplification and p53 overexpression. Alu and LINE-1 methylation status was significantly different between breast cancer subtypes, and the HER2 enriched subtype had lowest methylation levels. In survival analyses, low Alu methylation status tended to be associated with poor disease-free survival of the patients. Our findings suggest that LINE-1 hypomethylation is an early event and Alu hypomethylation is probably a late event during breast cancer progression, and prominent hypomethylation of Alu and LINE-1 in HER2 enriched subtype may be related to chromosomal instability of this specific subtype.

  9. 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.

  10. An Alu-based phylogeny of gibbons (hylobatidae).

    PubMed

    Meyer, Thomas J; McLain, Adam T; Oldenburg, J Michael; Faulk, Christopher; Bourgeois, Matthew G; Conlin, Erin M; Mootnick, Alan R; de Jong, Pieter J; Roos, Christian; Carbone, Lucia; Batzer, Mark A

    2012-11-01

    Gibbons (Hylobatidae) are small, arboreal apes indigenous to Southeast Asia that diverged from other apes ∼15-18 Ma. Extant lineages radiated rapidly 6-10 Ma and are organized into four genera (Hylobates, Hoolock, Symphalangus, and Nomascus) consisting of 12-19 species. The use of short interspersed elements (SINEs) as phylogenetic markers has seen recent popularity due to several desirable characteristics: the ancestral state of a locus is known to be the absence of an element, rare potentially homoplasious events are relatively easy to resolve, and samples can be quickly and inexpensively genotyped. During radiation of primates, one particular family of SINEs, the Alu family, has proliferated in primate genomes. Nomascus leucogenys (northern white-cheeked gibbon) sequences were analyzed for repetitive content with RepeatMasker using a custom library. The sequences containing Alu elements identified as members of a gibbon-specific subfamily were then compared with orthologous positions in other primate genomes. A primate phylogenetic panel consisting of 18 primate species, including 13 gibbon species representing all four extant genera, was assayed for all loci, and a total of 125 gibbon-specific Alu insertions were identified. The resulting amplification patterns were used to generate a phylogenetic tree. We demonstrate significant support for Symphalangus as the most basal lineage within the family. Our findings also place Nomascus as a derived lineage, sister to Hoolock, with the Nomascus-Hoolock clade sister to Hylobates. Further, our analysis groups N. leucogenys and Nomascus siki as sister taxa to the exclusion of the other Nomascus species assayed. This study represents the first use of SINEs to determine the genus level phylogenetic relationships within the family Hylobatidae. These relationships have been resolved with robust support at most internal nodes, demonstrating the utility of SINE-based phylogenetic analysis. We postulate that hybridization

  11. 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.

  12. Assembly and characterization of novel Alu inserts detected from next-generation sequencing data

    PubMed Central

    Mustafa, Harun; David, Matei; Brudno, Michael

    2014-01-01

    Repetitive elements generally, and Alu inserts specifically are a large contributor to the recent evolution of the human genome. By assembling the sequences of novel Alu inserts using their respective subfamily consensus sequences as references, we found an exponential decay in the Alu subfamily call enrichment with increased number of sequence variants (Pearson correlation r=−0.68, p<0.0039). By mapping the sequences of these inserts to a human reference genome, we infer the reference Alu sources of a subset of the novel Alus, of which 85% were previously shown to be active. We also evaluate relationships between the loci of the novel inserts and their inferred sources. PMID:26442170

  13. Genome-wide analysis of the human Alu Yb-lineage

    PubMed Central

    2004-01-01

    The Alu Yb-lineage is a 'young' primarily human-specific group of short interspersed element (SINE) subfamilies that have integrated throughout the human genome. In this study, we have computationally screened the draft sequence of the human genome for Alu Yb-lineage subfamily members present on autosomal chromosomes. A total of 1,733 Yb Alu subfamily members have integrated into human autosomes. The average ages of Yb-lineage subfamilies, Yb7, Yb8 and Yb9, are estimated as 4.81, 2.39 and 2.32 million years, respectively. In order to determine the contribution of the Alu Yb-lineage to human genomic diversity, 1,202 loci were analysed using polymerase chain reaction (PCR)-based assays, which amplify the genomic regions containing individual Yb-lineage subfamily members. Approximately 20 per cent of the Yb-lineage Alu elements are polymorphic for insertion presence/absence in the human genome. Fewer than 0.5 per cent of the Yb loci also demonstrate insertions at orthologous positions in non-human primate genomes. Genomic sequencing of these unusual loci demonstrates that each of the orthologous loci from non-human primate genomes contains older Y, Sg and Sx Alu family members that have been altered, through various mechanisms, into Yb8 sequences. These data suggest that Alu Yb-lineage subfamily members are largely restricted to the human genome. The high copy number, level of insertion polymorphism and estimated age indicate that members of the Alu Yb elements will be useful in a wide range of genetic analyses. PMID:15588477

  14. Alu Insertion Polymorphisms and Human Evolution: Evidence for a Larger Population Size in Africa

    PubMed Central

    Stoneking, Mark; Fontius, Jennifer J.; Clifford, Stephanie L.; Soodyall, Himla; Arcot, Santosh S.; Saha, Nilmani; Jenkins, Trefor; Tahir, Mohammad A.; Deininger, Prescott L.; Batzer, Mark A.

    1997-01-01

    Alu insertion polymorphisms (polymorphisms consisting of the presence/absence of an Alu element at a particular chromosomal location) offer several advantages over other nuclear DNA polymorphisms for human evolution studies. First, they are typed by rapid, simple, PCR-based assays; second, they are stable polymorphisms—newly inserted Alu elements rarely undergo deletion; third, the presence of an Alu element represents identity by descent—the probability that different Alu elements would independently insert into the exact same chromosomal location is negligible; and fourth, the ancestral state is known with certainty to be the absence of an Alu element. We report here a study of 8 loci in 1500 individuals from 34 worldwide populations. African populations exhibit the most between-population differentiation, and the population tree is rooted in Africa; moreover, the estimated effective time of separation of African versus non-African populations is 137,000 ± 15,000 years ago, in accordance with other genetic data. However, a principal coordinates analysis indicates that populations from Sahul (Australia and New Guinea) are nearly as close to the hypothetical ancestor as are African populations, suggesting that there was an early expansion of tropical populations of our species. An analysis of heterozygosity versus genetic distance suggests that African populations have had a larger effective population size than non-African populations. Overall, these results support the African origin of modern humans in that an earlier expansion of the ancestors of African populations is indicated. PMID:9371742

  15. Patterns of Ancestral Human Diversity: An Analysis of Alu-Insertion and Restriction-Site Polymorphisms

    PubMed Central

    Watkins, W. S.; Ricker, C. E.; Bamshad, M. J.; Carroll, M. L.; Nguyen, S. V.; Batzer, M. A.; Harpending, H. C.; Rogers, A. R.; Jorde, L. B.

    2001-01-01

    We have analyzed 35 widely distributed, polymorphic Alu loci in 715 individuals from 31 world populations. The average frequency of Alu insertions (the derived state) is lowest in Africa (.42) but is higher and similar in India (.55), Europe (.56), and Asia (.57). A comparison with 30 restriction-site polymorphisms (RSPs) for which the ancestral state has been determined shows that the frequency of derived RSP alleles is also lower in Africa (.35) than it is in Asia (.45) and in Europe (.46). Neighbor-joining networks based on Alu insertions or RSPs are rooted in Africa and show African populations as separate from other populations, with high statistical support. Correlations between genetic distances based on Alu and nuclear RSPs, short tandem-repeat polymorphisms, and mtDNA, in the same individuals, are high and significant. For the 35 loci, Alu gene diversity and the diversity attributable to population subdivision is highest in Africa but is lower and similar in Europe and Asia. The distribution of ancestral alleles is consistent with an origin of early modern human populations in sub-Saharan Africa, the isolation and preservation of ancestral alleles within Africa, and an expansion out of Africa into Eurasia. This expansion is characterized by increasing frequencies of Alu inserts and by derived RSP alleles with reduced genetic diversity in non-African populations. PMID:11179020

  16. 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

  17. 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.

  18. Polymorphic Alu insertions and genetic diversity among African populations.

    PubMed

    Terreros, Maria C; Martinez, Laisel; Herrera, Rene J

    2005-10-01

    Thorough assessment of modern genetic diversity and interpopulation affinities within the African continent is essential for understanding the processes that have been at work during the course of worldwide human evolution. Regardless of whether autosomal, Y-chromosome, or mtDNA markers are used, allele- or haplotype-frequency data from African populations are necessary in setting the framework for the construction of global population phylogenies. In the present study we analyze genetic differentiation and population structure in a data set of nine African populations using 12 polymorphic Alu insertions (PAls). Furthermore, to place our findings within a global context, we also examined an equal number of non-African groups. Frequency data from 456 individuals presented for the first time in this work plus additional data obtained from the literature indicate an overall pattern of higher intrapopulation diversity in sub-Saharan populations than in northern Africa, a prominent differentiation between these two locations, an appreciably high degree of transcontinental admixture in Egypt, and significant discontinuity between Morocco and the Iberian peninsula. Moreover, the topologies of our phylogenetic analyses suggest that out of the studied sub-Saharan groups, the southern Bantu population of Sotho/ Tswana presents the highest level of antiquity, perhaps as a result of ancestral or acquired Khoisan genetic signals. Close affinities of eastern sub-Saharan populations with Egypt in the phylogenetic trees may indicate the existence of gene flow along the Nile River.

  19. Methylation Status of Alu and LINE-1 Interspersed Repetitive Sequences in Behcet's Disease Patients

    PubMed Central

    Yüksel, Şahru; Kucukazman, Selma Ozbek; Karataş, Gülten Sungur; Ozturk, Mehmet Akif; Prombhul, Sasiprapa; Hirankarn, Nattiya

    2016-01-01

    Behcet's Disease (BD) is a multisystem chronic inflammatory disease. The pathology is believed to involve both genetic susceptibility and environmental factors. Hypomethylation leading to activation of interspersed repetitive sequences (IRSs) such as LINE-1 and Alu contributes to the pathologies of autoimmune diseases and cancer. Herein, the epigenetic changes of IRSs in BD were evaluated using combined bisulfite restriction analysis-interspersed repetitive sequences (COBRA-IRS). DNA from neutrophils and peripheral blood mononuclear cells (PBMCs) of BD patients with ocular involvement that were in active or inactive states and healthy controls were used to analyze LINE-1 and Alu methylation levels. For Alu sequences, significant differences were observed in the frequency of uCuC alleles between PBMCs of patients and controls (p = 0.03), and between inactive patients and controls (p = 0.03). For neutrophils, the frequency of uCuC was significantly higher between patients and controls (p = 0.006) and between inactive patients and controls (p = 0.002). The partial methylation (uCmC + mCuC) frequencies of Alu between inactive patients and control samples also differed (p = 0.02). No statistically significant differences for LINE-1 were detected. Thus, changes in the methylation level of IRS elements might contribute to the pathogenesis of BD. The role of Alu transcripts in BD should be investigated further. PMID:27123441

  20. Linkage disequilibrium among RFLPs at the insulin-receptor locus despite intervening alu repeat sequences

    SciTech Connect

    Elbein, S.C. )

    1992-11-01

    Multiple mutations of the insulin receptor (INSR) gene have been identified in individuals with extreme insulin resistance. These mutations have included recombination events between Alu repeat units in the tyrosine kinase-encoding [beta]-chain region of the gene. To evaluate the influence of Alu and dinucleotide repetitive sequences on recombination events within the insulin receptor gene, the author examined the degree of linkage disequilibrium between RFLP pairs spanning the gene. The author established 228 independent haplotypes for seven RFLPs (two each for PstI, RsaI, and SstI and one for MspI and 172 independent haplotypes which included an additional RFLP with BglII) from 19 pedigrees. These RFLPs span >130 kb of this gene, and it was previously demonstrated that multiple Alu sequences separate RFLP pairs. Observed haplotype frequencies deviated significantly from those predicted. Pairwise analysis of RFLP showed high levels of linkage disequilibrium among RFLP in the [beta]-chain region of the insulin receptor, but not between [alpha]-chain RFLPs and those of the [beta]-chain. Disequilibrium was present among [beta]-chain RFLPs, despite separation by one or more Alu repeat sequences. The very strong linkage disequilibrium which was present in sizable regions of the INSR gene despite the presence of both Alu and microsatellite repeats suggested that these regions do not have a major impact on recombinations at this locus. 25 refs., 1 fig., 5 tabs.

