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Sample records for prevalent antisense transcription

  1. Strand-specific community RNA-seq reveals prevalent and dynamic antisense transcription in human gut microbiota

    PubMed Central

    Bao, Guanhui; Wang, Mingjie; Doak, Thomas G.; Ye, Yuzhen

    2015-01-01

    Metagenomics and other meta-omics approaches (including metatranscriptomics) provide insights into the composition and function of microbial communities living in different environments or animal hosts. Metatranscriptomics research provides an unprecedented opportunity to examine gene regulation for many microbial species simultaneously, and more importantly, for the majority that are unculturable microbial species, in their natural environments (or hosts). Current analyses of metatranscriptomic datasets focus on the detection of gene expression levels and the study of the relationship between changes of gene expression and changes of environment. As a demonstration of utilizing metatranscriptomics beyond these common analyses, we developed a computational and statistical procedure to analyze the antisense transcripts in strand-specific metatranscriptomic datasets. Antisense RNAs encoded on the DNA strand opposite a gene’s CDS have the potential to form extensive base-pairing interactions with the corresponding sense RNA, and can have important regulatory functions. Most studies of antisense RNAs in bacteria are rather recent, are mostly based on transcriptome analysis, and have been applied mainly to single bacterial species. Application of our approaches to human gut-associated metatranscriptomic datasets allowed us to survey antisense transcription for a large number of bacterial species associated with human beings. The ratio of protein coding genes with antisense transcription ranges from 0 to 35.8% (median = 10.0%) among 47 species. Our results show that antisense transcription is dynamic, varying between human individuals. Functional enrichment analysis revealed a preference of certain gene functions for antisense transcription, and transposase genes are among the most prominent ones (but we also observed antisense transcription in bacterial house-keeping genes). PMID:26388849

  2. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    PubMed Central

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  3. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

    PubMed

    Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A; Trotter, Kevin W; Gilchrist, Daniel A; Bennett, Brian D; Burkholder, Adam B; Burd, Craig J; Fargo, David C; Archer, Trevor K

    2016-08-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  4. Natural antisense transcription from a comparative perspective.

    PubMed

    Piatek, Monica J; Henderson, Victoria; Zynad, Hany S; Werner, Andreas

    2016-08-01

    Natural antisense transcripts (NATs) can interfere with the expression of complementary sense transcripts with exquisite specificity. We have previously cloned NATs of Slc34a loci (encoding Na-phosphate transporters) from fish and mouse. Here we report the cloning of a human SLC34A1-related NAT that represents an alternatively spliced PFN3 transcript (Profilin3). The transcript is predominantly expressed in testis. Phylogenetic comparison suggests two distinct mechanisms producing Slc34a-related NATs: Alternative splicing of a transcript from a protein coding downstream gene (Pfn3, human/mouse) and transcription from the bi-directional promoter (Rbpja, zebrafish). Expression analysis suggested independent regulation of the complementary Slc34a mRNAs. Analysis of randomly selected bi-directionally transcribed human/mouse loci revealed limited phylogenetic conservation and independent regulation of NATs. They were reduced on X chromosomes and clustered in regions that escape inactivation. Locus structure and expression pattern suggest a NATs-associated regulatory mechanisms in testis unrelated to the physiological role of the sense transcript encoded protein. PMID:27241791

  5. Natural Antisense Transcripts and Long Non-Coding RNA in Neurospora crassa

    PubMed Central

    Arthanari, Yamini; Heintzen, Christian; Griffiths-Jones, Sam; Crosthwaite, Susan K.

    2014-01-01

    The prevalence of long non-coding RNAs (lncRNA) and natural antisense transcripts (NATs) has been reported in a variety of organisms. While a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. Here, we use RNA sequencing data from the ABI SOLiD platform to identify lncRNA and NATs obtained from samples of the filamentous fungus Neurospora crassa grown under different light and temperature conditions. We identify 939 novel lncRNAs, of which 477 are antisense to annotated genes. Across the whole dataset, the extent of overlap between sense and antisense transcripts is large: 371 sense/antisense transcripts are complementary over 500 nts or more and 236 overlap by more than 1000 nts. Most prevalent are 3′ end overlaps between convergently transcribed sense/antisense pairs, but examples of divergently transcribed pairs and nested transcripts are also present. We confirm the expression of a subset of sense/antisense transcript pairs by qPCR. We examine the size, types of overlap and expression levels under the different environmental stimuli of light and temperature, and identify 11 lncRNAs that are up-regulated in response to light. We also find differences in transcript length and the position of introns between protein-coding transcripts that have antisense expression and transcripts with no antisense expression. These results demonstrate the ability of N. crassa lncRNAs and NATs to be regulated by different environmental stimuli and provide the scope for further investigation into the function of NATs. PMID:24621812

  6. Natural antisense transcripts associated with salinity response in alfalfa

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural antisense transcripts (NATs) are long non-coding RNAs (lncRNAs) complimentary to the messenger (sense) RNA (Wang et al. 2014). Many of them are involved in regulation of their own sense transcripts thus playing pivotal biological roles in all processes of organismal development and responses...

  7. Bacterial antisense RNAs are mainly the product of transcriptional noise.

    PubMed

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I; Serrano, Luis; Lluch-Senar, Maria

    2016-03-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  8. Bacterial antisense RNAs are mainly the product of transcriptional noise

    PubMed Central

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

    2016-01-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  9. Bimodal expression of PHO84 is modulated by early termination of antisense transcription

    PubMed Central

    Castelnuovo, Manuele; Rahman, Samir; Guffanti, Elisa; Infantino, Valentina; Stutz, Françoise; Zenklusen, Daniel

    2016-01-01

    Many S. cerevisiae genes encode antisense transcripts some of which are unstable and degraded by the exosome component Rrp6. Loss of Rrp6 results in the accumulation of long PHO84 antisense RNAs and repression of sense transcription through PHO84 promoter deacetylation. We used single molecule resolution fluorescent in situ hybridization (smFISH) to investigate antisense-mediated transcription regulation. We show that PHO84 antisense RNA acts as a bimodal switch, where continuous low frequency antisense transcription represses sense expression within individual cells. Surprisingly, antisense RNAs do not accumulate at the PHO84 gene but are exported to the cytoplasm. Furthermore, loss of Rrp6, rather than stabilizing PHO84 antisense RNA, promotes antisense elongation by reducing its early transcription termination by Nrd1-Nab3-Sen1. These observations suggest that PHO84 silencing results from constant low frequency antisense transcription through the promoter rather than its static accumulation at the repressed gene. PMID:23770821

  10. Extensive Polycistronism and Antisense Transcription in the Mammalian Hox Clusters

    PubMed Central

    Mainguy, Gaëll; Koster, Jan; Woltering, Joost; Jansen, Hans; Durston, Antony

    2007-01-01

    The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70%) have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp) overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb). This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering. PMID:17406680

  11. Protein-coding cis-natural antisense transcripts have high and broad expression in Arabidopsis.

    PubMed

    Zhan, Shuhua; Lukens, Lewis

    2013-04-01

    Pairs of genes within eukaryotic genomes are often located on opposite DNA strands such that transcription generates cis-natural sense antisense transcripts (cis-NATs). This orientation of genes has been associated with the biogenesis of splice variants and natural antisense small RNAs. Here, in an analysis of currently available data, we report that within Arabidopsis (Arabidopsis thaliana), protein-coding cis-NATs are also characterized by high abundance, high coexpression, and broad expression. Our results suggest that a permissive chromatin environment may have led to the proximity of these genes. Compared with other genes, cis-NAT-encoding genes have enriched low-nucleosome-density regions, high levels of histone H3 lysine-9 acetylation, and low levels of H3 lysine-27 trimethylation. Promoters associated with broadly expressed genes are preferentially found in the 5' regulatory sequences of cis-NAT-encoding genes. Our results further suggest that natural antisense small RNA production from cis-NATs is limited. Small RNAs sequenced from natural antisense small RNA biogenesis mutants including dcl1, dcl2, dcl3, and rdr6 map to cis-NATs as frequently as small RNAs sequenced from wild-type plants. Future work will investigate if the positive transcriptional regulation of overlapping protein-coding genes contributes to the prevalence of these genes within other eukaryotic genomes. PMID:23457227

  12. Regulation of Antisense Transcription by NuA4 Histone Acetyltransferase and Other Chromatin Regulatory Factors.

    PubMed

    Uprety, Bhawana; Kaja, Amala; Ferdoush, Jannatul; Sen, Rwik; Bhaumik, Sukesh R

    2016-01-01

    NuA4 histone lysine (K) acetyltransferase (KAT) promotes transcriptional initiation of TATA-binding protein (TBP)-associated factor (TAF)-dependent ribosomal protein genes. TAFs have also been recently found to enhance antisense transcription from the 3' end of the GAL10 coding sequence. However, it remains unknown whether, like sense transcription of the ribosomal protein genes, TAF-dependent antisense transcription of GAL10 also requires NuA4 KAT. Here, we show that NuA4 KAT associates with the GAL10 antisense transcription initiation site at the 3' end of the coding sequence. Such association of NuA4 KAT depends on the Reb1p-binding site that recruits Reb1p activator to the GAL10 antisense transcription initiation site. Targeted recruitment of NuA4 KAT to the GAL10 antisense transcription initiation site promotes GAL10 antisense transcription. Like NuA4 KAT, histone H3 K4/36 methyltransferases and histone H2B ubiquitin conjugase facilitate GAL10 antisense transcription, while the Swi/Snf and SAGA chromatin remodeling/modification factors are dispensable for antisense, but not sense, transcription of GAL10. Taken together, our results demonstrate for the first time the roles of NuA4 KAT and other chromatin regulatory factors in controlling antisense transcription, thus illuminating chromatin regulation of antisense transcription. PMID:26755557

  13. Open reading frames provide a rich pool of potential natural antisense transcripts in fungal genomes.

    PubMed

    Steigele, Stephan; Nieselt, Kay

    2005-01-01

    Natural antisense transcripts are reported from all kingdoms of life and several recent reports of genomewide screens indicate that they are widely distributed. These transcripts seem to be involved in various biological functions and may govern the expression of their respective sense partner. Very little, however, is known about the degree of evolutionary conservation of antisense transcripts. Furthermore, none of the earlier analyses has studied whether antisense relationships are solely dual or involved in more complex relationships. Here we present a systematic screen for cis- and trans-located antisense transcripts based on open reading frames (ORFs) from five fungal species. The relative number of ORFs involved in antisense relationships varies greatly between the five species. In addition, other significant differences are found between the species, such as the mean length of the antisense region. The majority of trans-located antisense transcripts is found to be involved in complex relationships, resulting in highly connected networks. The analysis of the degree of evolutionary conservation of antisense transcripts shows that most antisense transcripts have no ortholog in any other species. An annotation of antisense transcripts based on Gene Ontology directs to common terms and shows that proteins of genes involved in antisense relationships preferentially localize to the nucleus with common functions in the regulation or maintenance of nucleic acids. PMID:16147987

  14. Pseudogenes as an alternative source of natural antisense transcripts

    PubMed Central

    2010-01-01

    Background Naturally occurring antisense transcripts (NATs) are non-coding RNAs that may regulate the activity of sense transcripts to which they bind because of complementarity. NATs that are not located in the gene they regulate (trans-NATs) have better chances to evolve than cis-NATs, which is evident when the sense strand of the cis-NAT is part of a protein coding gene. However, the generation of a trans-NAT requires the formation of a relatively large region of complementarity to the gene it regulates. Results Pseudogene formation may be one evolutionary mechanism that generates trans-NATs to the parental gene. For example, this could occur if the parental gene is regulated by a cis-NAT that is copied as a trans-NAT in the pseudogene. To support this we identified human pseudogenes with a trans-NAT to the parental gene in their antisense strand by analysis of the database of expressed sequence tags (ESTs). We found that the mutations that appeared in these trans-NATs after the pseudogene formation do not show the flat distribution that would be expected in a non functional transcript. Instead, we found higher similarity to the parental gene in a region nearby the 3' end of the trans-NATs. Conclusions Our results do not imply a functional relation of the trans-NAT arising from pseudogenes over their respective parental genes but add evidence for it and stress the importance of duplication mechanisms of genetic material in the generation of non-coding RNAs. We also provide a plausible explanation for the large transcripts that can be found in the antisense strand of some pseudogenes. PMID:21047404

  15. Characteristics of Antisense Transcript Promoters and the Regulation of Their Activity

    PubMed Central

    Lin, Shudai; Zhang, Li; Luo, Wen; Zhang, Xiquan

    2015-01-01

    Recently, an increasing number of studies on natural antisense transcripts have been reported, especially regarding their classification, temporal and spatial expression patterns, regulatory functions and mechanisms. It is well established that natural antisense transcripts are produced from the strand opposite to the strand encoding a protein. Despite the pivotal roles of natural antisense transcripts in regulating the expression of target genes, the transcriptional mechanisms initiated by antisense promoters (ASPs) remain unknown. To date, nearly all of the studies conducted on this topic have focused on the ASP of a single gene of interest, whereas no study has systematically analyzed the locations of ASPs in the genome, ASP activity, or factors influencing this activity. This review focuses on elaborating on and summarizing the characteristics of ASPs to extend our knowledge about the mechanisms of antisense transcript initiation. PMID:26703594

  16. Cis-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach

    PubMed Central

    Bordoy, Antoni E.; Chatterjee, Anushree

    2015-01-01

    Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of cis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards

  17. Strand-Specific RNA-Seq Reveals Ordered Patterns of Sense and Antisense Transcription in Bacillus anthracis

    PubMed Central

    Passalacqua, Karla D.; Varadarajan, Anjana; Weist, Charlotte; Ondov, Brian D.; Byrd, Benjamin; Read, Timothy D.; Bergman, Nicholas H.

    2012-01-01

    Background Although genome-wide transcriptional analysis has been used for many years to study bacterial gene expression, many aspects of the bacterial transcriptome remain undefined. One example is antisense transcription, which has been observed in a number of bacteria, though the function of antisense transcripts, and their distribution across the bacterial genome, is still unclear. Methodology/Principal Findings Single-stranded RNA-seq results revealed a widespread and non-random pattern of antisense transcription covering more than two thirds of the B. anthracis genome. Our analysis revealed a variety of antisense structural patterns, suggesting multiple mechanisms of antisense transcription. The data revealed several instances of sense and antisense expression changes in different growth conditions, suggesting that antisense transcription may play a role in the ways in which B. anthracis responds to its environment. Significantly, genome-wide antisense expression occurred at consistently higher levels on the lagging strand, while the leading strand showed very little antisense activity. Intrasample gene expression comparisons revealed a gene dosage effect in all growth conditions, where genes farthest from the origin showed the lowest overall range of expression for both sense and antisense directed transcription. Additionally, transcription from both strands was verified using a novel strand-specific assay. The variety of structural patterns we observed in antisense transcription suggests multiple mechanisms for this phenomenon, suggesting that some antisense transcription may play a role in regulating the expression of key genes, while some may be due to chromosome replication dynamics and transcriptional noise. Conclusions/Significance Although the variety of structural patterns we observed in antisense transcription suggest multiple mechanisms for antisense expression, our data also clearly indicate that antisense transcription may play a genome-wide role

  18. Natural antisense transcripts regulate the neuronal stress response and excitability

    PubMed Central

    Zheng, Xingguo; Valakh, Vera; DiAntonio, Aaron; Ben-Shahar, Yehuda

    2014-01-01

    Neurons regulate ionic fluxes across their plasma membrane to maintain their excitable properties under varying environmental conditions. However, the mechanisms that regulate ion channels abundance remain poorly understood. Here we show that pickpocket 29 (ppk29), a gene that encodes a Drosophila degenerin/epithelial sodium channel (DEG/ENaC), regulates neuronal excitability via a protein-independent mechanism. We demonstrate that the mRNA 3′UTR of ppk29 affects neuronal firing rates and associated heat-induced seizures by acting as a natural antisense transcript (NAT) that regulates the neuronal mRNA levels of seizure (sei), the Drosophila homolog of the human Ether-à-go-go Related Gene (hERG) potassium channel. We find that the regulatory impact of ppk29 mRNA on sei is independent of the sodium channel it encodes. Thus, our studies reveal a novel mRNA dependent mechanism for the regulation of neuronal excitability that is independent of protein-coding capacity. DOI: http://dx.doi.org/10.7554/eLife.01849.001 PMID:24642409

  19. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

    PubMed

    Kramer, Nicholas J; Carlomagno, Yari; Zhang, Yong-Jie; Almeida, Sandra; Cook, Casey N; Gendron, Tania F; Prudencio, Mercedes; Van Blitterswijk, Marka; Belzil, Veronique; Couthouis, Julien; Paul, Joseph West; Goodman, Lindsey D; Daughrity, Lillian; Chew, Jeannie; Garrett, Aliesha; Pregent, Luc; Jansen-West, Karen; Tabassian, Lilia J; Rademakers, Rosa; Boylan, Kevin; Graff-Radford, Neill R; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Boeve, Bradley F; Deng, Ning; Feng, Yanan; Cheng, Tzu-Hao; Dickson, Dennis W; Cohen, Stanley N; Bonini, Nancy M; Link, Christopher D; Gao, Fen-Biao; Petrucelli, Leonard; Gitler, Aaron D

    2016-08-12

    An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately. PMID:27516603

  20. Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe.