  1. Polymorphic Alu insertions in six Brazilian African-derived populations.

    PubMed

    Cotrim, Nelson Henderson; Auricchio, Maria Teresa B M; Vicente, João Pedro; Otto, Paulo A; Mingroni-Netto, Regina Célia

    2004-01-01

    At least 25 African-derived populations (quilombo remnants) are believed to exist in the Ribeira River Valley, located in the southern part of São Paulo State, Brazil. We studied four Alu polymorphic loci (APO, ACE, TPA25, and FXIIIB) in individuals belonging to six quilombo remnants in addition to individuals sampled from the city of São Paulo. The allelic frequencies observed in the quilombo remnants were similar to those previously observed in African-derived populations from Central and North America. Genetic variability indexes (Fst and Gst values) in our quilombos were higher than the reported values for the majority of other populations analyzed for the same kind of markers, but lower than the variability usually observed in Amerindian groups. The observed high degree of genetic differentiation may be due to genetic drift, especially the founder effect. Our results suggest that these populations behave genetically as semi-isolates. The degree of genetic variability within populations was larger than among them, a finding described in other studies. In the neighbor-joining tree, some of the Brazilian quilombos clustered with the African and African-derived populations (São Pedro and Galvão), others with the Europeans (Pilões, Maria Rosa, and Abobral). Pedro Cubas was placed in an isolated branch. Principal component analysis was also performed and confirmed the trends observed in the neighbor-joining tree. Overall, the quilombos showed a higher degree of gene flow than average when compared to other worldwide populations, but similar to other African-derived populations.

  2. Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3.

    PubMed

    Gu, Shen; Yuan, Bo; Campbell, Ian M; Beck, Christine R; Carvalho, Claudia M B; Nagamani, Sandesh C S; Erez, Ayelet; Patel, Ankita; Bacino, Carlos A; Shaw, Chad A; Stankiewicz, Paweł; Cheung, Sau Wai; Bi, Weimin; Lupski, James R

    2015-07-15

    Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu-Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu-Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process.

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

    PubMed

    Hashimoto, Masakazu; Takemoto, Tatsuya

    2015-06-11

    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.

  4. 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

  5. Evolution of a pseudogene: exclusive survival of a functional mitochondrial nad7 gene supports Haplomitrium as the earliest liverwort lineage and proposes a secondary loss of RNA editing in Marchantiidae.

    PubMed

    Groth-Malonek, Milena; Wahrmund, Ute; Polsakiewicz, Monika; Knoop, Volker

    2007-04-01

    Gene transfer from the mitochondrion into the nucleus is a corollary of the endosymbiont hypothesis. The frequent and independent transfer of genes for mitochondrial ribosomal proteins is well documented with many examples in angiosperms, whereas transfer of genes for components of the respiratory chain is a rarity. A notable exception is the nad7 gene, encoding subunit 7 of complex I, in the liverwort Marchantia polymorpha, which resides as a full-length, intron-carrying and transcribed, but nonspliced pseudogene in the chondriome, whereas its functional counterpart is nuclear encoded. To elucidate the patterns of pseudogene degeneration, we have investigated the mitochondrial nad7 locus in 12 other liverworts of broad phylogenetic distribution. We find that the mitochondrial nad7 gene is nonfunctional in 11 of them. However, the modes of pseudogene degeneration vary: whereas point mutations, accompanied by single-nucleotide indels, predominantly introduce stop codons into the reading frame in marchantiid liverworts, larger indels introduce frameshifts in the simple thalloid and leafy jungermanniid taxa. Most notably, however, the mitochondrial nad7 reading frame appears to be intact in the isolated liverwort genus Haplomitrium. Its functional expression is shown by cDNA analysis identifying typical RNA-editing events to reconstitute conserved codon identities and also confirming functional splicing of the 2 liverwort-specific group II introns. We interpret our results 1) to indicate the presence of a functional mitochondrial nad7 gene in the earliest land plants and strongly supporting a basal placement of Haplomitrium among the liverworts, 2) to indicate different modes of pseudogene degeneration and chondriome evolution in the later branching liverwort clades, 3) to suggest a surprisingly long maintenance of a nonfunctional gene in the presumed oldest group of land plants, and 4) to support the model of a secondary loss of RNA-editing activity in marchantiid

  6. 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

  7. The ABA-deficiency suppressor locus HAS2 encodes the PPR protein LOI1/MEF11 involved in mitochondrial RNA editing.

    PubMed

    Sechet, Julien; Roux, Camille; Plessis, Anne; Effroy, Delphine; Frey, Anne; Perreau, François; Biniek, Catherine; Krieger-Liszkay, Anja; Macherel, David; North, Helen M; Mireau, Hakim; Marion-Poll, Annie

    2015-04-01

    The hot ABA-deficiency suppressor2 (has2) mutation increases drought tolerance and the ABA sensitivity of stomata closure and seed germination. Here we report that the HAS2 locus encodes the mitochondrial editing factor11 (MEF11), also known as lovastatin insensitive1. has2/mef11 mutants exhibited phenotypes very similar to the ABA-hypersensitive mutant, hai1-1 pp2ca-1 hab1-1 abi1-2, which is impaired in four genes encoding type 2C protein phosphatases (PP2C) that act as upstream negative regulators of the ABA signaling cascade. Like pp2c, mef11 plants were more resistant to progressive water stress and seed germination was more sensitive to paclobutrazol (a gibberellin biosynthesis inhibitor) as well as mannitol and NaCl, compared with the wild-type plants. Phenotypic alterations in mef11 were associated with the lack of editing of transcripts for the mitochondrial cytochrome c maturation FN2 (ccmFN2) gene, which encodes a cytochrome c-heme lyase subunit involved in cytochrome c biogenesis. Although the abundance of electron transfer chain complexes was not affected, their dysfunction could be deduced from increased respiration and altered production of hydrogen peroxide and nitric oxide in mef11 seeds. As minor defects in mitochondrial respiration affect ABA signaling, this suggests an essential role for ABA in mitochondrial retrograde regulation.

  8. Quantification of Unmethylated Alu (QUAlu): a tool to assess global hypomethylation in routine clinical samples

    PubMed Central

    Buj, Raquel; Mallona, Izaskun; Díez-Villanueva, Anna; Barrera, Víctor; Mauricio, Dídac; Puig-Domingo, Manel; Reverter, Jordi L.; Matias-Guiu, Xavier; Azuara, Daniel; Ramírez, Jose L.; Alonso, Sergio; Rosell, Rafael; Capellà, Gabriel; Perucho, Manuel; Robledo, Mercedes; Peinado, Miguel A.; Jordà, Mireia

    2016-01-01

    Hypomethylation of DNA is a hallmark of cancer and its analysis as tumor biomarker has been proposed, but its determination in clinical settings is hampered by lack of standardized methodologies. Here, we present QUAlu (Quantification of Unmethylated Alu), a new technique to estimate the Percentage of UnMethylated Alu (PUMA) as a surrogate for global hypomethylation. QUAlu consists in the measurement by qPCR of Alu repeats after digestion of genomic DNA with isoschizomers with differential sensitivity to DNA methylation. QUAlu performance has been evaluated for reproducibility, trueness and specificity, and validated by deep sequencing. As a proof of use, QUAlu has been applied to a broad variety of pathological examination specimens covering five cancer types. Major findings of the preliminary application of QUAlu to clinical samples include: (1) all normal tissues displayed similar PUMA; (2) tumors showed variable PUMA with the highest levels in lung and colon and the lowest in thyroid cancer; (3) stools from colon cancer patients presented higher PUMA than those from control individuals; (4) lung squamous cell carcinomas showed higher PUMA than lung adenocarcinomas, and an increasing hypomethylation trend associated with smoking habits. In conclusion, QUAlu is a simple and robust method to determine Alu hypomethylation in human biospecimens and may be easily implemented in research and clinical settings. PMID:26859682

  9. Genetic variation among world populations: inferences from 100 Alu insertion polymorphisms.

    PubMed

    Watkins, W Scott; Rogers, Alan R; Ostler, Christopher T; Wooding, Steve; Bamshad, Michael J; Brassington, Anna-Marie E; Carroll, Marion L; Nguyen, Son V; Walker, Jerilyn A; Prasad, B V Ravi; Reddy, P Govinda; Das, Pradipta K; Batzer, Mark A; Jorde, Lynn B

    2003-07-01

    We examine the distribution and structure of human genetic diversity for 710 individuals representing 31 populations from Africa, East Asia, Europe, and India using 100 Alu insertion polymorphisms from all 22 autosomes. Alu diversity is highest in Africans (0.349) and lowest in Europeans (0.297). Alu insertion frequency is lowest in Africans (0.463) and higher in Indians (0.544), E. Asians (0.557), and Europeans (0.559). Large genetic distances are observed among African populations and between African and non-African populations. The root of a neighbor-joining network is located closest to the African populations. These findings are consistent with an African origin of modern humans and with a bottleneck effect in the human populations that left Africa to colonize the rest of the world. Genetic distances among all pairs of populations show a significant product-moment correlation with geographic distances (r = 0.69, P < 0.00001). F(ST), the proportion of genetic diversity attributable to population subdivision is 0.141 for Africans/E. Asians/Europeans, 0.047 for E. Asians/Indians/Europeans, and 0.090 for all 31 populations. Resampling analyses show that approximately 50 Alu polymorphisms are sufficient to obtain accurate and reliable genetic distance estimates. These analyses also demonstrate that markers with higher F(ST) values have greater resolving power and produce more consistent genetic distance estimates.

  10. Enrichment analysis of Alu elements with different spatial chromatin proximity in the human genome.

    PubMed

    Gu, Zhuoya; Jin, Ke; Crabbe, M James C; Zhang, Yang; Liu, Xiaolin; Huang, Yanyan; Hua, Mengyi; Nan, Peng; Zhang, Zhaolei; Zhong, Yang

    2016-04-01

    Transposable elements (TEs) have no longer been totally considered as "junk DNA" for quite a time since the continual discoveries of their multifunctional roles in eukaryote genomes. As one of the most important and abundant TEs that still active in human genome, Alu, a SINE family, has demonstrated its indispensable regulatory functions at sequence level, but its spatial roles are still unclear. Technologies based on 3C (chromosome conformation capture) have revealed the mysterious three-dimensional structure of chromatin, and make it possible to study the distal chromatin interaction in the genome. To find the role TE playing in distal regulation in human genome, we compiled the new released Hi-C data, TE annotation, histone marker annotations, and the genome-wide methylation data to operate correlation analysis, and found that the density of Alu elements showed a strong positive correlation with the level of chromatin interactions (hESC: r = 0.9, P < 2.2 × 10(16); IMR90 fibroblasts: r = 0.94, P < 2.2 × 10(16)) and also have a significant positive correlation with some remote functional DNA elements like enhancers and promoters (Enhancer: hESC: r = 0.997, P = 2.3 × 10(-4); IMR90: r = 0.934, P = 2 × 10(-2); Promoter: hESC: r = 0.995, P = 3.8 × 10(-4); IMR90: r = 0.996, P = 3.2 × 10(-4)). Further investigation involving GC content and methylation status showed the GC content of Alu covered sequences shared a similar pattern with that of the overall sequence, suggesting that Alu elements also function as the GC nucleotide and CpG site provider. In all, our results suggest that the Alu elements may act as an alternative parameter to evaluate the Hi-C data, which is confirmed by the correlation analysis of Alu elements and histone markers. Moreover, the GC-rich Alu sequence can bring high GC content and methylation flexibility to the regions with more distal chromatin contact, regulating the transcription of tissue-specific genes. PMID:26861146

  11. Alu elements in primates are preferentially lost from areas of high GC content

    PubMed Central

    Brookfield, John FY

    2013-01-01

    The currently-accepted dogma when analysing human Alu transposable elements is that ‘young’ Alu elements are found in low GC regions and ‘old’ Alus in high GC regions. The correlation between high GC regions and high gene frequency regions make this observation particularly difficult to explain. Although a number of studies have tackled the problem, no analysis has definitively explained the reason for this trend. These observations have been made by relying on the subfamily as a proxy for age of an element. In this study, we suggest that this is a misleading assumption and instead analyse the relationship between the taxonomic distribution of an individual element and its surrounding GC environment. An analysis of 103906 Alu elements across 6 human chromosomes was carried out, using the presence of orthologous Alu elements in other primate species as a proxy for age. We show that the previously-reported effect of GC content correlating with subfamily age is not reflected by the ages of the individual elements. Instead, elements are preferentially lost from areas of high GC content over time. The correlation between GC content and subfamily may be due to a change in insertion bias in the young subfamilies. The link between Alu subfamily age and GC region was made due to an over-simplification of the data and is incorrect. We suggest that use of subfamilies as a proxy for age is inappropriate and that the analysis of ortholog presence in other primate species provides a deeper insight into the data. PMID:23717800

  12. 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,…

  13. 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

  14. Alu-mediated diverse and complex pathogenic copy-number variants within human chromosome 17 at p13.3

    PubMed Central

    Gu, Shen; Yuan, Bo; Campbell, Ian M.; Beck, Christine R.; Carvalho, Claudia M.B.; Nagamani, Sandesh C.S.; Erez, Ayelet; Patel, Ankita; Bacino, Carlos A.; Shaw, Chad A.; Stankiewicz, Paweł; Cheung, Sau Wai; Bi, Weimin; Lupski, James R.

    2015-01-01

    Alu repetitive elements are known to be major contributors to genome instability by generating Alu-mediated copy-number variants (CNVs). Most of the reported Alu-mediated CNVs are simple deletions and duplications, and the mechanism underlying Alu–Alu-mediated rearrangement has been attributed to non-allelic homologous recombination (NAHR). Chromosome 17 at the p13.3 genomic region lacks extensive low-copy repeat architecture; however, it is highly enriched for Alu repetitive elements, with a fraction of 30% of total sequence annotated in the human reference genome, compared with the 10% genome-wide and 18% on chromosome 17. We conducted mechanistic studies of the 17p13.3 CNVs by performing high-density oligonucleotide array comparative genomic hybridization, specifically interrogating the 17p13.3 region with ∼150 bp per probe density; CNV breakpoint junctions were mapped to nucleotide resolution by polymerase chain reaction and Sanger sequencing. Studied rearrangements include 5 interstitial deletions, 14 tandem duplications, 7 terminal deletions and 13 complex genomic rearrangements (CGRs). Within the 17p13.3 region, Alu–Alu-mediated rearrangements were identified in 80% of the interstitial deletions, 46% of the tandem duplications and 50% of the CGRs, indicating that this mechanism was a major contributor for formation of breakpoint junctions. Our studies suggest that Alu repetitive elements facilitate formation of non-recurrent CNVs, CGRs and other structural aberrations of chromosome 17 at p13.3. The common observation of Alu-mediated rearrangement in CGRs and breakpoint junction sequences analysis further demonstrates that this type of mechanism is unlikely attributed to NAHR, but rather may be due to a recombination-coupled DNA replicative repair process. PMID:25908615

  15. Detection of Alu sequences and mtDNA in comets using padlock probes.

    PubMed

    Shaposhnikov, Sergey; Larsson, Chatarina; Henriksson, Sara; Collins, Andrew; Nilsson, Mats

    2006-07-01

    Single cell gel electrophoresis, or the comet assay, is widely used to measure DNA damage and repair. However, the behaviour of the DNA under the conditions used for the comet assay is not fully understood. In developing a method for studying specific gene sequences within comets, using 'padlock probes' (circularizable oligonucleotide probes), we have first applied probes that hybridize to Alu repetitive elements and to mitochondrial DNA (mtDNA). During the sequence of stages in the comet assay, mtDNA progressively disperses into the surrounding agarose gel, showing no tendency to remain with nuclear DNA in the comets. In contrast, Alu probes remain associated with both tail and head DNA. PMID:16940044

  16. The coding region of the human c-mos pseudogene contains Alu repeat insertions.

    PubMed

    Zabarovsky, E R; Chumakov, I M; Prassolov, V S; Kisselev, L L

    1984-10-01

    We have determined the nucleotide sequence of an 841-bp fragment derived from a segment of the human genome previously cloned by Chumakov et al. [Gene 17 (1982) 19-26] and Zabarovsky et al. [Gene 23 (1983) 379-384] and containing regions homologous to the viral mos gene probe. This sequence displays homology with part of the coding region of the human and murine c-mos genes, contains several termination codons, and is interrupted by two Alu-family elements flanked by short direct repeats. Probably, the progenitor of the human c-mos gene was duplicated approximately at the time of mammalian divergence, was converted to a pseudogene, and acquired insertions of two Alu elements.

  17. Identification of homogeneously staining regions by G-banding and chromosome microdissection, and FISH marker selection using human Alu sequence primers in a scleractinian coral Coelastrea aspera Verrill, 1866 (Cnidaria)

    PubMed Central

    Taguchi, Takahiro; Kubota, Satoshi; Mezaki, Takuma; Tagami, Erika; Sekida, Satoko; Nakachi, Shu; Okuda, Kazuo; Tominaga, Akira

    2016-01-01

    Abstract Karyotype analysis was performed on the scleractinian coral Coelastrea aspera Verrill, 1866, commonly found along temperate coasts in Japan (30–35°N) and in coastal waters in the Indian and Pacific oceans. G-banding of Coelastrea aspera was successfully performed, although the banding pattern was not as clear as that in mammals. The karyogram clearly revealed that this coral had a homogeneously staining region (hsr) in chromosome 11. This hsr consisted of ribosomal RNA (rRNA) related genes, which was demonstrated by fluorescence in situ hybridization (FISH) with probes generated using 28S ribosomal DNA (rDNA) primers and those generated through chromosome microdissection. In addition, we conducted silver-stained nucleolus organizer region (Ag-NOR) analysis and found Ag depositions in the interphase nuclei but not on rRNA gene loci and hsr(s) in the mitotic stage. The hsr of this coral was observed in approximately 50% of the metaphase spreads analyzed. This may explain the diversity of coral rDNA based on the molecular study of sequence analysis. Furthermore, it was discovered that human telomere and Alu repeated sequences were present in this Coelastrea aspera. Probes derived from human Alu sequences are expected to play an important role in the classification of corals. Overall, our data can be of great value in discriminating among scleractinian coral species and understanding their genetics, including chromosomal evolution. PMID:27186338

  18. Identification of homogeneously staining regions by G-banding and chromosome microdissection, and FISH marker selection using human Alu sequence primers in a scleractinian coral Coelastrea aspera Verrill, 1866 (Cnidaria).

    PubMed

    Taguchi, Takahiro; Kubota, Satoshi; Mezaki, Takuma; Tagami, Erika; Sekida, Satoko; Nakachi, Shu; Okuda, Kazuo; Tominaga, Akira

    2016-01-01

    Karyotype analysis was performed on the scleractinian coral Coelastrea aspera Verrill, 1866, commonly found along temperate coasts in Japan (30-35°N) and in coastal waters in the Indian and Pacific oceans. G-banding of Coelastrea aspera was successfully performed, although the banding pattern was not as clear as that in mammals. The karyogram clearly revealed that this coral had a homogeneously staining region (hsr) in chromosome 11. This hsr consisted of ribosomal RNA (rRNA) related genes, which was demonstrated by fluorescence in situ hybridization (FISH) with probes generated using 28S ribosomal DNA (rDNA) primers and those generated through chromosome microdissection. In addition, we conducted silver-stained nucleolus organizer region (Ag-NOR) analysis and found Ag depositions in the interphase nuclei but not on rRNA gene loci and hsr(s) in the mitotic stage. The hsr of this coral was observed in approximately 50% of the metaphase spreads analyzed. This may explain the diversity of coral rDNA based on the molecular study of sequence analysis. Furthermore, it was discovered that human telomere and Alu repeated sequences were present in this Coelastrea aspera. Probes derived from human Alu sequences are expected to play an important role in the classification of corals. Overall, our data can be of great value in discriminating among scleractinian coral species and understanding their genetics, including chromosomal evolution. PMID:27186338

  19. 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…

  20. 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…

  1. 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…

  2. Polymorphic Alu Insertion/Deletion in Different Caste and Tribal Populations from South India

    PubMed Central

    Chinniah, Rathika; Vijayan, Murali; Thirunavukkarasu, Manikandan; Mani, Dhivakar; Raju, Kamaraj; Ravi, Padma Malini; Sivanadham, Ramgopal; C, Kandeepan; N, Mahalakshmi; Karuppiah, Balakrishnan

    2016-01-01

    Seven human-specific Alu markers were studied in 574 unrelated individuals from 10 endogamous groups and 2 hill tribes of Tamil Nadu and Kerala states. DNA was isolated, amplified by PCR-SSP, and subjected to agarose gel electrophoresis, and genotypes were assigned for various Alu loci. Average heterozygosity among caste populations was in the range of 0.292–0.468. Among tribes, the average heterozygosity was higher for Paliyan (0.3759) than for Kani (0.2915). Frequency differences were prominent in all loci studied except Alu CD4. For Alu CD4, the frequency was 0.0363 in Yadavas, a traditional pastoral and herd maintaining population, and 0.2439 in Narikuravars, a nomadic gypsy population. The overall genetic difference (Gst) of 12 populations (castes and tribes) studied was 3.6%, which corresponds to the Gst values of 3.6% recorded earlier for Western Asian populations. Thus, our study confirms the genetic similarities between West Asian populations and South Indian castes and tribes and supported the large scale coastal migrations from Africa into India through West Asia. However, the average genetic difference (Gst) of Kani and Paliyan tribes with other South Indian tribes studied earlier was 8.3%. The average Gst of combined South and North Indian Tribes (CSNIT) was 9.5%. Neighbor joining tree constructed showed close proximity of Kani and Paliyan tribal groups to the other two South Indian tribes, Toda and Irula of Nilgiri hills studied earlier. Further, the analysis revealed the affinities among populations and confirmed the presence of North and South India specific lineages. Our findings have documented the highly diverse (micro differentiated) nature of South Indian tribes, predominantly due to isolation, than the endogamous population groups of South India. Thus, our study firmly established the genetic relationship of South Indian castes and tribes and supported the proposed large scale ancestral migrations from Africa, particularly into South India

  3. 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.