    PubMed

    Bitton, Danny A; Grallert, Agnes; Scutt, Paul J; Yates, Tim; Li, Yaoyong; Bradford, James R; Hey, Yvonne; Pepper, Stuart D; Hagan, Iain M; Miller, Crispin J

    2011-01-01

    Strand-specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3' termination and bidirectional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent 'horsetail' stage of meiosis. Antisense production had no impact at any of these loci when the RNA interference (RNAi) machinery was removed. Thus, far from being simply 'genome chatter', this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe. PMID:22186733

  1. Programmed fluctuations in sense/antisense transcript ratios drive sexual differentiation in S. pombe

    PubMed Central

    Bitton, Danny A; Grallert, Agnes; Scutt, Paul J; Yates, Tim; Li, Yaoyong; Bradford, James R; Hey, Yvonne; Pepper, Stuart D; Hagan, Iain M; Miller, Crispin J

    2011-01-01

    Strand-specific RNA sequencing of S. pombe revealed a highly structured programme of ncRNA expression at over 600 loci. Waves of antisense transcription accompanied sexual differentiation. A substantial proportion of ncRNA arose from mechanisms previously considered to be largely artefactual, including improper 3′ termination and bidirectional transcription. Constitutive induction of the entire spk1+, spo4+, dis1+ and spo6+ antisense transcripts from an integrated, ectopic, locus disrupted their respective meiotic functions. This ability of antisense transcripts to disrupt gene function when expressed in trans suggests that cis production at native loci during sexual differentiation may also control gene function. Consistently, insertion of a marker gene adjacent to the dis1+ antisense start site mimicked ectopic antisense expression in reducing the levels of this microtubule regulator and abolishing the microtubule-dependent ‘horsetail' stage of meiosis. Antisense production had no impact at any of these loci when the RNA interference (RNAi) machinery was removed. Thus, far from being simply ‘genome chatter', this extensive ncRNA landscape constitutes a fundamental component in the controls that drive the complex programme of sexual differentiation in S. pombe. PMID:22186733

  2. Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

    PubMed Central

    2011-01-01

    Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression. PMID:22129310

  3. Upstream Anti-sense Promoters are Hubs of Transcription Factor Binding and Active Histone Modifications

    PubMed Central

    Scruggs, Benjamin S.; Gilchrist, Daniel A.; Nechaev, Sergei; Muse, Ginger W.; Burkholder, Adam; Fargo, David C.; Adelman, Karen

    2015-01-01

    SUMMARY Anti-sense transcription originating upstream of mammalian protein-coding genes is a well-documented phenomenon, but remarkably little is known about the regulation or function of anti-sense promoters and the non-coding RNAs they generate. Here we define at nucleotide resolution the divergent transcription start sites (TSSs) near mouse mRNA genes. We find that coupled sense and anti-sense TSSs precisely define the boundaries of a nucleosome-depleted region (NDR) that is highly enriched in transcription factor (TF) motifs. Notably, as the distance between sense and anti-sense TSSs increases, so does the size of the NDR, the level of signal-dependent TF binding and gene activation. We further discover a group of anti-sense TSSs in macrophages with an enhancer-like chromatin signature. Interestingly, this signature identifies divergent promoters that are activated during immune challenge. We propose that anti-sense promoters serve as platforms for TF binding and establishment of active chromatin to further regulate or enhance sense-strand mRNA expression. PMID:26028540

  4. The Role of Transcription Factors at Antisense-Expressing Gene Pairs in Yeast

    PubMed Central

    Mostovoy, Yulia; Thiemicke, Alexander; Hsu, Tiffany Y.; Brem, Rachel B.

    2016-01-01

    Genes encoded close to one another on the chromosome are often coexpressed, by a mechanism and regulatory logic that remain poorly understood. We surveyed the yeast genome for tandem gene pairs oriented tail-to-head at which expression antisense to the upstream gene was conserved across species. The intergenic region at most such tandem pairs is a bidirectional promoter, shared by the downstream gene mRNA and the upstream antisense transcript. Genomic analyses of these intergenic loci revealed distinctive patterns of transcription factor regulation. Mutation of a given transcription factor verified its role as a regulator in trans of tandem gene pair loci, including the proximally initiating upstream antisense transcript and downstream mRNA and the distally initiating upstream mRNA. To investigate cis-regulatory activity at such a locus, we focused on the stress-induced NAD(P)H dehydratase YKL151C and its downstream neighbor, the metabolic enzyme GPM1. Previous work has implicated the region between these genes in regulation of GPM1 expression; our mutation experiments established its function in rich medium as a repressor in cis of the distally initiating YKL151C sense RNA, and an activator of the proximally initiating YKL151C antisense RNA. Wild-type expression of all three transcripts required the transcription factor Gcr2. Thus, at this locus, the intergenic region serves as a focal point of regulatory input, driving antisense expression and mediating the coordinated regulation of YKL151C and GPM1. Together, our findings implicate transcription factors in the joint control of neighboring genes specialized to opposing conditions and the antisense transcripts expressed between them. PMID:27190003

  5. Intragenic pausing and anti-sense transcription within the murine c-myc locus.

    PubMed Central

    Nepveu, A; Marcu, K B

    1986-01-01

    We present a detailed analysis of strand-specific transcription in different regions of the murine c-myc locus. In normal and transformed cell lines, RNA polymerase II directed transcription occurs in the sense and anti-sense direction. Three noncontiguous regions show a high level of transcription in the anti-sense orientation: upstream of the first exon, within the first intron and in the 3' part of the gene (intron 2 and exon 3). In a cell line carrying a c-myc amplification (54c12), anti-sense transcription is not uniformly increased throughout the locus and is differentially affected by inhibition of protein synthesis. These results suggest that anti-sense transcription in various parts of the locus is independently regulated. In the sense orientation, transcriptional activity is higher in the first exon than in the rest of the gene indicating that transcription pauses near the 3' end of the first exon. The extent of this intragenic pausing varies among different cell lines and is most severe in cells with a c-myc amplification. Transcription initiation and pausing are both negatively regulated by labile proteins. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3024965

  6. An HIV-Encoded Antisense Long Noncoding RNA Epigenetically Regulates Viral Transcription

    PubMed Central

    Saayman, Sheena; Ackley, Amanda; Turner, Anne-Marie W; Famiglietti, Marylinda; Bosque, Alberto; Clemson, Matthew; Planelles, Vicente; Morris, Kevin V

    2014-01-01

    The abundance of long noncoding RNAs (lncRNAs) and their wide range of functional roles in human cells are fast becoming realized. Importantly, lncRNAs have been identified as epigenetic modulators and consequently play a pivotal role in the regulation of gene expression. A human immunodeficiency virus-encoded antisense RNA transcript has recently been reported and we sought to characterize this RNA and determine its potential role in viral transcription regulation. The intrinsic properties of this human immunodeficiency virus-expressed lncRNA were characterized and the data presented here suggest that it functions as an epigenetic brake to modulate viral transcription. Suppression of this long antisense transcript with small single-stranded antisense RNAs resulted in the activation of viral gene expression. This lncRNA was found to localize to the 5′ long-term repeats (LTR) and to usurp components of endogenous cellular pathways that are involved in lncRNA directed epigenetic gene silencing. Collectively, we find that this viral expressed antisense lncRNA is involved in modulating human immunodeficiency virus gene expression and that this regulatory effect is due to an alteration in the epigenetic landscape at the viral promoter. PMID:24576854

  7. Mining SAGE data allows large-scale, sensitive screening of antisense transcript expression.

    PubMed

    Quéré, Ronan; Manchon, Laurent; Lejeune, Mireille; Clément, Oliver; Pierrat, Fabien; Bonafoux, Béatrice; Commes, Thérèse; Piquemal, David; Marti, Jacques

    2004-01-01

    As a growing number of complementary transcripts, susceptible to exert various regulatory functions, are being found in eukaryotes, high throughput analytical methods are needed to investigate their expression in multiple biological samples. Serial Analysis of Gene Expression (SAGE), based on the enumeration of directionally reliable short cDNA sequences (tags), is capable of revealing antisense transcripts. We initially detected them by observing tags that mapped on to the reverse complement of known mRNAs. The presence of such tags in individual SAGE libraries suggested that SAGE datasets contain latent information on antisense transcripts. We raised a collection of virtual tags for mining these data. Tag pairs were assembled by searching for complementarities between 24-nt long sequences centered on the potential SAGE-anchoring sites of well-annotated human expressed sequences. An analysis of their presence in a large collection of published SAGE libraries revealed transcripts expressed at high levels from both strands of two adjacent, oppositely oriented, transcription units. In other cases, the respective transcripts of such cis-oriented genes displayed a mutually exclusive expression pattern or were co-expressed in a small number of libraries. Other tag pairs revealed overlapping transcripts of trans-encoded unique genes. Finally, we isolated a group of tags shared by multiple transcripts. Most of them mapped on to retroelements, essentially represented in humans by Alu sequences inserted in opposite orientations in the 3'UTR of otherwise different mRNAs. Registering these tags in separate files makes possible computational searches focused on unique sense-antisense pairs. The method developed in the present work shows that SAGE datasets constitute a major resource of rapidly investigating with high sensitivity the expression of antisense transcripts, so that a single tag may be detected in one library when screening a large number of biological samples. PMID

  8. Regulation of the NPT gene by a naturally occurring antisense transcript.

    PubMed

    Werner, Andreas; Preston-Fayers, Keziah; Dehmelt, Leif; Nalbant, Perihan

    2002-01-01

    The epithelial Na/Pi cotransporter (NaPi-II) is instrumental in maintaining phosphate (Pi) homeostasis in vertebrates. Hormones and metabolic factors (PTH, Pi availability) that acutely influence renal Pi excretion have been demonstrated to target NaPi-II expression. Upon stimulation, newly synthesized transporter molecules become integrated into the brush-border membrane to increase the Vmax of Pi uptake; reduction of Pi reabsorption is achieved by endocytosis of NaPi-II followed by lysosomal degradation of the protein. The long-term regulation of the protein is less well studied. Only recently, regulatory elements for vitamin D3 and Pi have been identified in the promoter region of the npt gene. However, signaling pathways leading to the activation of these regulatory sequences need to be established. Other reports suggested messenger RNA stability to play a role in the medium range regulation of NaPi-II expression. Recent findings in our laboratory added to the complex picture of npt gene regulation. We have identified npt-related endogenous antisense transcripts from mouse, zebrafish, and winter flounder. The two fish transcripts have been cloned and characterized; the mouse homolog has only very recently been detected. The transcripts are devoid of an open reading frame and appear in different splice forms. The evolutionary conservation of bidirectional transcription of the npt gene implies a regulatory function for the antisense transcript. In order to test the functional consequences of bidirectional transcription, we coexpressed sense and the antisense transcripts from zebrafish in Xenopus oocytes. Pi transport activity was reduced as a result of the presence of antisense RNA. Re-extraction of the RNA from injected oocytes followed by Northern blot revealed that the coexpression had no significant effect on the stability of either transcript. We concluded that the antisense mRNA interfered with the translation of the transporter if coexpressed in the

  9. PlantNATsDB: a comprehensive database of plant natural antisense transcripts

    PubMed Central

    Chen, Dijun; Yuan, Chunhui; Zhang, Jian; Zhang, Zhao; Bai, Lin; Meng, Yijun; Chen, Ling-Ling; Chen, Ming

    2012-01-01

    Natural antisense transcripts (NATs), as one type of regulatory RNAs, occur prevalently in plant genomes and play significant roles in physiological and pathological processes. Although their important biological functions have been reported widely, a comprehensive database is lacking up to now. Consequently, we constructed a plant NAT database (PlantNATsDB) involving approximately 2 million NAT pairs in 69 plant species. GO annotation and high-throughput small RNA sequencing data currently available were integrated to investigate the biological function of NATs. PlantNATsDB provides various user-friendly web interfaces to facilitate the presentation of NATs and an integrated, graphical network browser to display the complex networks formed by different NATs. Moreover, a ‘Gene Set Analysis’ module based on GO annotation was designed to dig out the statistical significantly overrepresented GO categories from the specific NAT network. PlantNATsDB is currently the most comprehensive resource of NATs in the plant kingdom, which can serve as a reference database to investigate the regulatory function of NATs. The PlantNATsDB is freely available at http://bis.zju.edu.cn/pnatdb/. PMID:22058132

  10. A novel transforming growth factor beta2 antisense transcript in mammalian lung.

    PubMed Central

    Coker, R K; Laurent, G J; Dabbagh, K; Dawson, J; McAnulty, R J

    1998-01-01

    Transforming growth factor (TGF) beta2 gene expression was examined in murine, rat and human lung by in situ hybridization with riboprobes. Hybridization signal was observed in a variety of cells with the sense probe, and Northern-blot analysis with this probe demonstrated the presence of a novel 3.5 kb transcript. This first report suggesting the existance of a natural TGFbeta2 antisense transcript raises the possibility that such a transcript may play a role in regulating TGFbeta2 production. PMID:9601055

  11. The zebrafish progranulin gene family and antisense transcripts

    PubMed Central

    Cadieux, Benoît; Chitramuthu, Babykumari P; Baranowski, David; Bennett, Hugh PJ

    2005-01-01

    Background Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor) that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. Results The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn) genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial compartments of various organs

  12. Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Natural antisense transcripts (NATs) are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded) or a different locus (trans-encoded). They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation....

  13. Revealing natural antisense transcripts from Plasmodium vivax isolates: evidence of genome regulation in complicated malaria.

    PubMed

    Boopathi, P A; Subudhi, Amit Kumar; Garg, Shilpi; Middha, Sheetal; Acharya, Jyoti; Pakalapati, Deepak; Saxena, Vishal; Aiyaz, Mohammed; Chand, Bipin; Mugasimangalam, Raja C; Kochar, Sanjay K; Sirohi, Parmendra; Kochar, Dhanpat K; Das, Ashis

    2013-12-01

    Plasmodium vivax is the most geographically widespread human malaria parasite causing approximately 130-435 million infections annually. It is an economic burden in many parts of the world and poses a public health challenge along with the other Plasmodium sp. The biology of this parasite is less studied and poorly understood, in spite of these facts. Emerging evidence of severe complications due to infections by this parasite provides an impetus to focus research on the same. Investigating the parasite directly from infected patients is the best way to study its biology and pathogenic mechanisms. Gene expression studies of this parasite directly obtained from the patients has provided evidence of gene regulation resulting in varying amount of transcript levels in the different blood stages. The mechanisms regulating gene expression in malaria parasites are not well understood. Discovery of Natural Antisense Transcripts (NATs) in Plasmodium falciparum has suggested that these might play an important role in regulating gene expression. We report here the genome-wide occurrence of NATs in P. vivax parasites from patients with differing clinical symptoms. A total of 1348 NATs against annotated gene loci have been detected using a custom designed microarray with strand specific probes. Majority of NATs identified from this study shows positive correlation with the expression pattern of the sense (S) transcript. Our data also shows condition specific expression patterns of varying S and antisense (AS) transcript levels. Genes with AS transcripts enrich to various biological processes. To our knowledge this is the first report on the presence of NATs from P. vivax obtained from infected patients with different disease complications. The data suggests differential regulation of gene expression in diverse clinical conditions, as shown by differing sense/antisense ratios and would lead to future detailed investigations of gene regulation. PMID:24121022

  14. Transcription of rat mitochondrial NADH-dehydrogenase subunits. Presence of antisense and precursor RNA species.

    PubMed

    Tullo, A; Tanzariello, F; D'Erchia, A M; Nardelli, M; Papeo, P A; Sbisà, E; Saccone, C

    1994-10-31

    We have characterized the transcriptional pattern of the rat mitochondrial ND6-containing region in vivo. We have identified a stable polyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of an antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a possible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable than the L transcripts. We have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNA(Glu). The mapping of the ND1 5' end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the expression of the mt genome are much more numerous and complex than those already described in the literature. PMID:7957896

  15. Transcriptional regulation of Oct4 by a long non-coding RNA antisense to Oct4-pseudogene 5.

    PubMed

    Hawkins, Peter G; Morris, Kevin V

    2010-11-01

    Long non-coding RNAs (lncRNAs) have been shown to epigenetically regulate certain genes in human cells. Here we report evidence for the involvement of an antisense lncRNA in the transcriptional regulation of the pluripotency-associated factor Oct4. When an lncRNA antisense to Oct4-pseudogene 5 was suppressed, transcription of Oct4 and Oct4 pseudogenes 4 and 5 was observed to increase. This increase correlated with a loss of silent state epigenetic marks and the histone methyltransferase Ezh2 at the Oct4 promoter. We observed this lncRNA to interact with nucleolin and PURA, a 35 kD single-stranded DNA and RNA binding protein, and found that these proteins may act to negatively regulate this antisense transcript. PMID:21151833

  16. R-loop stabilization represses antisense transcription at the Arabidopsis FLC locus.

    PubMed

    Sun, Qianwen; Csorba, Tibor; Skourti-Stathaki, Konstantina; Proudfoot, Nicholas J; Dean, Caroline

    2013-05-01

    Roles for long noncoding RNAs (lncRNAs) in gene expression are emerging, but regulation of the lncRNA itself is poorly understood. We have identified a homeodomain protein, AtNDX, that regulates COOLAIR, a set of antisense transcripts originating from the 3' end of Arabidopsis FLOWERING LOCUS C (FLC). AtNDX associates with single-stranded DNA rather than double-stranded DNA non-sequence-specifically in vitro, and localizes to a heterochromatic region in the COOLAIR promoter in vivo. Single-stranded DNA was detected in vivo as part of an RNA-DNA hybrid, or R-loop, that covers the COOLAIR promoter. R-loop stabilization mediated by AtNDX inhibits COOLAIR transcription, which in turn modifies FLC expression. Differential stabilization of R-loops could be a general mechanism influencing gene expression in many organisms. PMID:23641115

  17. The expression of ELK transcription factors in adult DRG: Novel isoforms, antisense transcripts and upregulation by nerve damage.