  4. Heritability and heteromorphic distributions of AluI chromosome banding variants in twins

    SciTech Connect

    Huang, B.; Meyer, J.M.; Jackson-Cook, C.K.

    1995-07-03

    The heritability and heteromorphic appearance of chromosomal banding patterns induced through in situ digestion with the restriction enzyme AluI were studied by analyzing the chromosomes of 25 monozygotic and 25 dizygotic twin pairs selected at random from a juvenile twin registry. A total of 19 AluI banding variants were found to be heteromorphic, with the pericentromeric region of chromosome 3 and the satellites of chromosome 22 being most and least heteromorphic, respectively. As expected, the correlations of the semi-quantitative scores for each of the chromosomal variants were significantly higher between MZ twin pairs (ranging from 0.48 to 0.95) than DZ twin pairs (ranging from -0.02 to 0.69), suggesting that genetic factors play an important role in their appearance. This finding was confirmed in a model fitting analysis in which the heritabilities of the AluI-induced chromosome variants were found to range from 70 to 96% for 12/13 heteromorphisms studied. These consistent findings are significant in that these variants may be useful for family studies in clinical genetics. 23 refs., 6 figs., 3 tabs.

  5. Design and implementation of low power clock gated 64-bit ALU on ultra scale FPGA

    NASA Astrophysics Data System (ADS)

    Gupta, Ashutosh; Murgai, Shruti; Gulati, Anmol; Kumar, Pradeep

    2016-03-01

    64-bit energy efficient Arithmetic and Logic Unit using negative latch based clock gating technique is designed in this paper. The 64-bit ALU is designed using multiplexer based full adder cell. We have designed a 64-bit ALU with a gated clock. We have used negative latch based circuit for generating gated clock. This gated clock is used to control the multiplexer based 64-bit ALU. The circuit has been synthesized on kintex FPGA through Xilinx ISE Design Suite 14.7 using 28 nm technology in Verilog HDL. The circuit has been simulated on Modelsim 10.3c. The design is verified using System Verilog on QuestaSim in UVM environment. We have achieved 74.07%, 92. 93% and 95.53% reduction in total clock power, 89.73%, 91.35% and 92.85% reduction in I/Os power, 67.14%, 62.84% and 74.34% reduction in dynamic power and 25.47%, 29.05% and 46.13% reduction in total supply power at 20 MHz, 200 MHz and 2 GHz frequency respectively. The power has been calculated using XPower Analyzer tool of Xilinx ISE Design Suite 14.3.

  6. The current source of human Alu retroposons is a conserved gene shared with Old World monkey

    SciTech Connect

    Britten, R.J.; Stout, D.B.; Davidson, E.H. )

    1989-05-01

    A significant fraction of human Alu repeated sequences are members of the precise, recently inserted class. A cloned member of this class has been used as a probe for interspecies hybridization and thermal stability determination. The probe was reassociated with human, mandrill, and spider monkey DNA under conditions such that only almost perfectly matching duplexes could form. Equally precise hybrids were formed with human and mandrill DNA (Old World monkey) but not with spider monkey DNA (New World). These measurements as well as reassociation kinetics show the presence in mandrill DNA of many precise class Alu sequences that are very similar or identical in quantity and sequence to those in human DNA. Human and mandrill are moderately distant species with a single-copy DNA divergence of about 6%. Nevertheless, their recently inserted Alu sequences arise by retroposition of transcripts of source genes with nearly identical sequences. Apparently a gene present in our common ancestor at the time of branching was inherited and highly conserved in sequence in both the lineage of Old World monkeys and the lineage of apes and man.

  7. Retrotransposon Alu is enriched in the epichromatin of HL-60 cells.

    PubMed

    Olins, Ada L; Ishaque, Naveed; Chotewutmontri, Sasithorn; Langowski, Jörg; Olins, Donald E

    2014-01-01

    Epichromatin, the surface of chromatin facing the nuclear envelope in an interphase nucleus, reveals a "rim" staining pattern with specific mouse monoclonal antibodies against histone H2A/H2B/DNA and phosphatidylserine epitopes. Employing a modified ChIP-Seq procedure on undifferentiated and differentiated human leukemic (HL-60/S4) cells,>95% of assembled epichromatin regions overlapped with Alu retrotransposons. They also exhibited enrichment of the AluS subfamily and of Alu oligomers. Furthermore, mapping epichromatin regions to the human chromosomes revealed highly similar localization patterns in the various cell states and with the different antibodies. Comparisons with available epigenetic databases suggested that epichromatin is neither "classical" heterochromatin nor highly expressing genes, implying another function at the surface of interphase chromatin. A modified chromatin immunoprecipitation procedure (xxChIP) was developed because the studied antibodies react generally with mononucleosomes and lysed chromatin. A second fixation is necessary to securely attach the antibodies to the epichromatin epitopes of the intact nucleus. PMID:24824428

  8. 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

  9. 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.

  10. Three RFLPs defining a haplotype associated with the common mutation in a human medium-chain acyl-CoA dehydrogenase (MCAD) deficiency occur in Alu repeats

    SciTech Connect

    Zhifang Zhang; Yeqing Zhou; Kelly, D.P.; Strauss, A.W. St. Louis Children's Hospital, MO ); Kolvraa, S.; Gregersen, N. )

    1993-06-01

    Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is a common inborn error of fatty-acid oxidation and may cause sudden infant death. Previous studies revealed that (i) homozygosity for an A-to-G mutation at nucleotide 985 of the mRNA coding region (A985G) is an extremely common cause of MCAD deficiency and (ii) MCAD deficiency is strongly associated with a particular haplotype for RFLPs for BanII, PstI, and TaqI. TaqI allele 2 is always associated with the A985G mutation in human MCAD deficiency. In this study, the authors have delineated the molecular basis of the RFLPs for PstI, BamHI, and TaqI in the human MCAD gene. Their results prove that the three RFLPs are caused by point mutations in the 8 kb of DNA encompassing exons 8--10 of the human MCAD gene. The TaqI polymorphism is caused by a C-to-A substitution 392 bp upstream of the exon 8, and the PstI and BamHI polymorphisms are due to T-to-C and G-to-A substitutions, respectively, which are 727 and 931 bp downstream of exon 10, respectively. All three RFLPs lie within Alu repetitive sequences. Comparison of intronic sequences immediately following exon 10 from two normal individuals with different haplotypes showed that this region contains densely packed Alu repeats and is highly polymorphic. The results are consistent both with a founder effect as the cause of the high prevalence of a single (A985G) mutation in MCAD deficiency and with its association with a particular haplotype for these intragenic RFLPs. 27 refs., 6 figs., 1 tab.

  11. Microlunatus cavernae sp. nov., a novel actinobacterium isolated from Alu ancient cave, Yunnan, South-West China.

    PubMed

    Cheng, Juan; Chen, Wei; Huo-Zhang, Bing; Nimaichand, Salam; Zhou, En-Min; Lu, Xin-Hua; Klenk, Hans-Peter; Li, Wen-Jun

    2013-07-01

    A Gram-positive, coccoid, non-endospore-forming actinobacterium, designated YIM C01117(T), was isolated from a soil sample collected from Alu ancient cave, Yunnan province, south-west China. Based on the 16S rRNA gene sequence analysis, strain YIM C01117(T) was shown to belong to the genus Microlunatus, with highest sequence similarity of 97.4 % to Microlunatus soli DSM 21800(T). The whole genomic DNA relatedness as shown by the DNA-DNA hybridization study between YIM C01117(T) and M. soli DSM 21800(T) had a low value (47 ± 2 %). Strain YIM C01117(T) was determined to contain LL-diaminopimelic acid with Gly, Glu and Ala amino acids (A3γ' type) in the cell wall. Whole-cell hydrolysates were found to contain glucose, galactose, mannose and ribose. The major polar lipids were determined to be phosphatidylglycerol and diphosphatidylglycerol. The predominant menaquinone system present is MK-9(H4), while the major fatty acids were identified to be anteiso-C15:0 (24.1 %), iso-C16:0 (22.3 %) and iso-C15:0 (11.4 %). The G+C content of the genomic DNA was determined to be 65.9 mol%. The chemotaxonomic and genotypic data support the affiliation of the strain YIM C01117(T) to the genus Microlunatus. The results of physiological and biochemical tests allow strain YIM C01117(T) to be differentiated phenotypically from recognized Microlunatus species. Strain YIM C01117(T) is therefore considered to represent a novel species of the genus Microlunatus, for which the name Microlunatus cavernae sp. nov. is proposed. The type strain is YIM C01117(T) (= DSM 26248(T) = JCM 18536(T)).

  12. 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

  13. Aberrant methylation and associated transcriptional mobilization of Alu elements contributes to genomic instability in hypoxia.

    PubMed

    Pal, Arnab; Srivastava, Tapasya; Sharma, Manish K; Mehndiratta, Mohit; Das, Prerna; Sinha, Subrata; Chattopadhyay, Parthaprasad

    2010-11-01

    Hypoxia is an integral part of tumorigenesis and contributes extensively to the neoplastic phenotype including drug resistance and genomic instability. It has also been reported that hypoxia results in global demethylation. Because a majority of the cytosine-phosphate-guanine (CpG) islands are found within the repeat elements of DNA, and are usually methylated under normoxic conditions, we suggested that retrotransposable Alu or short interspersed nuclear elements (SINEs) which show altered methylation and associated changes of gene expression during hypoxia, could be associated with genomic instability. U87MG glioblastoma cells were cultured in 0.1% O₂ for 6 weeks and compared with cells cultured in 21% O₂ for the same duration. Real-time PCR analysis showed a significant increase in SINE and reverse transcriptase coding long interspersed nuclear element (LINE) transcripts during hypoxia. Sequencing of bisulphite treated DNA as well as the Combined Bisulfite Restriction Analysis (COBRA) assay showed that the SINE loci studied underwent significant hypomethylation though there was patchy hypermethylation at a few sites. The inter-alu PCR profile of DNA from cells cultured under 6-week hypoxia, its 4-week revert back to normoxia and 6-week normoxia showed several changes in the band pattern indicating increased alu mediated genomic alteration. Our results show that aberrant methylation leading to increased transcription of SINE and reverse transcriptase associated LINE elements could lead to increased genomic instability in hypoxia. This might be a cause of genetic heterogeneity in tumours especially in variegated hypoxic environment and lead to a development of foci of more aggressive tumour cells.

  14. Design of Wallace tree multiplier and other components of a quantum ALU using reversible TSG gate

    NASA Astrophysics Data System (ADS)

    Thapliyal, Himanshu; Srinivas, M. B.

    2006-05-01

    This paper presents the design of a novel modified Wallace tree multiplier, using the reversible TSG gate proposed by the authors earlier. The novelty of the TSG gate is that it can also work singly as a reversible full adder. The TSG gate is also used in this paper to design various other reversible arithmetic and logical components that can be assembled to realize a primitive reversible/quantum ALU. It is also shown that these components are optimal, in terms of number of reversible gates and garbage outputs, compared to other designs existing in literature.

  15. Flash x-ray test of the CMOS/SOS SCP-STAR ALU

    SciTech Connect

    Brucker, G.J.; Kertesz, C.; Miller, M.; Oey, K.K.

    1984-12-01

    This paper reports on the measurement of the x-ray upset dose rate for a subsystem which was designed to represent the ALU part of the SCP-STAR spacecraft computer. This computer was fabricated totally with radhard, CMOS/SOS devices. The test was conducted at the OWL flash x-ray facility of Physics International. The results showed that the upset dose rate for the subsystem was 9.8 x 10/sup 10/ and 1.7 x 10/sup 11/ rads(Si)/s during dynamic and static modes of operation, respectively. Subsystem interactions did not compromise the upset levels of the discrete components.

  16. Trans-editing of mischarged tRNAs

    PubMed Central

    Ahel, Ivan; Korencic, Dragana; Ibba, Michael; Söll, Dieter

    2003-01-01

    Aminoacyl-tRNA synthetases (aaRSs) are multidomain proteins that specifically attach amino acids to their cognate tRNAs. Their most conserved, and presumably evolutionarily oldest, domains are the catalytic cores, which activate amino acids and transfer them to the 3′ ends of tRNAs. Additional domains appended to or inserted in the body of aaRSs increase efficiency and specificity of the aminoacylation process, either by providing additional tRNA contacts, or by hydrolyzing noncognate amino acid products (cis-editing). Here, we report specific tRNA-dependent trans-editing by aaRS-like proteins that reciprocate the editing domains of aaRSs, but not the remainder of the corresponding enzyme. A freestanding homologue of the prolyl-tRNA synthetase-editing domain, the PrdX protein from Clostridium sticklandii, efficiently and specifically hydrolyzes Ala-tRNAPro. Similarly, autonomous alanyl-tRNA synthetase-editing domain homologues (AlaX proteins) from Methanosarcina barkeri and Sulfolobus solfataricus hydrolyze Ser-tRNAAla and Gly-tRNAAla substrates. The discovery of autonomous editing proteins efficient in hydrolyzing misacylated products provides a direct link between ancestral aaRSs consisting solely of the catalytic core and extant enzymes to which functionally independent modules are appended. PMID:14663147

  17. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells

    PubMed Central

    Klawitter, Sabine; Fuchs, Nina V.; Upton, Kyle R.; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J.; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J. Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J.; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L.; Faulkner, Geoffrey J.; Schumann, Gerald G.

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs. PMID:26743714

  18. Analysis of the regions flanking the human insulin gene and sequence of an Alu family member.

    PubMed Central

    Bell, G I; Pictet, R; Rutter, W J

    1980-01-01

    The regions around the human insulin gene have been studied by heteroduplex, hybridization and sequence analysis. These studies indicated that there is a region of heterogeneous length located approximately 700 bp before the 5' end of the gene; and that the 19 kb of cloned DNA which includes the 1430 bp insulin gene as well as 5650 bp before and 11,500 bp after the gene is single copy sequence except for 500 bp located 6000 bp from the 3' end of the gene. This 500 bp segment contains a member of the Alu family of dispersed middle repetitive sequences as well as another less highly repeated homopolymeric segment. The sequence of this region was determined. This Alu repeat is bordered by 19 bp direct repeats and also contains an 83 bp sequence which is present twice. The regions flanking the human and rat I insulin genes were compared by heteroduplex analysis to localize homologous sequences in the flanking regions which could be involved in the regulation of insulin biosynthesis. The homology between the two genes is restricted to the region encoding preproinsulin and a short region of approximately 60 bp flanking the 5' side of the genes. Images PMID:6253909

  19. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

    PubMed

    Klawitter, Sabine; Fuchs, Nina V; Upton, Kyle R; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L; Faulkner, Geoffrey J; Schumann, Gerald G

    2016-01-08

    Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs.

  20. Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

    PubMed

    Klawitter, Sabine; Fuchs, Nina V; Upton, Kyle R; Muñoz-Lopez, Martin; Shukla, Ruchi; Wang, Jichang; Garcia-Cañadas, Marta; Lopez-Ruiz, Cesar; Gerhardt, Daniel J; Sebe, Attila; Grabundzija, Ivana; Merkert, Sylvia; Gerdes, Patricia; Pulgarin, J Andres; Bock, Anja; Held, Ulrike; Witthuhn, Anett; Haase, Alexandra; Sarkadi, Balázs; Löwer, Johannes; Wolvetang, Ernst J; Martin, Ulrich; Ivics, Zoltán; Izsvák, Zsuzsanna; Garcia-Perez, Jose L; Faulkner, Geoffrey J; Schumann, Gerald G

    2016-01-01

    Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established. Here we apply retrotransposon capture sequencing to eight hiPSC lines and three human embryonic stem cell (hESC) lines, revealing endogenous L1, Alu and SINE-VNTR-Alu (SVA) mobilization during reprogramming and pluripotent stem cell cultivation. Surprisingly, 4/7 de novo L1 insertions are full length and 6/11 retrotransposition events occurred in protein-coding genes expressed in pluripotent stem cells. We further demonstrate that an intronic L1 insertion in the CADPS2 gene is acquired during hiPSC cultivation and disrupts CADPS2 expression. These experiments elucidate endogenous retrotransposition, and its potential consequences, in hiPSCs and hESCs. PMID:26743714

  1. Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts.

    PubMed

    Caudron-Herger, Maïwen; Pankert, Teresa; Rippe, Karsten

    2016-05-01

    The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression.

  2. Regulation of nucleolus assembly by non-coding RNA polymerase II transcripts.

    PubMed

    Caudron-Herger, Maïwen; Pankert, Teresa; Rippe, Karsten

    2016-05-01

    The nucleolus is a nuclear subcompartment for tightly regulated rRNA production and ribosome subunit biogenesis. It also acts as a cellular stress sensor and can release enriched factors in response to cellular stimuli. Accordingly, the content and structure of the nucleolus change dynamically, which is particularly evident during cell cycle progression: the nucleolus completely disassembles during mitosis and reassembles in interphase. Although the mechanisms that drive nucleolar (re)organization have been the subject of a number of studies, they are only partly understood. Recently, we identified Alu element-containing RNA polymerase II transcripts (aluRNAs) as important for nucleolar structure and rRNA synthesis. Integrating these findings with studies on the liquid droplet-like nature of the nucleolus leads us to propose a model on how RNA polymerase II transcripts could regulate the assembly of the nucleolus in response to external stimuli and during cell cycle progression. PMID:27416361

  3. Evolution of an Alu DNA element of type Sx in the lineage of primates and the origin of an associated tetranucleotide microsatellite.

    PubMed

    Crouau-Roy, B; Clisson, I

    2000-08-01

    A 394-bp DNA fragment, which in human is on chromosome 6 near the MOG (myelin oligodendrocyte glycoprotein) gene and encompasses an Alu element and an associated tetranucleotide microsatellite, was sequenced from a large range of primate species to follow its evolutionary divergence and to understand the origin of the microsatellite. This Alu element is found at the same orthologous position in all primates sequenced, but the tetranucleotide repeat is present only in Catarrhini between the 3'-oligo(dA) of the Alu element and the 3' flanking direct repeat. Little intraspecific variation was found. Sequence identity values for this orthologous primate Alu averaged 90% (82-99%) with transitions comprising between 70% and 100% of the observed nucleotide substitutions. Although the insertion of the Alu element predates the separation of these species, the original sequence of the site of integration can still be identified. This identification of the direct repeats suggests an active role of the oligo(dA) of the Alu element in the origin of the tetranucleotide repeats. The microsatellite probably appeared after the insertion of the Alu element, early in the lineage leading to the common ancestor of the hominoids and the Old World monkeys.

  4. Gain of a New Exon by a Lineage-Specific Alu Element-Integration Event in the BCS1L Gene during Primate Evolution

    PubMed Central

    Park, Sang-Je; Kim, Young-Hyun; Lee, Sang-Rae; Choe, Se-Hee; Kim, Myung-Jin; Kim, Sun-Uk; Kim, Ji-Su; Sim, Bo-Woong; Song, Bong-Seok; Jeong, Kang-Jin; Jin, Yeung-Bae; Lee, Youngjeon; Park, Young-Ho; Park, Young Il; Huh, Jae-Won; Chang, Kyu-Tae

    2015-01-01

    BCS1L gene encodes mitochondrial protein and is a member of conserved AAA protein family. This gene is involved in the incorporation of Rieske FeS and Qcr10p into complex III of respiratory chain. In our previous study, AluYRa2-derived alternative transcript in rhesus monkey genome was identified. However, this transcript has not been reported in human genome. In present study, we conducted evolutionary analysis of AluYRa2-exonized transcript with various primate genomic DNAs and cDNAs from humans, rhesus monkeys, and crab-eating monkeys. Remarkably, our results show that AluYRa2 element has only been integrated into genomes of Macaca species. This Macaca lineage-specific integration of AluYRa2 element led to exonization event in the first intron region of BCS1L gene by producing a conserved 3′ splice site. Intriguingly, in rhesus and crab-eating monkeys, more diverse transcript variants by alternative splicing (AS) events, including exon skipping and different 5′ splice sites from humans, were identified. Alignment of amino acid sequences revealed that AluYRa2-exonized transcript has short N-terminal peptides. Therefore, AS events play a major role in the generation of various transcripts and proteins during primate evolution. In particular, lineage-specific integration of Alu elements and species-specific Alu-derived exonization events could be important sources of gene diversification in primates. PMID:26537194

  5. 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.

  6. Transcultural Counseling. Second edition.

    ERIC Educational Resources Information Center

    McFadden, John, Ed.