    PubMed

    Kerr, Niall; Pintzas, Alexander; Holmes, Fiona; Hobson, Sally-Ann; Pope, Robert; Wallace, Mark; Wasylyk, Christine; Wasylyk, Bohdan; Wynick, David

    2010-06-01

    ELK transcription factors are known to be expressed in a number of regions in the nervous system. We show by RT-PCR that the previously described Elk1, Elk3/Elk3b/Elk3c and Elk4 mRNAs are expressed in adult dorsal root ganglia (DRG), together with the novel alternatively spliced isoforms Elk1b, Elk3d and Elk4c/Elk4d/Elk4e. These isoforms are also expressed in brain, heart, kidney and testis. In contrast to Elk3 protein, the novel Elk3d isoform is cytoplasmic, fails to bind ETS binding sites and yet can activate transcription by an indirect mechanism. The Elk3 and Elk4 genes are overlapped by co-expressed Pctk2 (Cdk17) and Mfsd4 genes, respectively, with the potential formation of Elk3/Pctaire2 and Elk4/Mfsd4 sense-antisense mRNA heteroduplexes. After peripheral nerve injury the Elk3 mRNA isoforms are each upregulated approximately 2.3-fold in DRG (P<0.005), whereas the natural antisense Pctaire2 isoforms show only a small increase (21%, P<0.01) and Elk1 and Elk4 mRNAs are unchanged. PMID:20304071

  18. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis

    PubMed Central

    Donaldson, Michael E; Saville, Barry J

    2013-01-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense–antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis. PMID:23650872

  19. Rcs signalling-activated transcription of rcsA induces strong anti-sense transcription of upstream fliPQR flagellar genes from a weak intergenic promoter: regulatory roles for the anti-sense transcript in virulence and motility.

    PubMed

    Wang, Qingfeng; Harshey, Rasika M

    2009-10-01

    In Salmonella enterica, an activated Rcs signalling system inhibits initiation of transcription of the flhD master operon. Under these conditions, where motility is shut down, microarray experiments showed an increased RNA signal for three flagellar genes -fliPQR- located upstream of rcsA. We show here that it is the anti-sense (AS) strand of these genes that is transcribed, originating at a weak promoter in the intergenic region between fliR and rcsA. RcsA is an auxiliary regulator for the Rcs system, whose transcription is dependent on the response regulator RcsB. Rcs-activated rightward transcription, but not translation, of rcsA is required for stimulation of leftward AS transcription. Our results implicate a combined action of RcsB and rcsA transcription in activating the AS promoter, likely by modulating DNA superhelicity in the intergenic region. We show that the AS transcript regulates many genes in the Rcs regulon, including SPI-1 and SPI-2 virulence and stress-response genes. In the wild-type strain the AS transcript is present in low amounts, independent of Rcs signalling. Here, AS transcription modulates complementary sense RNA levels and impacts swarming motility. It appears that the flagellar AS transcript has been co-opted by the Rcs system to regulate virulence. PMID:19703110

  20. Rcs signaling-activated transcription of rcsA induces strong anti-sense transcription of upstream fliPQR flagellar genes from a weak intergenic promoter: regulatory roles for the anti-sense transcript in virulence and motility

    PubMed Central

    Wang, Qingfeng; Harshey, Rasika M.

    2009-01-01

    Summary In Salmonella enterica, an activated Rcs signaling system inhibits initiation of transcription of the flhD master operon. Under these conditions, where motility is shut down, microarray experiments showed an increased RNA signal for three flagellar genes - fliPQR - located upstream of rcsA. We show here that it is the anti-sense (AS) strand of these genes that is transcribed, originating at a weak promoter in the intergenic region between fliR and rcsA. RcsA is an auxiliary regulator for the Rcs system, whose transcription is dependent on the response regulator RcsB. Rcs-activated rightward transcription, but not translation, of rcsA is required for stimulation of leftward AS transcription. Our results implicate a combined action of RcsB and rcsA transcription in activating the AS promoter, likely by modulating DNA superhelicity in the intergenic region. We show that the AS transcript regulates many genes in the Rcs regulon, including SPI-1 and SPI-2 virulence and stress-response genes. In the wild-type strain the AS transcript is present in low amounts, independent of Rcs signaling. Here, AS transcription modulates complementary sense RNA levels and impacts swarming motility. It appears that the flagellar AS transcript has been co-opted by the Rcs system to regulate virulence. PMID:19703110

  1. Hormone-Dependent Expression of a Steroidogenic Acute Regulatory Protein Natural Antisense Transcript in MA-10 Mouse Tumor Leydig Cells

    PubMed Central

    Castillo, Ana Fernanda; Fan, Jinjiang; Papadopoulos, Vassilios; Podestá, Ernesto J.

    2011-01-01

    Cholesterol transport is essential for many physiological processes, including steroidogenesis. In steroidogenic cells hormone-induced cholesterol transport is controlled by a protein complex that includes steroidogenic acute regulatory protein (StAR). Star is expressed as 3.5-, 2.8-, and 1.6-kb transcripts that differ only in their 3′-untranslated regions. Because these transcripts share the same promoter, mRNA stability may be involved in their differential regulation and expression. Recently, the identification of natural antisense transcripts (NATs) has added another level of regulation to eukaryotic gene expression. Here we identified a new NAT that is complementary to the spliced Star mRNA sequence. Using 5′ and 3′ RACE, strand-specific RT-PCR, and ribonuclease protection assays, we demonstrated that Star NAT is expressed in MA-10 Leydig cells and steroidogenic murine tissues. Furthermore, we established that human chorionic gonadotropin stimulates Star NAT expression via cAMP. Our results show that sense-antisense Star RNAs may be coordinately regulated since they are co-expressed in MA-10 cells. Overexpression of Star NAT had a differential effect on the expression of the different Star sense transcripts following cAMP stimulation. Meanwhile, the levels of StAR protein and progesterone production were downregulated in the presence of Star NAT. Our data identify antisense transcription as an additional mechanism involved in the regulation of steroid biosynthesis. PMID:21829656

  2. Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes.

    PubMed

    Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

    2015-09-30

    Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3' maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. PMID:26209135

  3. Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes

    PubMed Central

    Parent, Jean-Sébastien; Jauvion, Vincent; Bouché, Nicolas; Béclin, Christophe; Hachet, Mélanie; Zytnicki, Matthias; Vaucheret, Hervé

    2015-01-01

    Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3′ maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS. PMID:26209135

  4. RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation

    NASA Astrophysics Data System (ADS)

    Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé

    2009-06-01

    Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA-RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners.

  5. Genome-wide view of natural antisense transcripts in Arabidopsis thaliana

    PubMed Central

    Yuan, Chunhui; Wang, Jingjing; Harrison, Andrew P.; Meng, Xianwen; Chen, Dijun; Chen, Ming

    2015-01-01

    Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications. PMID:25922535

  6. Genome-wide view of natural antisense transcripts in Arabidopsis thaliana.

    PubMed

    Yuan, Chunhui; Wang, Jingjing; Harrison, Andrew P; Meng, Xianwen; Chen, Dijun; Chen, Ming

    2015-06-01

    Natural antisense transcripts (NATs) are endogenous transcripts that can form double-stranded RNA structures. Many protein-coding genes (PCs) and non-protein-coding genes (NPCs) tend to form cis-NATs and trans-NATs, respectively. In this work, we identified 4,080 cis-NATs and 2,491 trans-NATs genome-widely in Arabidopsis. Of these, 5,385 NAT-siRNAs were detected from the small RNA sequencing data. NAT-siRNAs are typically 21nt, and are processed by Dicer-like 1 (DCL1)/DCL2 and RDR6 and function in epigenetically activated situations, or 24nt, suggesting these are processed by DCL3 and RDR2 and function in environment stress. NAT-siRNAs are significantly derived from PC/PC pairs of trans-NATs and NPC/NPC pairs of cis-NATs. Furthermore, NAT pair genes typically have similar pattern of epigenetic status. Cis-NATs tend to be marked by euchromatic modifications, whereas trans-NATs tend to be marked by heterochromatic modifications. PMID:25922535

  7. Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain

    NASA Technical Reports Server (NTRS)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.

  8. Tuning growth cycles of Brassica crops via natural antisense transcripts of BrFLC.

    PubMed

    Li, Xiaorong; Zhang, Shaofeng; Bai, Jinjuan; He, Yuke

    2016-03-01

    Several oilseed and vegetable crops of Brassica are biennials that require a prolonged winter cold for flowering, a process called vernalization. FLOWERING LOCUS C (FLC) is a central repressor of flowering. Here, we report that the overexpression of natural antisense transcripts (NATs) of Brassica rapa FLC (BrFLC) greatly shortens plant growth cycles. In rapid-, medium- and slow-cycling crop types, there are four copies of the BrFLC genes, which show extensive variation in sequences and expression levels. In Bre, a biennial crop type that requires vernalization, five NATs derived from the BrFLC2 locus are rapidly induced under cold conditions, while all four BrFLC genes are gradually down-regulated. The transgenic Bre lines overexpressing a long NAT of BrFLC2 do not require vernalization, resulting in a gradient of shortened growth cycles. Among them, a subset of lines both flower and set seeds as early as Yellow sarson, an annual crop type in which all four BrFLC genes have non-sense mutations and are nonfunctional in flowering repression. Our results demonstrate that the growth cycles of biennial crops of Brassica can be altered by changing the expression levels of BrFLC2 NATs. Thus, BrFLC2 NATs and their transgenic lines are useful for the genetic manipulation of crop growth cycles. PMID:26250982

  9. Does the linear Sry transcript function as a ceRNA for miR-138? The sense of antisense

    PubMed Central

    Granados-Riveron, Javier Tadeo; Aquino-Jarquin, Guillermo

    2014-01-01

    Recently, the sex determining region Y ( Sry) and the cerebellar degeneration-related protein 1 ( CDR1as) RNA transcripts have been described to function as a new class of post-transcriptional regulatory RNAs that behave as circular endogenous RNA sponges for the micro RNAs (miRNAs) miR-138 and miR-7, respectively. A special feature of the Sry gene is its ability to generate linear and circular transcripts, both transcribed in the sense orientation. Here we remark that both sense (e.g. Sry RNA) and antisense (e.g. CDR1as) transcripts could circularize and behave as miRNAs sponges, and importantly, that also protein-coding segments of mRNAs could also assume this role. Thus, it is reasonable to think that the linear Sry sense transcript could additionally act as a miRNA sponge, or as an endogenous competing RNA for miR-138. PMID:25580223

  10. The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

    PubMed Central

    Verhaart, Ingrid E C; van Vliet-van den Dool, Laura; Sipkens, Jessica A; de Kimpe, Sjef J; Kolfschoten, Ingrid G M; van Deutekom, Judith C T; Liefaard, Lia; Ridings, Jim E; Hood, Steve R; Aartsma-Rus, Annemieke

    2014-01-01

    Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD) to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides) will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks). Oligonucleotide half-life was longer in heart (~65 days) compared with that in skeletal muscle, liver, and kidney (~35 days). Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days). Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD. PMID:24549299

  11. Antisense RNA Controls LRP1 Sense Transcript Expression Through Interaction With a Chromatin-Associated Protein, HMGB2

    PubMed Central

    Yamanaka, Yasunari; Faghihi, Mohammad Ali; Magistri, Marco; Alvarez-Garcia, Oscar; Lotz, Martin; Wahlestedt, Claes

    2015-01-01

    SUMMARY Long non-coding RNAs (lncRNAs) including natural antisense transcripts (NATs) are expressed more extensively than previously anticipated, and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here we identify a NAT of Low-density lipoprotein receptor-related protein 1 (Lrp1), referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to High mobility group box 2 (Hmgb2) and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein, and increase Lrp1 expression by enhancing Hmgb2 activity. qRT-PCR analysis of Alzheimer’s disease brain samples and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a new regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion. PMID:25937287

  12. In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts

    PubMed Central

    Ling, King-Hwa; Brautigan, Peter J.; Moore, Sarah; Fraser, Rachel; Leong, Melody Pui-Yee; Leong, Jia-Wen; Zainal Abidin, Shahidee; Lee, Han-Chung; Cheah, Pike-See; Raison, Joy M.; Babic, Milena; Lee, Young Kyung; Daish, Tasman; Mattiske, Deidre M.; Mann, Jeffrey R.; Adelson, David L.; Thomas, Paul Q.; Hahn, Christopher N.; Scott, Hamish S.

    2016-01-01

    SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1. PMID:26958646

  13. In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts.

    PubMed

    Ling, King-Hwa; Brautigan, Peter J; Moore, Sarah; Fraser, Rachel; Leong, Melody Pui-Yee; Leong, Jia-Wen; Zainal Abidin, Shahidee; Lee, Han-Chung; Cheah, Pike-See; Raison, Joy M; Babic, Milena; Lee, Young Kyung; Daish, Tasman; Mattiske, Deidre M; Mann, Jeffrey R; Adelson, David L; Thomas, Paul Q; Hahn, Christopher N; Scott, Hamish S

    2016-06-01

    SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1. PMID:26958646

  14. Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis.

    PubMed

    Borsani, Omar; Zhu, Jianhua; Verslues, Paul E; Sunkar, Ramanjulu; Zhu, Jian-Kang

    2005-12-29

    In higher eukaryotes, miRNAs and siRNAs guide translational inhibition, mRNA cleavage, or chromatin regulation. We found that the antisense overlapping gene pair of Delta(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH), a stress-related gene, and SRO5, a gene of unknown function, generates two types of siRNAs. When both transcripts are present, a 24-nt siRNA is formed by a biogenesis pathway dependent on DCL2, RDR6, SGS3, and NRPD1A. Initial cleavage of the P5CDH transcript guided by the 24-nt siRNA establishes a phase for the subsequent generation of 21-nt siRNAs by DCL1 and further cleavage of P5CDH transcripts. The expression of SRO5 is induced by salt, and this induction is required to initiate siRNA formation. Our data suggest that the P5CDH and SRO5 proteins are also functionally related, and that the P5CDH-SRO5 gene pair defines a mode of siRNA function and biogenesis that may be applied to other natural cis-antisense gene pairs in eukaryotic genomes. PMID:16377568

  15. Regulation of Nav1.7: A Conserved SCN9A Natural Antisense Transcript Expressed in Dorsal Root Ganglia

    PubMed Central

    Koenig, Jennifer; Werdehausen, Robert; Linley, John E.; Habib, Abdella M.; Vernon, Jeffrey; Lolignier, Stephane; Eijkelkamp, Niels; Zhao, Jing; Okorokov, Andrei L.; Woods, C. Geoffrey; Wood, John N.; Cox, James J.

    2015-01-01

    The Nav1.7 voltage-gated sodium channel, encoded by SCN9A, is critical for human pain perception yet the transcriptional and post-transcriptional mechanisms that regulate this gene are still incompletely understood. Here, we describe a novel natural antisense transcript (NAT) for SCN9A that is conserved in humans and mice. The NAT has a similar tissue expression pattern to the sense gene and is alternatively spliced within dorsal root ganglia. The human and mouse NATs exist in cis with the sense gene in a tail-to-tail orientation and both share sequences that are complementary to the terminal exon of SCN9A/Scn9a. Overexpression analyses of the human NAT in human embryonic kidney (HEK293A) and human neuroblastoma (SH-SY5Y) cell lines show that it can function to downregulate Nav1.7 mRNA, protein levels and currents. The NAT may play an important role in regulating human pain thresholds and is a potential candidate gene for individuals with chronic pain disorders that map to the SCN9A locus, such as Inherited Primary Erythromelalgia, Paroxysmal Extreme Pain Disorder and Painful Small Fibre Neuropathy, but who do not contain mutations in the sense gene. Our results strongly suggest the SCN9A NAT as a prime candidate for new therapies based upon augmentation of existing antisense RNAs in the treatment of chronic pain conditions in man. PMID:26035178

  16. Characterization of Natural Antisense Transcript, Sclerotia Development and Secondary Metabolism by Strand-Specific RNA Sequencing of Aspergillus flavus

    PubMed Central

    Yin, Chao; Guo, Yong; Lin, Ying; Pan, Li; Wang, Bin

    2014-01-01

    Aspergillus flavus has received much attention owing to its severe impact on agriculture and fermented products induced by aflatoxin. Sclerotia morphogenesis is an important process related to A. flavus reproduction and aflatoxin biosynthesis. In order to obtain an extensive transcriptome profile of A. flavus and provide a comprehensive understanding of these physiological processes, the isolated mRNA of A. flavus CA43 cultures was subjected to high-throughput strand-specific RNA sequencing (ssRNA-seq). Our ssRNA-seq data profiled widespread transcription across the A. flavus genome, quantified vast transcripts (73% of total genes) and annotated precise transcript structures, including untranslated regions, upstream open reading frames (ORFs), alternative splicing variants and novel transcripts. We propose natural antisense transcripts in A. flavus might regulate gene expression mainly on the post-transcriptional level. This regulation might be relevant to tune biological processes such as aflatoxin biosynthesis and sclerotia development. Gene Ontology annotation of differentially expressed genes between the mycelia and sclerotia cultures indicated sclerotia development was related closely to A. flavus reproduction. Additionally, we have established the transcriptional profile of aflatoxin biosynthesis and its regulation model. We identified potential genes linking sclerotia development and aflatoxin biosynthesis. These genes could be used as targets for controlled regulation of aflatoxigenic strains of A. flavus. PMID:24849659

  17. Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice

    PubMed Central

    2012-01-01

    Background Cis-natural antisense transcripts (cis-NATs) are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs), which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq) to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa). Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set), 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions Our study profiles an

  18. ChiTaRS 2.1--an improved database of the chimeric transcripts and RNA-seq data with novel sense-antisense chimeric RNA transcripts.

    PubMed

    Frenkel-Morgenstern, Milana; Gorohovski, Alessandro; Vucenovic, Dunja; Maestre, Lorena; Valencia, Alfonso

    2015-01-01

    Chimeric RNAs that comprise two or more different transcripts have been identified in many cancers and among the Expressed Sequence Tags (ESTs) isolated from different organisms; they might represent functional proteins and produce different disease phenotypes. The ChiTaRS 2.1 database of chimeric transcripts and RNA-Seq data (http://chitars.bioinfo.cnio.es/) is the second version of the ChiTaRS database and includes improvements in content and functionality. Chimeras from eight organisms have been collated including novel sense-antisense (SAS) chimeras resulting from the slippage of the sense and anti-sense intragenic regions. The new database version collects more than 29,000 chimeric transcripts and indicates the expression and tissue specificity for 333 entries confirmed by RNA-seq reads mapping the chimeric junction sites. User interface allows for rapid and easy analysis of evolutionary conservation of fusions, literature references and experimental data supporting fusions in different organisms. More than 1428 cancer breakpoints have been automatically collected from public databases and manually verified to identify their correct cross-references, genomic sequences and junction sites. As a result, the ChiTaRS 2.1 collection of chimeras from eight organisms and human cancer breakpoints extends our understanding of the evolution of chimeric transcripts in eukaryotes as well as their functional role in carcinogenic processes. PMID:25414346

  19. Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis

    PubMed Central

    Villamizar, Olga; Chambers, Christopher B.; Mo, Yin-Yuan; Torry, Donald S.; Hofstrand, Reese; Riberdy, Janice M.; Persons, Derek A.; Wilber, Andrew

    2016-01-01

    This paper describes data related to a research article titled, “Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death” [1]. Long noncoding RNAs (lncRNAs) are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis). Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf) described in the research article. Also included are 5′ untranslated sequences (UTR) for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT)18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf. PMID:27141526

  20. Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis.

    PubMed

    Villamizar, Olga; Chambers, Christopher B; Mo, Yin-Yuan; Torry, Donald S; Hofstrand, Reese; Riberdy, Janice M; Persons, Derek A; Wilber, Andrew

    2016-06-01

    This paper describes data related to a research article titled, "Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death" [1]. Long noncoding RNAs (lncRNAs) are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis). Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf) described in the research article. Also included are 5' untranslated sequences (UTR) for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34(+) cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT)18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34(+) cells transduced using mock conditions or with lentivirus particles encoding for Saf. PMID:27141526

  1. Ikaros and RAG-2-Mediated Antisense Transcription Are Responsible for Lymphocyte-Specific Inactivation of NWC Promoter

    PubMed Central

    Kasztura, Monika; Śnieżewski, Łukasz; Janik, Sylwia; Kisielow, Paweł; Cebrat, Małgorzata

    2014-01-01

    Recombination activating gene-2 (RAG-2) and NWC are strongly evolutionarily conserved overlapping genes which are convergently transcribed. In non-lymphoid cells the NWC promoter is active whereas in lymphocytes it is inactive due to the DNA methylation. Analysing the mechanism responsible for lymphocyte-specific methylation and inactivation of NWC promoter we found that Ikaros, a lymphocyte-specific transcription factor, acts as a repressor of NWC promoter - thus identifying a new Ikaros target - but is insufficient for inducing its methylation which depends on the antisense transcription driven by RAG-2 promoter. Possible implications of these observations for understanding evolutionary mechanisms leading to lymphocyte specific expression of RAG genes are discussed. PMID:25198102

  2. Natural Antisense Transcript for Hyaluronan Synthase 2 (HAS2-AS1) Induces Transcription of HAS2 via Protein O-GlcNAcylation*

    PubMed Central

    Vigetti, Davide; Deleonibus, Sara; Moretto, Paola; Bowen, Timothy; Fischer, Jens W.; Grandoch, Maria; Oberhuber, Alexander; Love, Dona C.; Hanover, John A.; Cinquetti, Raffaella; Karousou, Eugenia; Viola, Manuela; D'Angelo, Maria Luisa; Hascall, Vincent C.; De Luca, Giancarlo; Passi, Alberto

    2014-01-01

    Changes in the microenvironment organization within vascular walls are critical events in the pathogenesis of vascular pathologies, including atherosclerosis and restenosis. Hyaluronan (HA) accumulation into artery walls supports vessel thickening and is involved in many cardiocirculatory diseases. Excessive cytosolic glucose can enter the hexosamine biosynthetic pathway, increase UDP-N-acetylglucosamine (UDP-GlcNAc) availability, and lead to modification of cytosolic proteins via O-linked attachment of the monosaccharide β-N-GlcNAc (O-GlcNAcylation) from UDP-GlcNAc by the enzyme O-GlcNAc transferase. As many cytoplasmic and nuclear proteins can be glycosylated by O-GlcNAc, we studied whether the expression of the HA synthases that synthesize HA could be controlled by O-GlcNAcylation in human aortic smooth muscle cells. Among the three HAS isoenzymes, only HAS2 mRNA increased after O-GlcNAcylation induced by glucosamine treatments or by inhibiting O-GlcNAc transferase with PUGNAC (O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate). We found that the natural antisense transcript of HAS2 (HAS2-AS1) was absolutely necessary to induce the transcription of the HAS2 gene. Moreover, we found that O-GlcNAcylation modulated HAS2-AS1 promoter activation by recruiting the NF-κB subunit p65, but not the HAS2 promoter, whereas HAS2-AS1 natural antisense transcript, working in cis, regulated HAS2 transcription by altering the chromatin structure around the HAS2 proximal promoter via O-GlcNAcylation and acetylation. These results indicate that HAS2 transcription can be finely regulated not only by recruiting transcription factors to the promoter as previously described but also by modulating chromatin accessibility by epigenetic modifications. PMID:25183006

  3. Long noncoding RNA FGFR3-AS1 promotes osteosarcoma growth through regulating its natural antisense transcript FGFR3.

    PubMed

    Sun, Jiabing; Wang, Xuming; Fu, Chunjiang; Wang, Xiaoyu; Zou, Jilong; Hua, Hanbing; Bi, Zhenggang

    2016-05-01

    Long noncoding RNAs (lncRNAs), a new class of RNAs with no protein-coding potential, have been reported to have crucial roles in the regulation of a variety of tumors. However, the functions and molecular mechanisms of lncRNAs to osteosarcoma are still largely unknown. The purpose of this study is to examine the expression, functions and molecular mechanisms of a new lncRNA FGFR3 antisense transcript 1 (FGFR3-AS1) in osteosarcoma. The expression of FGFR3-AS1 was examined by real-time quantitative PCR. The regulation of FGFR3 by FGFR3-AS1 was examined by RNase protection assay, real-time quantitative PCR, western blotting, and luciferase reporter assay. The effects of FGFR3-AS1 on osteosarcoma cell proliferation and cell cycle were determined by Cell Counting Kit-8, Ethynyl deoxyuridine incorporation assay and flow cytometry. FGFR3-AS1 was upregulated in osteosarcoma. Increased FGFR3-AS1 expression correlates with large tumor size, advanced Enneking stage, metastasis and poor survival. Through antisense pairing with FGFR3 3'UTR, FGFR3-AS1 increases FGFR3 mRNA stability and upregulates FGFR3 expression. The expression of FGFR3-AS1 and FGFR3 is positively correlated in osteosarcoma tissues. Knockdown of FGFR3-AS1 inhibits the proliferation and cell cycle progression of osteosarcoma cells in vitro. Moreover, knockdown of FGFR3-AS1 inhibits xenograft tumor growth of osteosarcoma cells in vivo. These data demonstrate the mechanisms of how antisense noncoding RNA regulate the expression of sense genes, and show the pivotal functions of FGFR3-AS1 in osteosarcoma. PMID:27022737

  4. Distinct transcripts are recognized by sense and antisense riboprobes for a member of the murine HSP70 gene family, HSP70.2, in various reproductive tissues

    NASA Technical Reports Server (NTRS)

    Murashov, A. K.; Wolgemuth, D. J.

    1996-01-01

    The expression of hsp70.2, an hsp70 gene family member, originally characterized by its high levels of expression in germ cells in the adult mouse testis, was detected in several other reproductive tissues, including epididymis, prostate, and seminal vesicles, as well as in extraembryonic tissues of mid-gestation fetuses. In addition, hybridization with RNA probes transcribed in the sense orientation surprisingly indicated the presence of slightly larger "antisense" transcripts in several tissues. The levels of antisense transcripts varied among the tissues, with the highest signal detected in the prostate and no signal being detectable in the testis. Consistent with these results, in situ hybridization analysis clearly localized the sense-orientation transcripts to pachytene spermatocytes, while no antisense-orientation transcripts were observed in adjacent sections of the same tubules. Our findings have thus shown that although hsp70.2 was expressed abundantly and in a highly stage-specific manner in the male germ line, it was also expressed in other murine tissues. Furthermore, we have made the surprising observation of antisense transcription of the hsp70.2 gene in several mouse tissues, revealing another level of complexity in the regulation and function of heat shock proteins.

  5. Natural genetic variation impacts expression levels of coding, non-coding, and antisense transcripts in fission yeast

    PubMed Central

    Clément-Ziza, Mathieu; Marsellach, Francesc X; Codlin, Sandra; Papadakis, Manos A; Reinhardt, Susanne; Rodríguez-López, María; Martin, Stuart; Marguerat, Samuel; Schmidt, Alexander; Lee, Eunhye; Workman, Christopher T; Bähler, Jürg; Beyer, Andreas

    2014-01-01

    Our current understanding of how natural genetic variation affects gene expression beyond well-annotated coding genes is still limited. The use of deep sequencing technologies for the study of expression quantitative trait loci (eQTLs) has the potential to close this gap. Here, we generated the first recombinant strain library for fission yeast and conducted an RNA-seq-based QTL study of the coding, non-coding, and antisense transcriptomes. We show that the frequency of distal effects (trans-eQTLs) greatly exceeds the number of local effects (cis-eQTLs) and that non-coding RNAs are as likely to be affected by eQTLs as protein-coding RNAs. We identified a genetic variation of swc5 that modifies the levels of 871 RNAs, with effects on both sense and antisense transcription, and show that this effect most likely goes through a compromised deposition of the histone variant H2A.Z. The strains, methods, and datasets generated here provide a rich resource for future studies. PMID:25432776

  6. Profiling of T helper cell-derived small RNAs reveals unique antisense transcripts and differential association of miRNAs with argonaute proteins 1 and 2

    PubMed Central

    Polikepahad, Sumanth; Corry, David B.

    2013-01-01

    RNA interference mediated through antisense transcripts is a fundamentally important mechanism regulating gene expression that remains incompletely understood. Here, we have used next-generation sequencing to determine from mouse CD4+ T cells the functional implications of antisense transcripts binding to argonaute (AGO) proteins that mediate RNA interference and post-transcriptional gene silencing. This effort identified 90 new microRNAs (miRNAs) and six endogenous hairpin RNA-derived small interfering RNAs (siRNAs) mapping to distinct introns. Unexpectedly, 69 miRNAs were expressed as non-canonical isomiRs as the dominant AGO-binding transcript, with extensive 3′ terminal nucleotide modifications. Furthermore, differential expression analysis between AGO1- and AGO2-bound miRNAs suggested preferential binding of isomiRs ending with 3′ adenine residues to AGO1 and 3′ uridine residues to AGO2. Analysis of the putative targets of all miRNAs suggested a striking preference for regulating transcription and transcription factors with additional evidence of a functional division of labor between AGO proteins in this regard. We further provide evidence that multiple mitochondrial genomic loci serve as the source of endogenous cis-natural antisense transcripts. These findings imply diversity in AGO protein function based on differential miRNA binding and indicate that RNA interference-based gene regulation is more complex than previously recognized. PMID:23185045

  7. Genomic SELEX for Hfq-binding RNAs identifies genomic aptamers predominantly in antisense transcripts

    PubMed Central

    Lorenz, C.; Gesell, T.; Zimmermann, B.; Schoeberl, U.; Bilusic, I.; Rajkowitsch, L.; Waldsich, C.; von Haeseler, A.; Schroeder, R.

    2010-01-01

    An unexpectedly high number of regulatory RNAs have been recently discovered that fine-tune the function of genes at all levels of expression. We employed Genomic SELEX, a method to identify protein-binding RNAs encoded in the genome, to search for further regulatory RNAs in Escherichia coli. We used the global regulator protein Hfq as bait, because it can interact with a large number of RNAs, promoting their interaction. The enriched SELEX pool was subjected to deep sequencing, and 8865 sequences were mapped to the E. coli genome. These short sequences represent genomic Hfq-aptamers and are part of potential regulatory elements within RNA molecules. The motif 5′-AAYAAYAA-3′ was enriched in the selected RNAs and confers low-nanomolar affinity to Hfq. The motif was confirmed to bind Hfq by DMS footprinting. The Hfq aptamers are 4-fold more frequent on the antisense strand of protein coding genes than on the sense strand. They were enriched opposite to translation start sites or opposite to intervening sequences between ORFs in operons. These results expand the repertoire of Hfq targets and also suggest that Hfq might regulate the expression of a large number of genes via interaction with cis-antisense RNAs. PMID:20348540

  8. Epitope-tagged yeast strains reveal promoter driven changes to 3'-end formation and convergent antisense-transcription from common 3' UTRs.