    New trends in transcultural theory, expanded cultural paradigms, innovative counseling techniques for working with diverse ethnic groups, and a comprehensive discussion of professional issues are presented in this second edition of a popular text. This edition is designed to support curriculum changes in counselor education programs to maximize…

  7. Association of the Alu insertion polymorphism in the progesterone receptor gene with breast cancer in a Mexican population

    PubMed Central

    Figuera, Luis E.; Flores-Ramos, Liliana Gómez; Puebla-Pérez, Ana María; Zúñiga-González, Guillermo Moisés

    2015-01-01

    Introduction The progesterone receptor (PR) gene plays an important role in reproduction-related events. Data on polymorphisms in the PR gene have revealed associations with cancer, particularly for the Alu insertion polymorphism, which has been suggested to affect progesterone receptor function and contribute to tumor promotion in the mammary gland. Material and methods We examined the role of the Alu insertion polymorphism in the PR gene by comparing the genotypes of 209 healthy Mexican women with those of 481 Mexican women with breast cancer (BC). Results The genotype frequencies observed in the controls and BC patients were 0% and 4% for T2/T2 (Alu insertion), 16% and 21% for T1/T2, and 84% and 75% for T1/T1 (Alu deletion), respectively. The obtained odds ratio (OR) was 1.7, with a 95% confidence interval (95% CI) of 1.1–2.6, p = 0.009, for the T1/T2–T2/T2 genotypes. The association was also evident when the distributions of the T1/T2–T2/T2 genotypes in patients in the following categories were compared: obesity grade II (OR = 1.81, 95% CI: 1.03–3.18, p = 0.039) and the chemotherapy response (OR = 1.91, 95% CI: 1.27–3.067, p = 0.002). Conclusions The T1/T2–T2/T2 genotypes of the Alu insertion polymorphism in the PR gene are associated with BC susceptibility in the analyzed Mexican population. PMID:26170848

  8. Gene flow and genetic structure in the Galician population (NW Spain) according to Alu insertions

    PubMed Central

    Varela, Tito A; Fariña, José; Diéguez, Lois Pérez; Lodeiro, Rosa

    2008-01-01

    Background The most recent Alu insertions reveal different degrees of polymorphism in human populations, and a series of characteristics that make them particularly suitable genetic markers for Human Biology studies. This has led these polymorphisms to be used to analyse the origin and phylogenetic relationships between contemporary human groups. This study analyses twelve Alu sequences in a sample of 216 individuals from the autochthonous population of Galicia (NW Spain), with the aim of studying their genetic structure and phylogenetic position with respect to the populations of Western and Central Europe and North Africa, research that is of special interest in revealing European population dynamics, given the peculiarities of the Galician population due to its geographical situation in western Europe, and its historical vicissitudes. Results The insertion frequencies of eleven of the Alu elements analysed were within the variability range of European populations, while Yb8NBC125 proved to be the lowest so far recorded to date in Europe. Taking the twelve polymorphisms into account, the GD value for the Galician population was 0.268. The comparative analyses carried out using the MDS, NJ and AMOVA methods reveal the existence of spatial heterogeneity, and identify three population groups that correspond to the geographic areas of Western-Central Europe, Eastern Mediterranean Europe and North Africa. Galicia is shown to be included in the Western-Central European cluster, together with other Spanish populations. When only considering populations from Mediterranean Europe, the Galician population revealed a degree of genetic flow similar to that of the majority of the populations from this geographic area. Conclusion The results of this study reveal that the Galician population, despite its geographic situation in the western edge of the European continent, occupies an intermediate position in relation to other European populations in general, and Iberian

  9. Terrestrial Sediment and Nutrient Fluxes to the Faga'alu Reefs in American Samoa

    NASA Astrophysics Data System (ADS)

    Messina, A. T.

    2013-12-01

    Land-based sources of pollution including increased sediment and nutrient fluxes to coastal waters have been identified and linked to degraded coral health in reef ecosystems adjacent to impacted streams such as Faga'alu in American Samoa. Monthly monitoring since 2002 has shown that Faga'alu stream has the highest turbidity of monitored streams on Tutuila, where degraded water quality is linked to lower reef health and fish biomass. To guide local and federal managers in mitigating land-based sources of pollution from agricultural, mining, urban, and residential areas, fluxes of sediment and nutrients were measured upstream and downstream of disturbed areas to identify and quantify significant pollution sources and guide mitigation efforts. Sediment flux from disturbed areas, mainly an open-pit aggregate quarry, contributed over 75% of the sediment loading to the bay. Faga'alu stream is characterized by flashy response to rainfall events and the total observed sediment yield was contributed by a small number of large storm events. Event-total sediment yield was more closely correlated with event-total discharge than event-total precipitation. It is hypothesized that the intensity of rainfall controls sediment yield for small events where sheetwash erosion from the quarry is more important. For larger events where easily available sediment is washed away in the first part of the storm it is hypothesized that increased sediment yield is due to streambank erosion and gullying from both disturbed and undisturbed areas. Based on sediment yield measurements and modeled sediment loading to the bay, recommendations for mitigation of land-based sources of pollution are focused on sediment mitigation at the quarry and runoff from the large impervious areas associated with the hospital. Measurements of sediment accumulation on the coral reef itself show sedimentation is controlled by sediment loading from the watershed and sediment scouring by increased wave and wind

  10. Electromagnetism, Second Edition

    NASA Astrophysics Data System (ADS)

    Grant, I. S.; Phillips, W. R.

    2003-09-01

    The Manchester Physics Series General Editors: D. J. Sandiford; F. Mandl; A. C. Phillips Department of Physics and Astronomy, University of Manchester Properties of Matter B. H. Flowers and E. Mendoza Optics Second Edition F. G. Smith and J. H. Thomson Statistical Physics Second Edition F. Mandl Electromagnetism Second Edition I. S. Grant and W. R. Phillips Statistics R. J. Barlow Solid State Physics Second Edition J. R. Hook and H. E. Hall Quantum Mechanics F. Mandl Particle Physics Second Edition B. R. Martin and G. Shaw the Physics of Stars Second Edition A. C. Phillips Computing for Scientists R. J. Barlow and A. R. Barnett Electromagnetism, Second Edition is suitable for a first course in electromagnetism, whilst also covering many topics frequently encountered in later courses. The material has been carefully arranged and allows for flexi-bility in its use for courses of different length and structure. A knowledge of calculus and an elementary knowledge of vectors is assumed, but the mathematical properties of the differential vector operators are described in sufficient detail for an introductory course, and their physical significance in the context of electromagnetism is emphasised. In this Second Edition the authors give a fuller treatment of circuit analysis and include a discussion of the dispersion of electromagnetic waves. Electromagnetism, Second Edition features: The application of the laws of electromagnetism to practical problems such as the behaviour of antennas, transmission lines and transformers. Sets of problems at the end of each chapter to help student understanding, with hints and solutions to the problems given at the end of the book. Optional "starred" sections containing more specialised and advanced material for the more ambitious reader. An Appendix with a thorough discussion of electromagnetic standards and units. Recommended by many institutions. Electromagnetism. Second Edition has also been adopted by the Open University as the

  11. A plant mitochondrial sequence transcribed in transgenic tobacco chloroplasts is not edited

    SciTech Connect

    Sutton, C.A.; Hanson, M.R.; Zoubenko, O.V.; Maliga, P.

    1995-03-01

    RNA editing occurs in two higher-plant organelles, chloroplasts, and mitochondria. Because chloroplasts and mitochondria exhibit some similarity in editing site selection, we investigated whether mitochondrial RNA sequences could be edited in chloroplasts. We produced transgenic tobacco plants that contained chimeric genes in which the second exon of a Petunia hybrida mitochondrial coxII gene was under the control of chloroplast gene regulatory sequences. coxII transcripts accumulated to low or high levels in transgenic chloroplasts containing chimeric genes with the plastid ribosomal protein gene rps16 or the rRNA operon promoter, respectively. Exon 2 of coxII was chosen because it carries seven editing sites and is edited in petunia mitochondria even when located in an abnormal context in an aberrant recombined gene. When editing of the coxII transcripts in transgenic chloroplasts was examined, no RNA editing at any of the usual sites was detected, nor was there any novel editing at any other sites. These results indicate that the RNA editing mechanisms of chloroplasts and mitochondria are not identical but must have at least some organelle-specific components. 33 refs., 5 figs.

  12. DNA sequences of Alu elements indicate a recent replacement of the human autosomal genetic complement

    SciTech Connect

    Knight, A.; Deininger, P.L.; Batzer, M.A.

    1996-04-30

    DNA sequences of neutral nuclear autosomal loci, compared across diverse human populations, provide a previously untapped perspective into the mode and tempo of the emergence of modern humans and a critical comparison with published clonally inherited mitochondrial DNA and Y chromosome measurements of human diversity. We obtained over 55 kilobases of sequence from three autosomal loci encompassing Alu repeats for representatives of diverse human populations as well as orthologous sequences for other hominoid species at one of these loci. Nucleotide diversity was exceedingly low. Most individuals and populations were identical. Only a single nucleotide difference distinguished presumed ancestral alleles from descendants. These results differ from those expected if alleles from divergent archaic populations were maintained through multiregional continuity. The observed virtual lack of sequence polymorphism is the signature of a recent single origin for modern humans, with general replacement of archaic populations. 47 refs., 2 figs., 1 tab.

  13. The human gene (CSNK2A1) coding for the casein kinase II subunit [alpha] is located on chromosome 20 and contains tandemly arranged Alu repeats

    SciTech Connect

    Wirkner, U.; Lichter, P.; Pyerin, W. ); Voss, H.; Ansorge, W. )

    1994-01-15

    The authors have isolated and characterized an 18.9-kb genomic clone representing a central portion of the human casein kinase II (CKII) subunit [alpha] gene (CSNK2A1). Using the whole clone as a probe, the gene was localized on chromosome 20p13. The clone contains eight exons whose sequences comprise bases 102 to 824 of the coding region of the human CKII[alpha]. The exon/intron splice junctions conform to the gt/ag rule. Three of the nine introns are located at positions corresponding to those in the CKII[alpha] gene of the nematode Caenorhabditis elegans. The introns contain eight complete and eight incomplete Alu repeats. Some of the Alu sequences are arranged in tandems of two or three, which seem to originate from insertions of younger Alu sequences into the poly(A) region of previously integrated Alu sequences, as indicated by flanking direct repeats. 50 refs., 5 figs., 1 tab.

  14. Terminal RNA uridylyltransferases of trypanosomes

    PubMed Central

    Aphasizhev, Ruslan; Aphasizheva, Inna

    2008-01-01

    Terminal RNA uridylyltransferases (TUTases) are functionally and structurally diverse nucleotidyl transferases that catalyze template-independent 3′ uridylylation of RNAs. Within the DNA polymerase β-type superfamily, TUTases are closely related to non-canonical poly(A) polymerases. Studies of U-insertion/deletion RNA editing in mitochondria of trypanosomatids identified the first TUTase proteins and their cellular functions: post-transcriptional uridylylation of guide RNAs by RNA editing TUTase 1 (RET1) and U-insertion mRNA editing by RNA editing TUTase 2 (RET2). The editing TUTases possess conserved catalytic and nucleotide base recognition domains, yet differ in quaternary structure, substrate specificity and processivity. The cytosolic TUTases TUT3 and TUT4 have also been identified in trypanosomes but their biological roles remain to be established. Structural analyses have revealed a mechanism of cognate nucleoside triphosphate selection by TUTases, which includes protein-UTP contacts as well as contribution of the RNA substrate. This review focuses on biological functions and structures of trypanosomal TUTases. PMID:18191648

  15. PredcircRNA: computational classification of circular RNA from other long non-coding RNA using hybrid features.

    PubMed

    Pan, Xiaoyong; Xiong, Kai

    2015-08-01

    Recently circular RNA (circularRNA) has been discovered as an increasingly important type of long non-coding RNA (lncRNA), playing an important role in gene regulation, such as functioning as miRNA sponges. So it is very promising to identify circularRNA transcripts from de novo assembled transcripts obtained by high-throughput sequencing, such as RNA-seq data. In this study, we presented a machine learning approach, named as PredcircRNA, focused on distinguishing circularRNA from other lncRNAs using multiple kernel learning. Firstly we extracted different sources of discriminative features, including graph features, conservation information and sequence compositions, ALU and tandem repeats, SNP densities and open reading frames (ORFs) from transcripts. Secondly, to better integrate features from different sources, we proposed a computational approach based on a multiple kernel learning framework to fuse those heterogeneous features. Our preliminary 5-fold cross-validation result showed that our proposed method can classify circularRNA from other types of lncRNAs with an accuracy of 0.778, sensitivity of 0.781, specificity of 0.770, precision of 0.784 and MCC of 0.554 in our constructed gold-standard dataset, respectively. Our feature importance analysis based on Random Forest illustrated some discriminative features, such as conservation features and a GTAG sequence motif. Our PredcircRNA tool is available for download at . PMID:26028480

  16. 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.

  17. Characterization and polymerase chain reaction (PCR) detection of an Alu deletion polymorphism in total linkage disequilibrium with myotonic dystrophy

    SciTech Connect

    Mahadevan, M.S. ); Foitzik, M.A. ); Surh, L.C.; Korneluk, R.G. Univ. of Ottawa )

    1993-02-01

    The mutation causing myotonic dystrophy has been identified as an unstable trinucleotide CRG repeat located in the 3[prime] untranslated region of a gene putatively encoding a serine-threonine protein kinase. The mutation has been reported to be in total linkage disequilibrium with an insertion/deletion polymorphism located within the kinase gene. To determine the nature of this polymorphism, we have sequenced this genomic fragment and have found that the sequence of this region consists of five consecutive Alu repeats. Further analysis suggests that the smaller of two alleles is actually due to a proposed deletion event that resulted in the loss of an equivalent of three Alu repeats. We have developed a PCR-based assay to detect this polymorphism, the closest, distal marker to the DM mutation. 12 refs., 2 figs.

  18. Alu Sb2 subfamily is present in all higher primates but was most succesfully amplified in humans

    SciTech Connect

    Richer, C.; Zietkiewicz, E.; Labuda, D.

    1994-09-01

    Alu repeats can be classified into subfamilies which amplified in primate genomes at different evolutionary time periods. A young Alu subfamily, Sb2, with a characteristic 7-nucleotide duplication at position 256, has been described in seven human loci. An Sb2 insertion found near the HD gene was unique to two HD families, indicating that Sb2 was still retropositionally active. Here, we have shown that the Sb2 insertion in the CHOL locus was similarly rare, being absent in 120 individuals of Caucasian, Oriental and Black origin. In contrast, Sb2 inserts in five other loci were found fixed (non-polymorphic), based on measurements in the same population sample, but absent from orthologous positions in higher apes. This suggest that Sb2 repeats spread relatively early in the human lineage following divergence from other primates and that these elements may be human-specific. By quantitative PCR, we investigated the presence of Sb2 sequences in different primate DNA, using one PCR primer anchored at the 5{prime} Alu-end and the other complementary to the duplicated Sb2-specific segment. With an Sb2-containing plasmid as a standard, we estimated the number of Sb2 repeats at 1500-1800 copies per human haploid equivalent; corresponding numbers in chimpanzee and gorilla were almost two orders of magnitude lower, while the signal observed in orangutan and gibbon DNAs was consistent with the presence of a single copy. The analysis of 22 human, 11 chimpanzee and 10 gorilla sequences indicates that the Alu Sb2 dispersed independently in these three primate lineages; gorilla consensus differs from the human Sb2 sequence by one position, while all chimpanzee repeats have their linker expanded by up to eight A-residues. Should they be thus considered as separate subfamilies? It is possible that sequence modifications with respect to the human consensus are responsible for poor retroposition of Sb2 in apes.

  19. 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

  20. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome

    SciTech Connect

    Minami, M.; Poussin, K.; Brechot, C.; Paterlini, P.

    1995-09-20

    The rapid and reproducible identification of new cellular DNA sequences is difficult to achieve with the currently available procedures. Here we describe a novel approach based on the polymerase chain reaction (PCR) using a primer specific to the known sequence and another directed to a human Alu repeat. To avoid undesirable amplifications between Alu sequences, primers are constructed with dUTPs and destroyed by uracil DNA glycosylase treatment after 10 initial cycles of amplification. Only desirable fragments are then further amplified with specific primers to the known region and to a tag sequence introduced in the Alu-specific primer. Using this protocol, we have successfully indentified cellular sequences flanking integrated hepatitis B virus DNA from the human genome of three hepatoma tissues. The method enables a direct specific amplification without any ligation or nonspecific annealing steps as required by previous PCR-based protocols. This rapid and straightforward approach will be a powerful tool for the study of viral integration sites, but is also widely applicable to other studies of the human genome. 39 refs., 4 figs.

  1. The Y Alu polymorphism in southern African populations and its relationship to other Y-specific polymorphisms

    SciTech Connect

    Spurdle, A.B.; Jenkins, T. ); Hammer, M.F. )

    1994-02-01

    Y-linked polymorphisms were studied in a number of African populations. The frequency of the alleles of a Y-specific Alu insertion polymorphism, termed the [open quotes]Y Alu polymorphism,[close quotes] was determined in 889 individuals from 23 different African population groups. A trend in frequency was observed, with the insert largely absent in Caucasoid populations, at intermediate frequency in the Khoisan, and at high frequency in Negroids. The insert predates diversification of Homo sapiens, since it occurs in all groups. The Alu insertion is believed to result from a unique mutation event, and comparisons between this and several other Y-linked polymorphisms were carried out in an attempt to validate their usefulness in population and evolutionary studies. The p21A1/Taql and pDP31/EcoRI polymorphisms and 49a/TaqI alleles were all shown to have arisen on more than one occasion, and evidence exists for a preraciation crossover event between the Y-linked pseudoautosomal XY275 locus and the Y chromosome pseudoautosomal boundary. 33 refs., 4 figs., 5 tabs.

  2. The genomic architecture of NLRP7 is Alu rich and predisposes to disease-associated large deletions.

    PubMed

    Reddy, Ramesh; Nguyen, Ngoc M P; Sarrabay, Guillaume; Rezaei, Maryam; Rivas, Mayra C G; Kavasoglu, Aysenur; Berkil, Hakan; Elshafey, Alaa; Nunez, Kristin P; Dreyfus, Hélène; Philippe, Merviel; Hadipour, Zahra; Durmaz, Asude; Eaton, Erin E; Schubert, Brittany; Ulker, Volkan; Hadipour, Fatemeh; Ahmadpour, Fatemeh; Touitou, Isabelle; Fardaei, Majid; Slim, Rima

    2016-10-01

    NLRP7 is a major gene responsible for recurrent hydatidiform moles. Here, we report 11 novel NLRP7 protein truncating variants, of which five deletions of more than 1-kb. We analyzed the transcriptional consequences of four variants. We demonstrate that one large homozygous deletion removes NLRP7 transcription start site and results in the complete absence of its transcripts in a patient in good health besides her reproductive problem. This observation strengthens existing data on the requirement of NLRP7 only for female reproduction. We show that two other variants affecting the splice acceptor of exon 6 lead to its in-frame skipping while another variant affecting the splice donor site of exon 9 leads to an in-frame insertion of 54 amino acids. Our characterization of the deletion breakpoints demonstrated that most of the breakpoints occurred within Alu repeats and the deletions were most likely mediated by microhomology events. Our data define a hotspot of Alu instability and deletions in intron 5 with six different breakpoints and rearrangements. Analysis of NLRP7 genomic sequences for repetitive elements demonstrated that Alu repeats represent 48% of its intronic sequences and these repeats seem to have been inserted into the common NLRP2/7 primate ancestor before its duplication into two genes.

  3. Inhibition of activated pericentromeric SINE/Alu repeat transcription in senescent human adult stem cells reinstates self-renewal

    PubMed Central

    Hostikka, Sirkka Liisa; Atallah, Michelle; Blackwell, Benjamin; Lee, Elbert; Cook, Peter J; Pasaniuc, Bogdan; Shariat, Goli; Halperin, Eran; Dobke, Marek; Rosenfeld, Michael G

    2011-01-01

    Cellular aging is linked to deficiencies in efficient repair of DNA double strand breaks and authentic genome maintenance at the chromatin level. Aging poses a significant threat to adult stem cell function by triggering persistent DNA damage and ultimately cellular senescence. Senescence is often considered to be an irreversible process. Moreover, critical genomic regions engaged in persistent DNA damage accumulation are unknown. Here we report that 65% of naturally occurring repairable DNA damage in self-renewing adult stem cells occurs within transposable elements. Upregulation of Alu retrotransposon transcription upon ex vivo aging causes nuclear cytotoxicity associated with the formation of persistent DNA damage foci and loss of efficient DNA repair in pericentric chromatin. This occurs due to a failure to recruit of condensin I and cohesin complexes. Our results demonstrate that the cytotoxicity of induced Alu repeats is functionally relevant for the human adult stem cell aging. Stable suppression of Alu transcription can reverse the senescent phenotype, reinstating the cells' self-renewing properties and increasing their plasticity by altering so-called “master” pluripotency regulators. PMID:21862875

  4. PAI: Predicting adenosine to inosine editing sites by using pseudo nucleotide compositions

    PubMed Central

    Chen, Wei; Feng, Pengmian; Ding, Hui; Lin, Hao

    2016-01-01

    The adenosine to inosine (A-to-I) editing is the most prevalent kind of RNA editing and involves in many biological processes. Accurate identification of A-to-I editing site is invaluable for better understanding its biological functions. Due to the limitations of experimental methods, in the present study, a support vector machine based-model, called PAI, is proposed to identify A-to-I editing site in D. melanogaster. In this model, RNA sequences are encoded by “pseudo dinucleotide composition” into which six RNA physiochemical properties were incorporated. PAI achieves promising performances in jackknife test and independent dataset test, indicating that it holds very high potential to become a useful tool for identifying A-to-I editing site. For the convenience of experimental scientists, a web-server was constructed for PAI and it is freely accessible at http://lin.uestc.edu.cn/server/PAI. PMID:27725762

  5. 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

  6. Genome Editing in Sugarcane: Challenges Ahead

    PubMed Central

    Mohan, Chakravarthi

    2016-01-01

    Genome editing opens new and unique opportunities for researchers to enhance crop production. Until 2013, the zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were the key tools used for genome editing applications. The advent of RNA-guided engineered nucleases - the type II clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 (CRISPR-associated) system from Streptococcus pyogenes holds great potential since it is simple, effective and more versatile than ZFNs and TALENs. CRISPR/Cas9 system has already been successfully employed in several crop plants. Use of these techniques is in its infant stage in sugarcane. Jung and Altpeter (2016) have reported TALEN mediated approach for the first time to reduce lignin content in sugarcane to make it amenable for biofuel production. This is so far the only report describing genome editing in sugarcane. Large genome size, polyploidy, low transformation efficiency, transgene silencing and lack of high throughput screening techniques are certainly great challenges for genome editing in sugarcane which would be discussed in detail in this review. PMID:27790238

  7. Sequences more than 500 base pairs upstream of the human U3 small nuclear RNA gene stimulate the synthesis of U3 RNA in frog oocytes

    SciTech Connect

    Suh, D.; Reddy, R. ); Wright, D. )

    1991-06-04

    Small nuclear RNA (snRNA) genes contain strong promoters capable of initiating transcription once every 4 s. Studies on the human U1 snRNA gene, carried out in other laboratories, showed that sequences within 400 bp of the 5' flanking region are sufficient for maximal levels of transcription both in vivo and in frog oocytes (reviewed in Dahlberg and Lund (1988)). The authors studied the expression of a human U3 snRNA gene by injecting 5' deletion mutants into frog oocytes. The results show that sequences more than 500 bp upstream of the U3 snRNA gene have a 2-3-fold stimulatory effect on the U3 snRNA synthesis. These results indicate that the human U3 snRNA gene is different from human U1 snRNA gene in containing regulatory elements more than 500 bp upstream. The U3 snRNA gene upstream sequences contain an AluI homologous sequence in the {minus}1,200 region; these AluI sequences were transcribed in vitro and in frog oocytes but were not detectable in Hela cells.