    PubMed

    Swaminathan, Angavai; Beilharz, Traude H

    2016-01-01

    Epitope-tagging by homologous recombination is ubiquitously used to study gene expression, protein localization and function in yeast. This is generally thought to insulate the regulation of gene expression to that mediated by the promoter and coding regions because native 3' UTR are replaced. Here we show that the 3' UTRs, CYC1 and ADH1, contain cryptic promoters that generate abundant convergent antisense-transcription in Saccharomyces cerevisiae. Moreover we show that aberrant, truncating 3' -end formation is often associated with regulated transcription in TAP-tagged strains. Importantly, the steady-state level of both 3' -truncated and antisense transcription products is locus dependent. Using TAP and GFP-tagged strains we show that the transcriptional state of the gene-of-interest induces changes to 3' -end formation by alternative polyadenylation and antisense transcription from a universal 3' UTR. This means that these 3' UTRs contains plastic features that can be molded to reflect the regulatory architecture of the locus rather than bringing their own regulatory paradigm to the gene-fusions as would be expected. Our work holds a cautionary note for studies utilizing tagged strains for quantitative biology, but also provides a new model for the study of promoter driven rewiring of 3' -end formation and regulatory non-coding transcription. PMID:26481348

  9. Staphylococcus aureus RNAIII coordinately represses the synthesis of virulence factors and the transcription regulator Rot by an antisense mechanism

    PubMed Central

    Boisset, Sandrine; Geissmann, Thomas; Huntzinger, Eric; Fechter, Pierre; Bendridi, Nadia; Possedko, Maria; Chevalier, Clément; Helfer, Anne Catherine; Benito, Yvonne; Jacquier, Alain; Gaspin, Christine; Vandenesch, François; Romby, Pascale

    2007-01-01

    RNAIII is the intracellular effector of the quorum-sensing system in Staphylococcus aureus. It is one of the largest regulatory RNAs (514 nucleotides long) that are known to control the expression of a large number of virulence genes. Here, we show that the 3′ domain of RNAIII coordinately represses at the post-transcriptional level, the expression of mRNAs that encode a class of virulence factors that act early in the infection process. We demonstrate that the 3′ domain acts primarily as an antisense RNA and rapidly anneals to these mRNAs, forming long RNA duplexes. The interaction between RNAIII and the mRNAs results in repression of translation initiation and triggers endoribonuclease III hydrolysis. These processes are followed by rapid depletion of the mRNA pool. In addition, we show that RNAIII and its 3′ domain mediate translational repression of rot mRNA through a limited number of base pairings involving two loop–loop interactions. Since Rot is a transcriptional regulatory protein, we proposed that RNAIII indirectly acts on many downstream genes, resulting in the activation of the synthesis of several exoproteins. These data emphasize the multitude of regulatory steps affected by RNAIII and its 3′ domain in establishing a network of S. aureus virulence factors. PMID:17545468

  10. A multifactor regulatory circuit involving H-NS, VirF and an antisense RNA modulates transcription of the virulence gene icsA of Shigella flexneri

    PubMed Central

    Tran, Chi Nhan; Giangrossi, Mara; Prosseda, Gianni; Brandi, Anna; Di Martino, Maria Letizia; Colonna, Bianca; Falconi, Maurizio

    2011-01-01

    The icsA gene of Shigella encodes a structural protein involved in colonization of the intestinal mucosa by bacteria. This gene is expressed upon invasion of the host and is controlled by a complex regulatory circuit involving the nucleoid protein H-NS, the AraC-like transcriptional activator VirF, and a 450 nt antisense RNA (RnaG) acting as transcriptional attenuator. We investigated on the interplay of these factors at the molecular level. DNase I footprints reveal that both H-NS and VirF bind to a region including the icsA and RnaG promoters. H-NS is shown to repress icsA transcription at 30°C but not at 37°C, suggesting a significant involvement of this protein in the temperature-regulated expression of icsA. We also demonstrate that VirF directly stimulates icsA transcription and is able to alleviate H-NS repression in vitro. According to these results, icsA expression is derepressed in hns- background and overexpressed when VirF is provided in trans. Moreover, we find that RnaG-mediated transcription attenuation depends on 80 nt at its 5′-end, a stretch carrying the antisense region. Bases engaged in the initial contact leading to sense–antisense pairing have been identified using synthetic RNA and DNA oligonucleotides designed to rebuild and mutagenize the two stem–loop motifs of the antisense region. PMID:21724612

  11. Global analysis of cis-natural antisense transcripts and their heat-responsive nat-siRNAs in Brassica rapa

    PubMed Central

    2013-01-01

    Background Brassica rapa includes several important leaf vegetable crops whose production is often damaged by high temperature. Cis-natural antisense transcripts (cis-NATs) and cis-NATs-derived small interfering RNAs (nat-siRNAs) play important roles in plant development and stress responses. However, genome-wide cis-NATs in B. rapa are not known. The NATs and nat-siRNAs that respond to heat stress have never been well studied in B. rapa. Here, we took advantage of RNA-seq and small RNA (sRNA) deep sequencing technology to identify cis-NATs and heat responsive nat-siRNAs in B. rapa. Results Analyses of four RNA sequencing datasets revealed 1031 cis-NATs B. rapa ssp. chinensis cv Wut and B. rapa ssp. pekinensis cv. Bre. Based on sequence homology between Arabidopsis thaliana and B. rapa, 303 conserved cis-NATs in B. rapa were found to correspond to 280 cis-NATs in Arabidopsis; the remaining 728 novel cis-NATs were identified as Brassica-specific ones. Using six sRNA libraries, 4846 nat-siRNAs derived from 150 cis-NATs were detected. Differential expression analysis revealed that nat-siRNAs derived from 12 cis-NATs were responsive to heat stress, and most of them showed strand bias. Real-time PCR indicated that most of the transcripts generating heat-responsive nat-siRNAs were upregulated under heat stress, while the transcripts from the opposite strands of the same loci were downregulated. Conclusions Our results provide the first subsets of genome-wide cis-NATs and heat-responsive nat-siRNAs in B. rapa; these sRNAs are potentially useful for the genetic improvement of heat tolerance in B. rapa and other crops. PMID:24320882

  12. Cutting Edge: A Natural Antisense Transcript, AS-IL1α, Controls Inducible Transcription of the Proinflammatory Cytokine IL-1α.

    PubMed

    Chan, Jennie; Atianand, Maninjay; Jiang, Zhaozhao; Carpenter, Susan; Aiello, Daniel; Elling, Roland; Fitzgerald, Katherine A; Caffrey, Daniel R

    2015-08-15

    Natural antisense transcripts (NATs) are a class of long noncoding RNAs (lncRNAs) that are complementary to other protein-coding genes. Although thousands of NATs are encoded by mammalian genomes, their functions in innate immunity are unknown. In this study, we identified and characterized a novel NAT, AS-IL1α, which is partially complementary to IL-1α. Similar to IL-1α, AS-IL1α is expressed at low levels in resting macrophages and is induced following infection with Listeria monocytogenes or stimulation with TLR ligands (Pam3CSK4, LPS, polyinosinic-polycytidylic acid). Inducible expression of IL-1α mRNA and protein were significantly reduced in macrophages expressing shRNA that target AS-IL1α. AS-IL1α is located in the nucleus and did not alter the stability of IL-1α mRNA. Instead, AS-IL1α was required for the recruitment of RNA polymerase II to the IL-1α promoter. In summary, our studies identify AS-IL1α as an important regulator of IL-1α transcription during the innate immune response. PMID:26179904

  13. Cis-Natural Antisense Transcripts Are Mainly Co-expressed with Their Sense Transcripts and Primarily Related to Energy Metabolic Pathways during Muscle Development

    PubMed Central

    Zhao, Yunxia; Hou, Ye; Zhao, Changzhi; Liu, Fei; Luan, Yu; Jing, Lu; Li, Xinyun; Zhu, Mengjin; Zhao, Shuhong

    2016-01-01

    Cis-natural antisense transcripts (cis-NATs) are a new class of RNAs identified in various species. However, the biological functions of cis-NATs are largely unknown. In this study, we investigated the transcriptional characteristics and functions of cis-NATs in the muscle tissue of lean Landrace and indigenous fatty Lantang pigs. In total, 3,306 cis-NATs of 2,469 annotated genes were identified in the muscle tissue of pigs. More than 1,300 cis-NATs correlated with their sense genes at the transcriptional level, and approximately 80% of them were co-expressed in the two breeds. Furthermore, over 1,200 differentially expressed cis-NATs were identified during muscle development. Function annotation showed that the cis-NATs participated in muscle development mainly by co-expressing with genes involved in energy metabolic pathways, including citrate cycle (TCA cycle), glycolysis or gluconeogenesis, mitochondrial activation and so on. Moreover, these cis-NATs and their sense genes abruptly increased at the transition from the late fetal stages to the early postnatal stages and then decreased along with muscle development. In conclusion, the cis-NATs in the muscle tissue of pigs were identified and determined to be mainly co-expressed with their sense genes. The co-expressed cis-NATs and their sense gene were primarily related to energy metabolic pathways during muscle development in pigs. Our results offered novel evidence on the roles of cis-NATs during the muscle development of pigs. PMID:27489504

  14. Cis-Natural Antisense Transcripts Are Mainly Co-expressed with Their Sense Transcripts and Primarily Related to Energy Metabolic Pathways during Muscle Development.

    PubMed

    Zhao, Yunxia; Hou, Ye; Zhao, Changzhi; Liu, Fei; Luan, Yu; Jing, Lu; Li, Xinyun; Zhu, Mengjin; Zhao, Shuhong

    2016-01-01

    Cis-natural antisense transcripts (cis-NATs) are a new class of RNAs identified in various species. However, the biological functions of cis-NATs are largely unknown. In this study, we investigated the transcriptional characteristics and functions of cis-NATs in the muscle tissue of lean Landrace and indigenous fatty Lantang pigs. In total, 3,306 cis-NATs of 2,469 annotated genes were identified in the muscle tissue of pigs. More than 1,300 cis-NATs correlated with their sense genes at the transcriptional level, and approximately 80% of them were co-expressed in the two breeds. Furthermore, over 1,200 differentially expressed cis-NATs were identified during muscle development. Function annotation showed that the cis-NATs participated in muscle development mainly by co-expressing with genes involved in energy metabolic pathways, including citrate cycle (TCA cycle), glycolysis or gluconeogenesis, mitochondrial activation and so on. Moreover, these cis-NATs and their sense genes abruptly increased at the transition from the late fetal stages to the early postnatal stages and then decreased along with muscle development. In conclusion, the cis-NATs in the muscle tissue of pigs were identified and determined to be mainly co-expressed with their sense genes. The co-expressed cis-NATs and their sense gene were primarily related to energy metabolic pathways during muscle development in pigs. Our results offered novel evidence on the roles of cis-NATs during the muscle development of pigs. PMID:27489504

  15. An antisense promoter in mouse L1 retrotransposon open reading frame-1 initiates expression of diverse fusion transcripts and limits retrotransposition

    PubMed Central

    Li, Jingfeng; Kannan, Manoj; Trivett, Anna L.; Liao, Hongling; Wu, Xiaolin; Akagi, Keiko; Symer, David E.

    2014-01-01

    Between 6 and 30% of human and mouse transcripts are initiated from transposable elements. However, the promoters driving such transcriptional activity are mostly unknown. We experimentally characterized an antisense (AS) promoter in mouse L1 retrotransposons for the first time, oriented antiparallel to the coding strand of L1 open reading frame-1. We found that AS transcription is mediated by RNA polymerase II. Rapid amplification of cDNA ends cloning mapped transcription start sites adjacent to the AS promoter. We identified >100 novel fusion transcripts, of which many were conserved across divergent mouse lineages, suggesting conservation of potential functions. To evaluate whether AS L1 transcription could regulate L1 retrotransposition, we replaced portions of native open reading frame-1 in donor elements by synonymously recoded sequences. The resulting L1 elements lacked AS promoter activity and retrotransposed more frequently than endogenous L1s. Overexpression of AS L1 transcripts also reduced L1 retrotransposition. This suppression of retrotransposition was largely independent of Dicer. Our experiments shed new light on how AS fusion transcripts are initiated from endogenous L1 elements across the mouse genome. Such AS transcription can contribute substantially both to natural transcriptional variation and to endogenous regulation of L1 retrotransposition. PMID:24493738

  16. Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals.

    PubMed

    Golovkin, Maxim; Reddy, Anireddy S N

    2003-08-01

    U1 small nuclear ribonucleoprotein (snRNP)-70K (U1-70K), a U1 snRNP-specific protein, is involved in the early stages of spliceosome formation. In non-plant systems, it is involved in constitutive and alternative splicing. It has been shown that U1snRNP is dispensable for in vitro splicing of some animal pre-mRNAs, and inactivation of U1-70K in yeast (Saccharomyces cerevisiae) is not lethal. As in yeast and humans (Homo sapiens), plant U1-70K is coded by a single gene. In this study, we blocked the expression of Arabidopsis U1-70K in petals and stamens by expressing U1-70K antisense transcript using the AP3 (APETALA3) promoter specific to these floral organs. Flowers of transgenic Arabidopsis plants expressing U1-70K antisense transcript showed partially developed stamens and petals that are arrested at different stages of development. In some transgenic lines, flowers have rudimentary petals and stamens and are male sterile. The severity of the phenotype is correlated with the level of the antisense transcript. Molecular analysis of transgenic plants has confirmed that the observed phenotype is not due to disruption of whorl-specific homeotic genes, AP3 or PISTILLATA, responsible for petal and stamen development. The AP3 transcript was not detected in transgenic flowers with severe phenotype. Flowers of Arabidopsis plants transformed with a reporter gene driven by the same promoter showed no abnormalities. These results show that U1-70K is necessary for the development of sepals and petals and is an essential gene in plants. PMID:12913145

  17. Promoter-restricted histone code, not the differentially methylated DNA regions or antisense transcripts, marks the imprinting status of IGF2R in human and mouse.

    PubMed

    Vu, Thanh H; Li, Tao; Hoffman, Andrew R

    2004-10-01

    Imprinting of the mouse Igf2r depends upon an intronic differentially methylated DNA region (DMR) and the presence of the Air antisense transcript. However, biallelic expression of mouse Igf2r in brain occurs despite the presence of Air, and biallelic expression of human IGF2R in peripheral tissues occurs despite the presence of an intronic DMR. We examined histone modifications throughout the mouse and human Igf2r/IGF2R using chromatin immuno-precipitation (ChIP) assays in combination with quantitative real time PCR. Methylation of Lys4 and Lys9 of histone H3 in the promoter regions marks the active and silenced alleles, respectively. We measured di- and tri-methyl Lys4 and Lys9 across the Igf2r and Air promoters. While both di- and tri-methyl Lys4 marked the active Igf2r and the active Air allele, tri-methyl Lys9, but not di-methyl Lys9, marked the suppressed Air allele. We show here that enrichment of parental allele-specific histone modifications in the promoter region, rather than the presence of DNA methylation or antisense transcription, correctly identifies the tissue- and species- specific imprinting status of Igf2r/IGF2R. We discuss these findings in light of recent progress in identifying specific components of the epigenetic marks in imprinted genes. PMID:15294879

  18. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

    PubMed

    Burel, Sebastien A; Hart, Christopher E; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-Hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M; Hung, Gene; Dan, Amy; Prakash, T P; Seth, Punit P; Swayze, Eric E; Bennett, C Frank; Crooke, Stanley T; Henry, Scott P

    2016-03-18

    High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

  19. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts

    PubMed Central

    Burel, Sebastien A.; Hart, Christopher E.; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M.; Hung, Gene; Dan, Amy; Prakash, T.P.; Seth, Punit P.; Swayze, Eric E.; Bennett, C. Frank; Crooke, Stanley T.; Henry, Scott P.

    2016-01-01

    High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

  20. A σD-dependent antisense transcript modulates expression of the cyclic-di-AMP hydrolase GdpP in Bacillus subtilis.

    PubMed

    Luo, Yun; Helmann, John D

    2012-11-01

    Cyclic-di-AMP (c-di-AMP) is an essential second messenger in Bacillus subtilis, and depletion leads to defects in the integrity of the cell wall. Levels of c-di-AMP are regulated by both the rates of synthesis (by diadenylate cyclases) and the rates of degradation (by the GdpP phosphodiesterase, formerly YybT). Little is known about the regulation of gdpP expression or GdpP activity, but mutations that inactivate GdpP lead to high-level resistance to β-lactam antibiotics. Here we demonstrate that expression of gdpP is regulated by a cis-acting antisense RNA (gdpP(as)) in vivo. Transcription of this antisense RNA is initiated in the middle of the gdp gene and is dependent on an alternative sigma factor, σ(D), previously associated with the expression of late flagellar genes, chemotaxis proteins and cell wall autolytic enzymes. Changes in σ(D) activity can modulate GdpP protein levels by ~2.5-fold, which may provide a mechanism for the cell to upregulate c-di-AMP levels in coordination with the activation of autolytic enzymes. PMID:22956758

  1. A σD-dependent antisense transcript modulates expression of the cyclic-di-AMP hydrolase GdpP in Bacillus subtilis

    PubMed Central

    Luo, Yun

    2012-01-01

    Cyclic-di-AMP (c-di-AMP) is an essential second messenger in Bacillus subtilis, and depletion leads to defects in the integrity of the cell wall. Levels of c-di-AMP are regulated by both the rates of synthesis (by diadenylate cyclases) and the rates of degradation (by the GdpP phosphodiesterase, formerly YybT). Little is known about the regulation of gdpP expression or GdpP activity, but mutations that inactivate GdpP lead to high-level resistance to β-lactam antibiotics. Here we demonstrate that expression of gdpP is regulated by a cis-acting antisense RNA (gdpPas) in vivo. Transcription of this antisense RNA is initiated in the middle of the gdp gene and is dependent on an alternative sigma factor, σD, previously associated with the expression of late flagellar genes, chemotaxis proteins and cell wall autolytic enzymes. Changes in σD activity can modulate GdpP protein levels by ~2.5-fold, which may provide a mechanism for the cell to upregulate c-di-AMP levels in coordination with the activation of autolytic enzymes. PMID:22956758

  2. CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells.

    PubMed

    Degner, Stephanie C; Verma-Gaur, Jiyoti; Wong, Timothy P; Bossen, Claudia; Iverson, G Michael; Torkamani, Ali; Vettermann, Christian; Lin, Yin C; Ju, Zhongliang; Schulz, Danae; Murre, Caroline S; Birshtein, Barbara K; Schork, Nicholas J; Schlissel, Mark S; Riblet, Roy; Murre, Cornelis; Feeney, Ann J

    2011-06-01

    Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the V(H) region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5' of DFL16 and the 3' regulatory region, and also the intronic enhancer (Eμ), creating a D(H)-J(H)-Eμ-C(H) domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D(H) region and parts of the V(H) locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus. PMID:21606361

  3. Human T-cell leukemia virus type 2 produces a spliced antisense transcript encoding a protein that lacks a classic bZIP domain but still inhibits Tax2-mediated transcription

    PubMed Central

    Halin, Marilène; Douceron, Estelle; Clerc, Isabelle; Journo, Chloé; Ko, Nga Ling; Landry, Sébastien; Murphy, Edward L.; Gessain, Antoine; Lemasson, Isabelle; Mesnard, Jean-Michel

    2009-01-01

    Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) retroviruses infect T lymphocytes. The minus strand of the HTLV-1 genome encodes HBZ, a protein that could play a role in the development of leukemia in infected patients. Herein, we demonstrate that the complementary strand of the HTLV-2 genome also encodes a protein that we named APH-2 for “antisense protein of HTLV-2.” APH-2 mRNA is spliced, polyadenylated, and initiates in the 3′-long terminal repeat at different positions. This transcript was detected in all HTLV-2–infected cell lines and short-term culture of lymphocytes obtained from HTLV-2 African patients tested and in 4 of 15 HTLV-2–infected blood donors. The APH-2 protein is 183 amino acids long, is localized in the cell nucleus, and is detected in vivo. Despite the lack of a consensus basic leucine zipper domain, APH-2 interacts with cyclic adenosine monophosphate-response element binding protein (CREB) and represses Tax2-mediated transcription in Tax2-expressing cells and in cells transfected with an HTLV-2 molecular clone. Altogether, our results demonstrate the existence of an antisense strand–encoded protein in HTLV-2, which could represent an important player in the development of disorders, such as lymphocytosis, which is frequently observed in HTLV-2 patients. PMID:19602711

  4. Human T-cell leukemia virus type 2 produces a spliced antisense transcript encoding a protein that lacks a classic bZIP domain but still inhibits Tax2-mediated transcription.