  8. Questioning the "melting pot": analysis of Alu inserts in three population samples from Uruguay.

    PubMed

    Hidalgo, Pedro C; Mut, Patricia; Ackermann, Elizabeth; Figueiro, Gonzalo; Sans, Monica

    2014-01-01

    The way that immigrants integrate into recipient societies has been discussed for decades, mainly from the perspective of the social sciences. Uruguay, as other American countries, received diffferent waves of European immigrants, although the details of the process of assimilation, when it did occur, are unclear. In this study we used genetic markers to understand the process experienced by the Basques, one of the major migration waves that populated Uruguay, and their relation to other immigrants, as well as to Native American and African descendants. For this purpose, we analyzed the allele frequencies of 10 ALU loci (A25, ACE, APOA1, B65, D1, F13B, PV92, TPA25, HS2.43, and HS4.65) in three samples from Uruguay (two of Basque descendants, one of non-Basque descendants) from two locations: Montevideo and Trinidad. No departure from Hardy-Weinberg expectations was observed, with the exceptions of the APOA1 and D1 loci in the non-Basque descendants' samples. Our data show that the major genetic contribution in the three samples comes from Europe (78-88%), with minor African (10-15%) and Native American (0-10%) contributions. Genetic distances reveal that Basque descendants from Trinidad cluster with Europeans, whereas both Montevideo samples cluster together and are separate from other populations, showing two diffferent types of integration, related to the general characteristics of each regional population.

  9. Human diversity in Jordan: polymorphic Alu insertions in general Jordanian and Bedouin groups.

    PubMed

    Zanetti, Daniela; Sadiq, May; Carreras-Torres, Robert; Khabour, Omar; Alkaraki, Almuthanna; Esteban, Esther; Via, Marc; Moral, Pedro

    2014-01-01

    Jordan, located in the Levant region, is an area crucial for the investigation of human migration between Africa and Eurasia. However, the genetic history of Jordanians has yet to be clarified, including the origin of the Bedouins today resident in Jordan. Here, we provide new genetic data on autosomal independent markers in two Jordanian population samples (Bedouins and the general population) to begin to examine the genetic diversity inside this country and to provide new information about the genetic position of these populations in the context of the Mediterranean and Middle East area. The markers analyzed were 18 Alu polymorphic insertions characterized by their identity by descent, known ancestral state (lack of insertion), and apparent selective neutrality. The results indicate significant genetic diffferences between Bedouins and general Jordanians (p = 0.038). Whereas Bedouins show a close genetic proximity to North Africans, general Jordanians appear genetically more similar to other Middle East populations. In general, these data are consistent with the hypothesis that Bedouins had an important role in the peopling of Jordan and constitute the original substrate of the current population. However, migration into Jordan in recent years likely has contributed to the diversity among current Jordanian population groups.

  10. A DYW-protein knockout in Physcomitrella affects two closely spaced mitochondrial editing sites and causes a severe developmental phenotype.

    PubMed

    Schallenberg-Rüdinger, Mareike; Kindgren, Peter; Zehrmann, Anja; Small, Ian; Knoop, Volker

    2013-11-01

    RNA-binding pentatricopeptide repeat (PPR) proteins carrying a carboxy-terminal DYW domain similar to cytidine deaminases have been characterized as site-specific factors for C-to-U RNA editing in plant organelles. Here we report that knockout of DYW-PPR_65 in Physcomitrella patens causes a severe developmental phenotype in the moss and specifically affects two editing sites located 18 nucleotides apart on the mitochondrial ccmFC mRNA. Intriguingly, PPR_71, another DYW-type PPR, had been identified previously as an editing factor specifically affecting only the downstream editing site, ccmFCeU122SF. The now characterized PPR_65 binds specifically only to the upstream target site, ccmFCeU103PS, in full agreement with a recent RNA-recognition code for PPR arrays. The functional interference between the two editing events may be caused by a combination of three factors: (i) the destabilization of an RNA secondary structure interfering with PPR_71 binding by prior binding of PPR_65; (ii) the resulting upstream C-U conversion; or (iii) a direct interaction between the two DYW proteins. Indeed, we find the Physcomitrella DYW-PPRs to interact in yeast-two-hybrid assays. The moss DYW-PPRs also interact yet more strongly with MORF (Multiple Organellar RNA editing Factor)/RIP (RNA editing factor interacting proteins) proteins of Arabidopsis known to be general editing factors in flowering plants, although MORF homologues are entirely absent in the moss. Finally, we demonstrate binding of Physcomitrella DYW-PPR_98, for which no KO lines could be raised, to its predicted target sequence upstream of editing site atp9eU92SL. Together with the functional characterization of DYW-PPR_65, this completes the assignment of RNA editing factors to all editing sites in the Physcomitrella mitochondrial transcriptome.

  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. 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.

  13. Authoritative Online Editions

    ERIC Educational Resources Information Center

    Benton, Thomas H.

    2007-01-01

    In this article, the author discusses how it is now very easy for anyone to find formerly hard-to-find books such as the works of Walt Whitman with the help of online booksellers. The author also describes the efforts made by various institutions to produce online editions of the works of major writers. One such prominent project is the archive…

  14. Aerospace Bibliography, Third Edition.

    ERIC Educational Resources Information Center

    National Aeronautics and Space Administration, Washington, DC.

    This third edition bibliography lists books and teaching aids related to aeronautics and space. Aeronautics titles are limited to aerospace-related research subjects, and books on astronomy to those directly related to space exploration. Also listed are pertinent references like pamphlets, films, film strips, booklets, charts, pictures,…

  15. Newspaper Editing: English, Journalism.

    ERIC Educational Resources Information Center

    Bullock, Johanna

    A course designed to groom editors for the newspaper is presented. Editing copy, copyreading and proofreading, principles of headlining, responsibility of the press, libel and slander laws, and problems of censorship are covered. Course objectives include the following: (1) The student will recognize and correct all newspaper items that do not…

  16. 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…

  17. Aerospace Bibliography. Sixth Edition.

    ERIC Educational Resources Information Center

    National Aerospace Education Council, Washington, DC.

    This sixth edition of the National Aeronautics and Space Administration's (NASA) bibliography presents an updated list of books, references, periodicals, and other educational materials related to space flight and space science. To find materials on a particular subject and for a specific reading level, users are advised to refer first to Part…

  18. 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.

  19. Alu and L1 sequence distributions in Xq24-q28 and their comparative utility in YAC contig assembly and verification

    SciTech Connect

    Porta, G.; Zucchi, I.; Schlessinger, D.; Hillier, L.; Green, P.; Nowotny, V.; D`Urso, M.

    1993-05-01

    The contents of Alu- and L1-containing TaqI restriction fragments were assessed by Southern blot analyses across YAC contigs already assembled by other means and localized within Xq24-q28. Fingerprinting patterns of YACs in contigs were concordant. Using software based on that of M. V. Olson et al. to analyze digitized data on fragment sizes, fingerprinting itself could establish matches among about 40% of a test group of 435 YACs. At 100-kb resolution, both repetitive elements were found throughout the region, with no apparent enrichment of Alu or L1 in DNA of G compared to that found in R bands. However, consistent with a random overall distribution, delimited regions of up to 100 kb contained clusters of repetitive elements. The local concentrations may help to account for the reported differential hybridization of Alu and L1 probes to segments of metaphase chromosomes. 40 refs., 6 figs., 2 tabs.

  20. Physical mapping of BK virus DNA with SacI, MboII, and AluI restriction endonucleases.

    PubMed Central

    Yang, R C; Wu, R

    1978-01-01

    A new restriction endonuclease, SacI from Streptomyces achromogenes cleaves BK virus (strain MM) DNA into 3 fragments, whereas MboII from Moraxella bovis and AluI from Arthrobacter luteus give 22 and 30 fragments, respectively. All these specific DNA fragments were ordered and mapped on the viral genome by two methods first, by the reciprocal digestion method using uniformly 32P-labeled DNA; and second, by the partial digestion technique using the single-end 32P-labeled DNA. This study, together with those reported earlier, defined the location of 90 cleavage sites on the BK virus DNA. Images PMID:215783

  1. LISP. Second edition

    SciTech Connect

    Winston, P.H.; Horn, B.K.

    1986-01-01

    In this edition, the authors redirect the orientation of the text towards the Common LISP dialect, an amalgam of the features of the dialects which have evolved from the LISP programming language. The authors introduce discussions of procedure abstraction and data abstraction; add an introduction to message-passing and object-centred programming, and have revised the chapters on mathematical examples, natural-language interfaces, symbolic pattern matching, and rule-based expert systems.

  2. Understanding Physics, First Edition

    NASA Astrophysics Data System (ADS)

    Cummings, Karen; Laws, Priscilla W.; Redish, Edward F.; Cooney, Patrick J.

    2004-03-01

    Built on the foundations of Halliday, Resnick, and Walker's Fundamentals of Physics Sixth Edition, this text is designed to work with interactive learning strategies that are increasingly being used in physics instruction (for example, microcomputer-based labs, interactive lectures, etc. ). In doing so, it incorporates new approaches based upon Physics Education Research (PER), aligns with courses that use computer-based laboratory tools, and promotes Activity Based Physics in lectures, labs, and recitations.

  3. 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.

  4. Interactive Metro Map Editing.

    PubMed

    Wang, Yu-Shuen; Peng, Wan-Yu

    2016-02-01

    Manual editing of a metro map is essential because many aesthetic and readability demands in map generation cannot be achieved by using a fully automatic method. In addition, a metro map should be updated when new metro lines are developed in a city. Considering that manually designing a metro map is time-consuming and requires expert skills, we present an interactive editing system that considers human knowledge and adjusts the layout to make it consistent with user expectations. In other words, only a few stations are controlled and the remaining stations are relocated by our system. Our system supports both curvilinear and octilinear layouts when creating metro maps. It solves an optimization problem, in which even spaces, route straightness, and maximum included angles at junctions are considered to obtain a curvilinear result. The system then rotates each edge to extend either vertically, horizontally, or diagonally while approximating the station positions provided by users to generate an octilinear layout. Experimental results, quantitative and qualitative evaluations, and user studies show that our editing system is easy to use and allows even non-professionals to design a metro map.

  5. Alu-alu recombination results in a duplication of seven exons in the lysyl hydroxylase gene in a patient with the t