    PubMed

    Halin, Marilène; Douceron, Estelle; Clerc, Isabelle; Journo, Chloé; Ko, Nga Ling; Landry, Sébastien; Murphy, Edward L; Gessain, Antoine; Lemasson, Isabelle; Mesnard, Jean-Michel; Barbeau, Benoît; Mahieux, Renaud

    2009-09-17

    Human T-cell leukemia virus type 1 (HTLV-1) and type 2 (HTLV-2) retroviruses infect T lymphocytes. The minus strand of the HTLV-1 genome encodes HBZ, a protein that could play a role in the development of leukemia in infected patients. Herein, we demonstrate that the complementary strand of the HTLV-2 genome also encodes a protein that we named APH-2 for "antisense protein of HTLV-2." APH-2 mRNA is spliced, polyadenylated, and initiates in the 3'-long terminal repeat at different positions. This transcript was detected in all HTLV-2-infected cell lines and short-term culture of lymphocytes obtained from HTLV-2 African patients tested and in 4 of 15 HTLV-2-infected blood donors. The APH-2 protein is 183 amino acids long, is localized in the cell nucleus, and is detected in vivo. Despite the lack of a consensus basic leucine zipper domain, APH-2 interacts with cyclic adenosine monophosphate-response element binding protein (CREB) and represses Tax2-mediated transcription in Tax2-expressing cells and in cells transfected with an HTLV-2 molecular clone. Altogether, our results demonstrate the existence of an antisense strand-encoded protein in HTLV-2, which could represent an important player in the development of disorders, such as lymphocytosis, which is frequently observed in HTLV-2 patients. PMID:19602711

  5. Comparison of hepatic transcription profiles of locked ribonucleic acid antisense oligonucleotides: evidence of distinct pathways contributing to non-target mediated toxicity in mice.

    PubMed

    Kakiuchi-Kiyota, Satoko; Koza-Taylor, Petra H; Mantena, Srinivasa R; Nelms, Linda F; Enayetallah, Ahmed E; Hollingshead, Brett D; Burdick, Andrew D; Reed, Lori A; Warneke, James A; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2014-03-01

    Development of LNA gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by non-target mediated hepatotoxicity issues. In the present study, we investigated hepatic transcription profiles of mice administered non-toxic and toxic LNA gapmers. After repeated administration, a toxic LNA gapmer (TS-2), but not a non-toxic LNA gapmer (NTS-1), caused hepatocyte necrosis and increased serum alanine aminotransferase levels. Microarray data revealed that, in addition to gene expression patterns consistent with hepatotoxicity, 17 genes in the clathrin-mediated endocytosis (CME) pathway were altered in the TS-2 group. TS-2 significantly down-regulated myosin 1E (Myo1E), which is involved in release of clathrin-coated pits from plasma membranes. To map the earliest transcription changes associated with LNA gapmer-induced hepatotoxicity, a second microarray analysis was performed using NTS-1, TS-2, and a severely toxic LNA gapmer (HTS-3) at 8, 16, and 72 h following a single administration in mice. The only histopathological change observed was minor hepatic hypertrophy in all LNA groups across time points. NTS-1, but not 2 toxic LNA gapmers, increased immune response genes at 8 and 16 h but not at 72 h. TS-2 significantly perturbed the CME pathway only at 72 h, while Myo1E levels were decreased at all time points. In contrast, HTS-3 modulated DNA damage pathway genes at 8 and 16 h and also modulated the CME pathway genes (but not Myo1E) at 16 h. Our results may suggest that different LNAs modulate distinct transcriptional genes and pathways contributing to non-target mediated hepatotoxicity in mice. PMID:24336348

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

    PubMed Central

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

    2013-01-01

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

  7. Quantitative Proteomics Analysis Reveals Novel Insights into Mechanisms of Action of Long Noncoding RNA Hox Transcript Antisense Intergenic RNA (HOTAIR) in HeLa Cells*

    PubMed Central

    Zheng, Peng; Xiong, Qian; Wu, Ying; Chen, Ying; Chen, Zhuo; Fleming, Joy; Gao, Ding; Bi, Lijun; Ge, Feng

    2015-01-01

    Long noncoding RNAs (lncRNAs), which have emerged in recent years as a new and crucial layer of gene regulators, regulate various biological processes such as carcinogenesis and metastasis. HOTAIR (Hox transcript antisense intergenic RNA), a lncRNA overexpressed in most human cancers, has been shown to be an oncogenic lncRNA. Here, we explored the role of HOTAIR in HeLa cells and searched for proteins regulated by HOTAIR. To understand the mechanism of action of HOTAIR from a systems perspective, we employed a quantitative proteomic strategy to systematically identify potential targets of HOTAIR. The expression of 170 proteins was significantly dys-regulated after inhibition of HOTAIR, implying that they could be potential targets of HOTAIR. Analysis of this data at the systems level revealed major changes in proteins involved in diverse cellular components, including the cytoskeleton and the respiratory chain. Further functional studies on vimentin (VIM), a key protein involved in the cytoskeleton, revealed that HOTAIR exerts its effects on migration and invasion of HeLa cells, at least in part, through the regulation of VIM expression. Inhibition of HOTAIR leads to mitochondrial dysfunction and ultrastructural alterations, suggesting a novel role of HOTAIR in maintaining mitochondrial function in cancer cells. Our results provide novel insights into the mechanisms underlying the function of HOTAIR in cancer cells. We expect that the methods used in this study will become an integral part of functional studies of lncRNAs. PMID:25762744

  8. Exploring Differentially Expressed Genes and Natural Antisense Transcripts in Sheep (Ovis aries) Skin with Different Wool Fiber Diameters by Digital Gene Expression Profiling

    PubMed Central

    Liu, Jianbin; Guo, Jian; Yuan, Chao; Feng, Ruilin; Niu, Chune; Sun, Xiaoping; Yang, Bohui

    2015-01-01

    Wool fiber diameter (WFD) is the most important economic trait of wool. However, the genes specifically controlling WFD remain elusive. In this study, the expression profiles of skin from two groups of Gansu Alpine merino sheep with different WFD (a super-fine wool group [FD = 18.0 ± 0.5 μm, n= 3] and a fine wool group [FD=23.0±0.5μm, n=3]) were analyzed using next-generation sequencing–based digital gene expression profiling. A total of 40 significant differentially expressed genes (DEGs) were detected, including 9 up-regulated genes and 31 down-regulated genes. Further expression profile analysis of natural antisense transcripts (NATs) showed that more than 30% of the genes presented in sheep skin expression profiles had NATs. A total of 7 NATs with significant differential expression were detected, and all were down-regulated. Among of 40 DEGs, 3 DEGs (AQP8, Bos d2, and SPRR) had significant NATs which were all significantly down-regulated in the super-fine wool group. In total of DEGs and NATs were summarized as 3 main GO categories and 38 subcategories. Among the molecular functions, cellular components and biological processes categories, binding, cell part and metabolic process were the most dominant subcategories, respectively. However, no significant enrichment of GO terms was found (corrected P-value >0.05). The pathways that were significantly enriched with significant DEGs and NATs were mainly the lipoic acid metabolism, bile secretion, salivary secretion and ribosome and phenylalanine metabolism pathways (P < 0.05). The results indicated that expression of NATs and gene transcripts were correlated, suggesting a role in gene regulation. The discovery of these DEGs and NATs could facilitate enhanced selection for super-fine wool sheep through gene-assisted selection or targeted gene manipulation in the future. PMID:26076016

  9. Sense antisense DNA strand?

    PubMed

    Boldogkói, Z; Kaliman, A V; Murvai, J; Fodor, I

    1994-01-01

    Recent evidence indicates that alphaherpesviruses express latency associated transcripts (LATs) from the antisense strand of immediate-early (IE) genes of the viral genome. It has been suggested that LATs containing extended open reading frames (ORFs), might be translated into (a) protein product(s). We found that a salient feature of some herpesvirus DNAs is a high GC preference at the third codon positions. The consequence of this feature is that the probability of a stop-codon appearing at two of the six reading frames of the DNA strand is very low. Therefore, the presence of an extended ORF does not necessarily mean that it is relevant to real translation. PMID:7810418

  10. Targeting Several CAG Expansion Diseases by a Single Antisense Oligonucleotide

    PubMed Central

    Evers, Melvin M.; Pepers, Barry A.; van Deutekom, Judith C. T.; Mulders, Susan A. M.; den Dunnen, Johan T.; Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B.; van Roon-Mom, Willeke M. C.

    2011-01-01

    To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2′-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)7, also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well. PMID:21909428

  11. Targeting several CAG expansion diseases by a single antisense oligonucleotide.

    PubMed

    Evers, Melvin M; Pepers, Barry A; van Deutekom, Judith C T; Mulders, Susan A M; den Dunnen, Johan T; Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B; van Roon-Mom, Willeke M C

    2011-01-01

    To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)(7), also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well. PMID:21909428

  12. The landscape of antisense gene expression in human cancers.

    PubMed

    Balbin, O Alejandro; Malik, Rohit; Dhanasekaran, Saravana M; Prensner, John R; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G; Nesvizhskii, Alexey I; Chinnaiyan, Arul M

    2015-07-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts' (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology. PMID:26063736

  13. Antisense Oligonucleotide Against GSK-3β in Brain of SAMP8 Mice Improves Learning and Memory and Decreases Oxidative Stress: Involvement of Transcription Factor Nrf2 and Implications for Alzheimer Disease

    PubMed Central

    Farr, Susan A.; Ripley, Jessica L.; Sultana, Rukhsana; Zhang, Zhaoshu; Niehoff, Michael L.; Platt, Thomas L.; Murphy, M. Paul; Morley, John E.; Kumar, Vijaya; Butterfield, D. Allan

    2014-01-01

    Glycogen synthase kinase (GSK) -3β is a multifunctional protein that has been implicated in the pathological characteristics of Alzheimer’s disease (AD), including the heightened levels of neurofibrillary tangles, amyloid-beta (Aβ) and neurodegeneration. In this study we used 12 month old SAMP8 mice, an AD model, to examine the effects GSK-3β may cause regarding the cognitive impairment and oxidative stress associated with AD. To suppress the level of GSK-3β, SAMP8 mice were treated with an antisense oligonucleotide (GAO) directed at this kinase. We measured a decreased level of GSK-3β in the cortex of the mice, indicating the success of the antisense treatment. Learning and memory assessments of the SAMP8 mice were tested post-antisense treatment using an aversive T-maze and object recognition test, both of which observably improved. In cortex samples of the SAMP8 mice, decreased levels of protein carbonyl and protein-bound HNE were measured indicating decreased oxidative stress. Nuclear factor erythroid -2-related factor 2 (Nrf2) is a transcription factor known to increase the level of many antioxidants, including glutathione-S transferase (GST), and is negatively regulated by the activity of GSK-3β. Our results indicated the increased nuclear localization of Nrf2 and level of GST, suggesting the increased activity of the transcription factor as a result of GSK-3β suppression, consistent with the decreased oxidative stress observed. Consistent with the improved learning and memory, and consistent with GSK-3b being a tau kinase, we observed decreased tau phosphorylation in brain of GAO-treated SAMP8 mice compared to that of RAO-treated SAMP8 mice. Lastly, we examined the ability of GAO to cross the blood-brain barrier and determined it to be possible. The results presented in this study demonstrate that reducing GSK-3 with a phosphorothionated antisense against GSK-3 improves learning and memory, reduces oxidative stress, possibly coincident with

  14. JACALIN-LECTIN LIKE1 Regulates the Nuclear Accumulation of GLYCINE-RICH RNA-BINDING PROTEIN7, Influencing the RNA Processing of FLOWERING LOCUS C Antisense Transcripts and Flowering Time in Arabidopsis1[OPEN

    PubMed Central

    Xiao, Jun; Li, Chunhua; Xu, Shujuan; Xing, Lijing; Xu, Yunyuan; Chong, Kang

    2015-01-01

    Lectins selectively recognize sugars or glycans for defense in living cells, but less is known about their roles in the development process and the functional network with other factors. Here, we show that Arabidopsis (Arabidopsis thaliana) JACALIN-LECTIN LIKE1 (AtJAC1) functions in flowering time control. Loss of function of AtJAC1 leads to precocious flowering, whereas overexpression of AtJAC1 causes delayed flowering. AtJAC1 influences flowering through regulation of the key flowering repressor gene FLOWERING LOCUS C (FLC). Genetic analysis revealed that AtJAC1’s function is mostly dependent on GLYCINE-RICH RNA-BINDING PROTEIN7 (GRP7), an upstream regulator of FLC. Biochemical and cell biological data indicated that AtJAC1 interacted physically with GRP7 specifically in the cytoplasm. AtJAC1 influences the nucleocytoplasmic distribution of GRP7, with predominant nuclear localization of GRP7 when AtJAC1 function is lost but retention of GRP7 in the cytoplasm when AtJAC1 is overexpressed. A temporal inducible assay suggested that AtJAC1’s regulation of flowering could be compromised by the nuclear accumulation of GRP7. In addition, GRP7 binds to the antisense precursor messenger RNA of FLC through a conserved RNA motif. Loss of GRP7 function leads to the elevation of total FLC antisense transcripts and reduced proximal-distal polyadenylation ratio, as well as histone methylation changes in the FLC gene body region and increased total functional sense FLC transcript. Attenuating the direct binding of GRP7 with competing artificial RNAs leads to changes of FLC antisense precursor messenger RNA processing and flowering transition. Taken together, our study indicates that AtJAC1 coordinates with GRP7 in shaping plant development through the regulation of RNA processing in Arabidopsis. PMID:26392261

  15. Complex sense-antisense architecture of TNFAIP1/POLDIP2 on 17q11.2 represents a novel transcriptional structural-functional gene module involved in breast cancer progression

    PubMed Central

    2010-01-01

    Background A sense-antisense gene pair (SAGP) is a gene pair where two oppositely transcribed genes share a common nucleotide sequence region. In eukaryotic genomes, SAGPs can be organized in complex sense-antisense architectures (CSAGAs) in which at least one sense gene shares loci with two or more antisense partners. As shown in several case studies, SAGPs may be involved in cancers, neurological diseases and complex syndromes. However, CSAGAs have not yet been characterized in the context of human disease or cancer. Results We characterize five genes (TMEM97, IFT20, TNFAIP1, POLDIP2 and TMEM199) organized in a CSAGA on 17q11.2 (we term this the TNFAIP1/POLDIP2 CSAGA) and demonstrate their strong and reproducible co-regulatory transcription pattern in breast cancer tumours. Genes of the TNFAIP1/POLDIP2 CSAGA are located inside the smallest region of recurrent amplification on 17q11.2 and their expression profile correlates with the DNA copy number of the region. Survival analysis of a group of 410 breast cancer patients revealed significant survival-associated individual genes and gene pairs in the TNFAIP1/POLDIP2 CSAGA. Moreover, several of the gene pairs associated with survival, demonstrated synergistic effects. Expression of genes-members of the TNFAIP1/POLDIP2 CSAGA also strongly correlated with expression of genes of ERBB2 core region of recurrent amplification on 17q12. We clearly demonstrate that the observed co-regulatory transcription profile of the TNFAIP1/POLDIP2 CSAGA is maintained not only by a DNA amplification mechanism, but also by chromatin remodelling and local transcription activation. Conclusion We have identified a novel TNFAIP1/POLDIP2 CSAGA and characterized its co-regulatory transcription profile in cancerous breast tissues. We suggest that the TNFAIP1/POLDIP2 CSAGA represents a clinically significant transcriptional structural-functional gene module associated with amplification of the genomic region on 17q11.2 and correlated with

  16. The landscape of antisense gene expression in human cancers

    PubMed Central

    Balbin, O. Alejandro; Malik, Rohit; Dhanasekaran, Saravana M.; Prensner, John R.; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G.; Nesvizhskii, Alexey I.; Chinnaiyan, Arul M.

    2015-01-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts’ (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology. PMID:26063736

  17. Development of Antisense Drugs for Dyslipidemia.

    PubMed

    Yamamoto, Tsuyoshi; Wada, Fumito; Harada-Shiba, Mariko

    2016-09-01

    Abnormal elevation of low-density lipoprotein (LDL) and triglyceride-rich lipoproteins in plasma as well as dysfunction of anti-atherogenic high-density lipoprotein (HDL) have both been recognized as essential components of the pathogenesis of atherosclerosis and are classified as dyslipidemia. This review describes the arc of development of antisense oligonucleotides for the treatment of dyslipidemia. Chemically-armed antisense candidates can act on various kinds of transcripts, including mRNA and miRNA, via several different endogenous antisense mechanisms, and have exhibited potent systemic anti-dyslipidemic effects. Here, we present specific cutting-edge technologies have recently been brought into antisense strategies, and describe how they have improved the potency of antisense drugs in regard to pharmacokinetics and pharmacodynamics. In addition, we discuss perspectives for the use of armed antisense oligonucleotides as new clinical options for dyslipidemia, in the light of outcomes of recent clinical trials and safety concerns indicated by several clinical and preclinical studies. PMID:27466159

  18. Functional correction by antisense therapy of a splicing mutation in the GALT gene.

    PubMed

    Coelho, Ana I; Lourenço, Sílvia; Trabuco, Matilde; Silva, Maria João; Oliveira, Anabela; Gaspar, Ana; Diogo, Luísa; Tavares de Almeida, Isabel; Vicente, João B; Rivera, Isabel

    2015-04-01

    In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314

  19. Antisense Therapy in Neurology

    PubMed Central

    Lee, Joshua J.A.; Yokota, Toshifumi

    2013-01-01

    Antisense therapy is an approach to fighting diseases using short DNA-like molecules called antisense oligonucleotides. Recently, antisense therapy has emerged as an exciting and promising strategy for the treatment of various neurodegenerative and neuromuscular disorders. Previous and ongoing pre-clinical and clinical trials have provided encouraging early results. Spinal muscular atrophy (SMA), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD), Fukuyama congenital muscular dystrophy (FCMD), dysferlinopathy (including limb-girdle muscular dystrophy 2B; LGMD2B, Miyoshi myopathy; MM, and distal myopathy with anterior tibial onset; DMAT), and myotonic dystrophy (DM) are all reported to be promising targets for antisense therapy. This paper focuses on the current progress of antisense therapies in neurology. PMID:25562650

  20. Does everything now make (anti)sense?

    PubMed

    Timmons, J A; Good, L

    2006-12-01

    The data generated by the FANTOM (Functional Annotation of Mouse) consortium, Compugen and Affymetrix have collectively provided evidence that most of the mammalian genomes are actively transcribed. The emergence of an antisense RNA world brings new practical complexities to the study and detection of gene expression. However, we also need to address the fundamental questions regarding the functional importance of these molecules. In this brief paper, we focus on non-coding natural antisense transcription, as it appears to be a potentially powerful mechanism for extending the complexity of the protein coding genome, which is currently unable to explain inter-species diversification. PMID:17073772

  1. Antisense oligonucleotides bound in the polysaccharide complex and the enhanced antisense effect due to the low hydrolysis.

    PubMed

    Mizu, Masami; Koumoto, Kazuya; Anada, Takahisa; Sakurai, Kazuo; Shinkai, Seiji

    2004-07-01

    Schizophyllan is a beta-(1-->3)-D-glucan and can form a novel complex with some single-chains of DNAs. As the preceding paper revealed, the polynucleotide bound in the complex is more stable to nuclease-mediated hydrolysis than the polynucleotide itself (i.e., naked polynucleotide). This paper examined possibility to apply this complex to an antisense DNA carrier, using an in vitro (cell-free) transcription/translation assay. In this assay, we used a plasmid DNA coding a green fluorescence protein (GFP) and an antisense DNA designed to hybridize the ribosome-binding site in the GFP-coded mRNA. When the antisense DNA was administered as the complex, a lower GFP expression efficiency (or higher antisense effect) is observed over naked DNA. This is because the antisense DNA in the complex is protected from the attack of deoxyribonuclease. When exonuclease I, which specifically hydrolyzes single DNA chains, was present in the GEP assay system, the antisense effect was not changed for the complex while being weakened in the naked antisense DNA system. These results imply that the exonuclease I cannot hydrolyze the antisense DNA in the complex, while it can hydrolyze naked DNA to reduce its antisense effect. PMID:14967546

  2. Antisense oligonucleotides as therapeutics for malignant diseases.

    PubMed

    Ho, P T; Parkinson, D R

    1997-04-01

    The continued progress in our understanding of the biology of neoplasia and in the identification, cloning, and sequencing of genes critical to tumor cell function permits the exploitation of this information to develop specific agents that may directly modulate the function of these genes or their protein products. Antisense oligonucleotides are being investigated as a potential therapeutic modality that takes direct advantage of molecular sequencing. The antisense approach uses short oligonucleotides designed to hybridize to a target mRNA transcript through Watson-Crick base pairing. The formation of this oligonucleotide: RNA heteroduplex results in mRNA inactivation and consequent inhibition of synthesis of the protein product. A fundamental attraction of the antisense approach is that this method potentially may be applied to any gene product, in theory, for the treatment of malignant and non-malignant diseases. However, this simple and attractive model has proven to be much more complex in practice. A number of important challenges in the preclinical development of antisense oligonucleotides have been identified, including stability, sequence length, cellular uptake, target sequence selection, appropriate negative controls, oligonucleotide: protein interactions, and cost of manufacture. Although the biological activity of an oligonucleotide against its molecular target is theoretically sequence-dependent, the animal pharmacokinetics and toxicology of phosphorothioate analogues directed against vastly disparate gene products appear relatively non-sequence-specific. In oncology, a number of clinical trials have been initiated with antisense oligonucleotides directed against molecular targets including: p53; bcl-2; raf kinase; protein kinase C-alpha; c-myb. The experience gained from these early clinical trials will be applicable to the next generation of antisense agents in development. These may include molecules with novel backbones or other structural

  3. Upping the Antisense Ante.

    ERIC Educational Resources Information Center

    Weiss, Rick

    1991-01-01

    Discussed is a designer-drug technology called antisense which blocks messenger RNA's ability to carry information to protein producing sites in the cell. The applications of this drug to AIDS research, cancer therapy, and other diseases are discussed. (KR)

  4. New methods as alternative or corrective measures for the pitfalls and artifacts of reverse transcription and polymerase chain reactions (RT-PCR) in cloning chimeric or antisense-accompanied RNA

    PubMed Central

    Yuan, Chengfu; Liu, Yongming; Yang, Min; Liao, D. Joshua

    2013-01-01

    We established new methods for cloning cDNA ends that start with reverse transcription (RT) and soon proceed with the synthesis of the second cDNA strand, avoiding manipulations of fragile RNA. Our 3′-end cloning method does not involve poly-dT primers and polymerase chain reactions (PCR), is low in efficiency but high in fidelity and can clone those RNAs without a poly-A tail. We also established a cDNA protection assay to supersede RNA protection assay. The protected cDNA can be amplified, cloned and sequenced, enhancing sensitivity and fidelity. We report that RT product using gene-specific primer (GSP) cannot be gene- or strand-specific because RNA sample contains endogenous random primers (ERP). The gene-specificity may be improved by adding a linker sequence at the 5′-end of the GSP to prime RT and using the linker as a primer in the ensuing PCR. The strand-specificity may be improved by using strand-specific DNA oligos in our protection assay. The CDK4 mRNA and TSPAN31 mRNA are transcribed from the opposite DNA strands and overlap at their 3′ ends. Using this relationship as a model, we found that the overlapped sequence might serve as a primer with its antisense as the template to create a wrong-template extension in RT or PCR. We infer that two unrelated RNAs or cDNAs overlapping at the 5′- or 3′-end might create a spurious chimera in this way, and many chimeras with a homologous sequence may be such artifacts. The ERP and overlapping antisense together set complex pitfalls, which one should be aware of. PMID:23618925

  5. Overexpression of members of the AP-1 transcriptional factor family from an early stage of renal carcinogenesis and inhibition of cell growth by AP-1 gene antisense oligonucleotides in the Tsc2 gene mutant (Eker) rat model.

    PubMed

    Urakami, S; Tsuchiya, H; Orimoto, K; Kobayashi, T; Igawa, M; Hino, O

    1997-12-01

    We previously isolated subtracted cDNA clones for genes having increased expression in Tsc2 gene mutant (Eker) rat renal carcinomas (RCs). Among them, fra-1 encoding a transcriptional factor activator protein 1 (AP-1) was identified. We have therefore investigated whether other members of the AP-1 transcription factor family might also be involved in renal carcinogenesis in the Eker rat model. In the present study, overexpression of fra-1, fra-2, c-jun, junB, and junD mRNAs was demonstrated in RCs by Northern blot analysis. Interestingly, AP-1 proteins were highly expressed even in the earliest preneoplastic lesions (e.g., phenotypically altered tubules) as suggested by immunohistochemistry. Moreover, 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE)-binding activity of AP-1 proteins was observed in RC cell extracts by electrophoretic mobility shift assay. As a next step, we transfected antisense oligonucleotides targeting AP-1 genes into RC cells and demonstrated that their growth was strongly inhibited. Thus, the data suggest that overexpression of AP-1 genes might play a crucial role in renal carcinogenesis in the Eker rat model. PMID:9405228

  6. Histone H3 Lysine 36 Trimethylation Is Established over the Xist Promoter by Antisense Tsix Transcription and Contributes to Repressing Xist Expression

    PubMed Central

    Ohhata, Tatsuya; Matsumoto, Mika; Leeb, Martin; Shibata, Shinwa; Sakai, Satoshi; Kitagawa, Kyoko; Niida, Hiroyuki

    2015-01-01

    One of the two X chromosomes in female mammals is inactivated by the noncoding Xist RNA. In mice, X chromosome inactivation (XCI) is regulated by the antisense RNA Tsix, which represses Xist on the active X chromosome. In the absence of Tsix, PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) is established over the Xist promoter. Simultaneous disruption of Tsix and PRC2 leads to derepression of Xist and in turn silencing of the single X chromosome in male embryonic stem cells. Here, we identified histone H3 lysine 36 trimethylation (H3K36me3) as a modification that is recruited by Tsix cotranscriptionally and extends over the Xist promoter. Reduction of H3K36me3 by expression of a mutated histone H3.3 with a substitution of methionine for lysine at position 36 causes a significant derepression of Xist. Moreover, depletion of the H3K36 methylase Setd2 leads to upregulation of Xist, suggesting H3K36me3 as a modification that contributes to the mechanism of Tsix function in regulating XCI. Furthermore, we found that reduction of H3K36me3 does not facilitate an increase in H3K27me3 over the Xist promoter, indicating that additional mechanisms exist by which Tsix blocks PRC2 recruitment to the Xist promoter. PMID:26370508

  7. rasiRNA pathway controls antisense expression of Drosophila telomeric retrotransposons in the nucleus

    PubMed Central

    Shpiz, Sergey; Kwon, Dmitry; Rozovsky, Yakov; Kalmykova, Alla

    2009-01-01

    Telomeres in Drosophila are maintained by the specialized telomeric retrotransposons HeT-A, TART and TAHRE. Sense transcripts of telomeric retroelements were shown to be the targets of a specialized RNA-interference mechanism, a repeat-associated short interfering (rasi)RNA-mediated system. Antisense rasiRNAs play a key role in this mechanism, highlighting the importance of antisense expression in retrotransposon silencing. Previously, bidirectional transcription was reported for the telomeric element TART. Here, we show that HeT-A is also bidirectionally transcribed, and HeT-A antisense transcription in ovaries is regulated by a promoter localized within its 3′ untranslated region. A remarkable feature of noncoding HeT-A antisense transcripts is the presence of multiple introns. We demonstrate that sense and antisense HeT-A-specific rasiRNAs are present in the same tissue, indicating that transcripts of both directions may be considered as natural targets of the rasiRNA pathway. We found that the expression of antisense transcripts of telomeric elements is regulated by the RNA silencing machinery, suggesting rasiRNA-mediated interplay between sense and antisense transcripts in the cell. Finally, this regulation occurs in the nucleus since disruption of the rasiRNA pathway leads to an accumulation of TART and HeT-A transcripts in germ cell nuclei. PMID:19036789

  8. Neighboring Gene Regulation by Antisense Long Non-Coding RNAs

    PubMed Central

    Villegas, Victoria E.; Zaphiropoulos, Peter G.

    2015-01-01

    Antisense transcription, considered until recently as transcriptional noise, is a very common phenomenon in human and eukaryotic transcriptomes, operating in two ways based on whether the antisense RNA acts in cis or in trans. This process can generate long non-coding RNAs (lncRNAs), one of the most diverse classes of cellular transcripts, which have demonstrated multifunctional roles in fundamental biological processes, including embryonic pluripotency, differentiation and development. Antisense lncRNAs have been shown to control nearly every level of gene regulation—pretranscriptional, transcriptional and posttranscriptional—through DNA–RNA, RNA–RNA or protein–RNA interactions. This review is centered on functional studies of antisense lncRNA-mediated regulation of neighboring gene expression. Specifically, it addresses how these transcripts interact with other biological molecules, nucleic acids and proteins, to regulate gene expression through chromatin remodeling at the pretranscriptional level and modulation of transcriptional and post-transcriptional processes by altering the sense mRNA structure or the cellular compartmental distribution, either in the nucleus or the cytoplasm. PMID:25654223

  9. Fast and accurate determination of sites along the FUT2 in vitro transcript that are accessible to antisense oligonucleotides by application of secondary structure predictions and RNase H in combination with MALDI-TOF mass spectrometry

    PubMed Central

    Gabler, Angelika; Krebs, Stefan; Seichter, Doris; Förster, Martin

    2003-01-01

    Alteration of gene expression by use of antisense oligonucleotides has considerable potential for therapeutic purposes and scientific studies. Although applied for almost 25 years, this technique is still associated with difficulties in finding antisense-effective regions along the target mRNA. This is mainly due to strong secondary structures preventing binding of antisense oligonucleotides and RNase H, playing a major role in antisense-mediated degradation of the mRNA. These difficulties make empirical testing of a large number of sequences complementary to various sites in the target mRNA a very lengthy and troublesome procedure. To overcome this problem, more recent strategies to find efficient antisense sites are based on secondary structure prediction and RNase H-dependent mechanisms. We were the first who directly combined these two strategies; antisense oligonucleotides complementary to predicted unpaired target mRNA regions were designed and hybridized to the corresponding RNAs. Incubation with RNase H led to cleavage of the RNA at the respective hybridization sites. Analysis of the RNA fragments by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, which has not been used in this context before, allowed exact determination of the cleavage site. Thus the technique described here is very promising when searching for effective antisense sites. PMID:12888531

  10. Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

    ERIC Educational Resources Information Center

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

  11. Selection of antisense oligodeoxynucleotides against glutathione S-transferase Mu.

    PubMed Central

    't Hoen, Peter A C; Out, Ruud; Commandeur, Jan N M; Vermeulen, Nico P E; van Batenburg, F H D; Manoharan, Muthiah; van Berkel, Theo J C; Biessen, Erik A L; Bijsterbosch, Martin K

    2002-01-01

    The aim of the present study was to identify functional antisense oligodeoxynucleotides (ODNs) against the rat glutathione S-transferase Mu (GSTM) isoforms, GSTM1 and GSTM2. These antisense ODNs would enable the study of the physiological consequences of GSTM deficiency. Because it has been suggested that the effectiveness of antisense ODNs is dependent on the secondary mRNA structures of their target sites, we made mRNA secondary structure predictions with two software packages, Mfold and STAR. The two programs produced only marginally similar structures, which can probably be attributed to differences in the algorithms used. The effectiveness of a set of 18 antisense ODNs was evaluated with a cell-free transcription/translation assay, and their activity was correlated with the predicted secondary RNA structures. Four phosphodiester ODNs specific for GSTM1, two ODNs specific for GSTM2, and four ODNs targeted at both GSTM isoforms were found to be potent, sequence-specific, and RNase H-dependent inhibitors of protein expression. The IC50 value of the most potent ODN was approximately 100 nM. Antisense ODNs targeted against regions that were predicted by STAR to be predominantly single stranded were more potent than antisense ODNs against double-stranded regions. Such a correlation was not found for the Mfold prediction. Our data suggest that simulation of the local folding of RNA facilitates the discovery of potent antisense sequences. In conclusion, we selected several promising antisense sequences, which, when synthesized as biologically stable oligonucleotides, can be applied for study of the physiological impact of reduced GSTM expression. PMID:12515389

  12. Morpholinos: Antisense and Sensibility.

    PubMed

    Blum, Martin; De Robertis, Edward M; Wallingford, John B; Niehrs, Christof

    2015-10-26

    For over 15 years, antisense morpholino oligonucleotides (MOs) have allowed developmental biologists to make key discoveries regarding developmental mechanisms in numerous model organisms. Recently, serious concerns have been raised as to the specificity of MO effects, and it has been recommended to discontinue their usage, despite the long experience of the scientific community with the MO tool in thousands of studies. Reviewing the many advantages afforded by MOs, we conclude that adequately controlled MOs should continue to be accepted as generic loss-of-function approach, as otherwise progress in developmental biology will greatly suffer. PMID:26506304

  13. Targeting Cancer with Antisense Oligomers

    SciTech Connect

    Hnatowich, DJ

    2008-10-28

    With financial assistance from the Department of Energy, we have shown definitively that radiolabeled antisense DNAs and other oligomers will accumulate in target cancer cells in vitro and in vivo by an antisense mechanism. We have also shown that the number of mRNA targets for our antisense oligomers in the cancer cell types that we have investigated so far is sufficient to provide and antisense image and/or radiotherapy of cancer in mice. These studies have been reported in about 10 publications. However our observation over the past several years has shown that radiolabeled antisense oligomers administered intravenously in their native and naked form will accumulate and be retained in target xenografts by an antisense mechanism but will also accumulate at high levels in normal organs such as liver, spleen and kidneys. We have investigated unsuccessfully several commercially available vectors. Thus the use of radiolabeled antisense oligomers for the imaging of cancer must await novel approaches to delivery. This laboratory has therefore pursued two new paths, optical imaging of tumor and Auger radiotherapy. We are developing a novel method of optical imaging tumor using antisense oligomers with a fluorophore is administered while hybridized with a shorter complementary oligomer with an inhibitor. In culture and in tumored mice that the duplex remains intact and thus nonfluorescent until it encounters its target mRNA at which time it dissociates and the antisense oligomer binds along with its fluorophore to the target. Simultaneous with the above, we have also observed, as have others, that antisense oligomers migrate rapidly and quantitatively to the nucleus upon crossing cell membranes. The Auger electron radiotherapy path results from this observation since the nuclear migration properties could be used effectively to bring and to retain in the nucleus an Auger emitting radionuclide such as 111In or 125I bound to the antisense oligomer. Since the object becomes

  14. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells

    SciTech Connect

    Sorscher, E.J.; Kirk, K.L.; Weaver, M.L.; Jilling, T.; Blalock, J.E.; LeBoeuf, R.D. )

    1991-09-01

    The authors have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropryl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment for 24 hr virtually abolished Cl{sup {minus}} transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br{sup {minus}} uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatments, but not after treatments for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl{sup {minus}} transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl{sup {minus}} permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl{sup {minus}} permeability.

  15. Antisense oligodeoxynucleotide to the cystic fibrosis gene inhibits anion transport in normal cultured sweat duct cells.

    PubMed Central

    Sorscher, E J; Kirk, K L; Weaver, M L; Jilling, T; Blalock, J E; LeBoeuf, R D

    1991-01-01

    We have tested the hypothesis that the cystic fibrosis (CF) gene product, called the CF transmembrane conductance regulator (CFTR), mediates anion transport in normal human sweat duct cells. Sweat duct cells in primary culture were treated with oligodeoxynucleotides that were antisense to the CFTR gene transcript in order to block the expression of the wild-type CFTR. Anion transport in CFTR transcript antisense-treated cells was then assessed with a halide-specific dye, 6-methoxy-N-(3-sulfopropyl)quinolinium, and fluorescent digital imaging microscopy to monitor halide influx and efflux from single sweat duct cells. Antisense oligodeoxynucleotide treatment (3.9 or 1.3 microM) for 24 hr virtually abolished Cl- transport in sweat duct cells compared with untreated cells or control cells treated with sense oligodeoxynucleotides. Br- uptake into sweat duct cells was also blocked after a 24-hr CFTR transcript antisense treatment, but not after treatment for only 4 hr. Lower concentrations of antisense oligodeoxynucleotides were less effective at inhibiting Cl- transport. These results indicate that oligodeoxynucleotides that are antisense to CFTR transcript inhibit sweat duct Cl- permeability in both a time-dependent and dose-dependent manner. This approach provides evidence that inhibition of the expression of the wild-type CFTR gene in a normal, untransfected epithelial cell results in an inhibition of Cl- permeability. Images PMID:1715578

  16. Viral escape from antisense RNA.

    PubMed

    Bull, J J; Jacobson, A; Badgett, M R; Molineux, I J

    1998-05-01

    RNA coliphage SP was propagated for several generations on a host expressing an inhibitory antisense RNA complementary to bases 31-270 of the positive-stranded genome. Phages evolved that escaped inhibition. Typically, these escape mutants contained 3-4 base substitutions, but different sequences were observed among different isolates. The mutations were located within three different types of structural features within the predicted secondary structure of SP genomic RNA: (i) hairpin loops; (ii) hairpin stems; and (iii) the 5' region of the phage genome complementary to the antisense molecule. Computer modelling of the mutant genomic RNAs showed that all of the substitutions within hairpin stems improved the Watson-Crick pairing of the stem. No major structural rearrangements were predicted for any of the mutant genomes, and most substitutions in coding regions did not alter the amino acid sequence. Although the evolved phage populations were polymorphic for substitutions, many substitutions appeared independently in two selected lines. The creation of a new, perfect, antisense RNA against an escape mutant resulted in the inhibition of that mutant but not of other escape mutants nor of the ancestral, unevolved phage. Thus, at least in this system, a population of viruses that evolved to escape from a single antisense RNA would require a cocktail of several antisense RNAs for inhibition. PMID:9643550

  17. Functional analysis of mce4A gene of Mycobacterium tuberculosis H37Rv using antisense approach.

    PubMed

    Chandolia, Amita; Rathor, Nisha; Sharma, Monika; Saini, Neeraj Kumar; Sinha, Rajesh; Malhotra, Pawan; Brahmachari, Vani; Bose, Mridula

    2014-01-01

    Antisense strategy is an attractive substitute for knockout mutations created for gene silencing. mce genes have been shown to be involved in mycobacterial uptake and intracellular survival. Here we report reduced expression of mce4A and mce1A genes of Mycobacterium tuberculosis using antisense technology. For this, 1.1 kb region of mce4A and mce1A was cloned in reverse orientation in pSD5 shuttle vector, resulting into antisense constructs pSD5-4AS and pSD5-1AS, respectively. In M. tuberculosis H37Rv approximately 60% reduction in Mce4A and 66% reduction in expression of Mce1A protein were observed. We also observed significantly reduced intracellular survival ability of both antisense strains in comparison to M. tuberculosis containing pSD5 alone. RT-PCR analysis showed antisense did not alter the transcription of upstream and downstream of mceA genes of the respective operon. The colony morphology, in vitro growth characteristics and drug susceptibility profile of the antisense construct remained unchanged. These results demonstrate that antisense can be a promising approach to assign function of a gene in a multiunit operon and could be suitably applied as a strategy. PMID:24556072

  18. Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances

    PubMed Central

    Chen, Wei-Hua; van Noort, Vera; Lluch-Senar, Maria; Hennrich, Marco L.; H. Wodke, Judith A.; Yus, Eva; Alibés, Andreu; Roma, Guglielmo; Mende, Daniel R.; Pesavento, Christina; Typas, Athanasios; Gavin, Anne-Claude; Serrano, Luis; Bork, Peer

    2016-01-01

    We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated ‘-omics’ data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. PMID:26773059

  19. Direct cloning of double-stranded RNAs from RNase protection analysis reveals processing patterns of C/D box snoRNAs and provides evidence for widespread antisense transcript expression

    PubMed Central

    Shen, Manli; Eyras, Eduardo; Wu, Jie; Khanna, Amit; Josiah, Serene; Rederstorff, Mathieu; Zhang, Michael Q.; Stamm, Stefan

    2011-01-01

    We describe a new method that allows cloning of double-stranded RNAs (dsRNAs) that are generated in RNase protection experiments. We demonstrate that the mouse C/D box snoRNA MBII-85 (SNORD116) is processed into at least five shorter RNAs using processing sites near known functional elements of C/D box snoRNAs. Surprisingly, the majority of cloned RNAs from RNase protection experiments were derived from endogenous cellular RNA, indicating widespread antisense expression. The cloned dsRNAs could be mapped to genome areas that show RNA expression on both DNA strands and partially overlapped with experimentally determined argonaute-binding sites. The data suggest a conserved processing pattern for some C/D box snoRNAs and abundant expression of longer, non-coding RNAs in the cell that can potentially form dsRNAs. PMID:21880592

  20. An egr-1 (zif268) Antisense Oligodeoxynucleotide Infused Into the Amygdala Disrupts Fear Conditioning

    PubMed Central

    Malkani, Seema; Wallace, Karin J.; Donley, Melanie P.; Rosen, Jeffrey B.

    2004-01-01

    Studies of gene expression following fear conditioning have demonstrated that the inducible transcription factor, egr-1, is increased in the lateral nucleus of the amygdala shortly following fear conditioning. These studies suggest that egr-1 and its protein product Egr-1 in the amygdala are important for learning and memory of fear. To directly test this hypothesis, an egr-1 antisense oligodeoxynucleotide (antisense-ODN) was injected bilaterally into the amygdala prior to contextual fear conditioning. The antisense-ODN reduced Egr-1 protein in the amygdala and interfered with fear conditioning. A 250-pmole dose produced an 11% decrease in Egr-1 protein and reduced long-term memory of fear as measured by freezing in a retention test 24 h after conditioning, but left shock-induced freezing intact. A larger 500-pmole dose produced a 25% reduction in Egr-1 protein and significantly decreased both freezing immediately following conditioning and freezing in the retention test. A nonsense-ODN had no effect on postshock or retention test freezing. In addition, 500 pmole of antisense-ODN infused prior to the retention test in previously trained rats did not reduce freezing, indicating that antisense-ODN did not suppress conditioned fear behavior. Finally, rats infused with 500 pmole of antisense-ODN displayed unconditioned fear to a predator odor, demonstrating that unconditioned freezing was unaffected by the antisense-ODN. The data indicate that the egr-1 antisense-ODN interferes with learning and memory processes of fear without affecting freezing behavior and suggests that the inducible transcription factor Egr-1 within the amygdala plays important functions in long-term learning and memory of fear. PMID:15466317

  1. Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.

    PubMed

    Rimessi, Paola; Fabris, Marina; Bovolenta, Matteo; Bassi, Elena; Falzarano, Sofia; Gualandi, Francesca; Rapezzi, Claudio; Coccolo, Fabio; Perrone, Daniela; Medici, Alessandro; Ferlini, Alessandra

    2010-09-01

    Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches. PMID:20486769

  2. MYCNOS functions as an antisense RNA regulating MYCN

    PubMed Central

    Vadie, Nadia; Saayman, Sheena; Lenox, Alexandra; Ackley, Amanda; Clemson, Mathew; Burdach, Jon; Hart, Jonathan; Vogt, Peter K; Morris, Kevin V

    2015-01-01

    Amplification or overexpression of neuronal MYC (MYCN) is associated with poor prognosis of human neuroblastoma. Three isoforms of the MYCN protein have been described as well as a protein encoded by an antisense transcript (MYCNOS) that originates from the opposite strand at the MYCN locus. Recent findings suggest that some antisense long non-coding RNAs (lncRNAs) can play a role in epigenetically regulating gene expression. Here we report that MYCNOS transcripts function as a modulator of the MYCN locus, affecting MYCN promoter usage and recruiting various proteins, including the Ras GTPase-activating protein-binding protein G3BP1, to the upstream MYCN promoter. Overexpression of MYCNOS results in a reduction of upstream MYCN promoter usage and increased MYCN expression, suggesting that the protein-coding MYCNOS also functions as a regulator of MYCN ultimately controlling MYCN transcriptional variants. The observations presented here demonstrate that protein-coding transcripts can regulate gene transcription and can tether regulatory proteins to target loci. PMID:26156430

  3. The Drosophila micropia retrotransposon encodes a testis-specific antisense RNA complementary to reverse transcriptase.

    PubMed Central

    Lankenau, S; Corces, V G; Lankenau, D H

    1994-01-01

    The micropia transposable element of Drosophila hydei is a long terminal repeat-containing retrotransposon present in both the autosomes and the Y chromosome. micropia expression gives rise to a complex set of sense and antisense RNAs transcribed primarily during spermatogenesis. The most abundant sense RNAs constitute an assortment of heterogeneous high-molecular-weight transcripts expressed as constituents of the Y-chromosomal lampbrush loops of primary spermatocytes. In addition, micropia encodes a full-length RNA that extends between the two long terminal repeats of the element. The major 1.0-kb antisense RNA characterized is complementary to the reverse transcriptase and RNase H coding regions of micropia. It is expressed from a testis-specific promoter during the primary spermatocyte stages and is detectable until spermatid elongation stages. Sequence comparison of this promoter with the 5' region of other testis-specific genes allows the conception of a conserved sequence that is responsible for this pattern of expression. A 284-bp fragment containing this sequence is able to drive testis-specific expression of the Escherichia coli lacZ gene in Drosophila melanogaster. This sequence is conserved in the micropia elements present in other Drosophila species that also encode an antisense RNA. The evolutionary conservation of micropia antisense RNA expression and the sequences responsible for its testis-specific transcription suggests a role for this antisense RNA in the control of germ line expression of the full-length transcript or transposon-encoded proteins. Images PMID:7509447

  4. Antisense approaches in prostate cancer.

    PubMed

    Chi, Kim N; Gleave, Martin E

    2004-06-01

    Patients with hormone refractory prostate cancer have limited treatment options and new therapies are urgently needed. Advances in the understanding of the molecular mechanisms implicated in prostate cancer progression have identified many potential therapeutic gene targets that are involved in apoptosis, growth factors, cell signalling and the androgen receptor (AR). Antisense oligonucleotides are short sequences of synthetic modified DNA that are designed to be complimentary to a selected gene's mRNA and thereby specifically inhibit expression of that gene. The antisense approach continues to hold promise as a therapeutic modality to target genes involved in cancer progression, especially those in which the gene products are not amenable to small molecule inhibition or antibodies. The current status and future direction of a number of antisense oligonucleotides targeting several genes, including BCL-2, BCL-XL, clusterin, the inhibitors of apoptosis (IAP) family, MDM2, protein kinase C-alpha, c-raf, insulin-like growth factor binding proteins and the AR, that have potential clinical use in prostate cancer are reviewed. PMID:15174974

  5. A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

    PubMed Central

    Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario

    2014-01-01

    Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines. PMID:24865486

  6. A simple three-step method for design and affinity testing of new antisense peptides: an example of erythropoietin.

    PubMed

    Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario

    2014-01-01

    Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide-receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope) as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense-antisense (epitope-paratope) peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s) could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines. PMID:24865486

  7. Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

    PubMed

    Popplewell, Linda J; Malerba, Alberto; Dickson, George

    2012-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. AO-induced exon skipping resulting in functional truncated dystrophin has been demonstrated in animal models of DMD both in vitro and in vivo, in DMD patient cells in vitro in culture, and in DMD muscle explants. The recent advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene. PMID:22454060

  8. Survey of variation in human transcription factors reveals prevalent DNA binding changes.

    PubMed

    Barrera, Luis A; Vedenko, Anastasia; Kurland, Jesse V; Rogers, Julia M; Gisselbrecht, Stephen S; Rossin, Elizabeth J; Woodard, Jaie; Mariani, Luca; Kock, Kian Hong; Inukai, Sachi; Siggers, Trevor; Shokri, Leila; Gordân, Raluca; Sahni, Nidhi; Cotsapas, Chris; Hao, Tong; Yi, Song; Kellis, Manolis; Daly, Mark J; Vidal, Marc; Hill, David E; Bulyk, Martha L

    2016-03-25

    Sequencing of exomes and genomes has revealed abundant genetic variation affecting the coding sequences of human transcription factors (TFs), but the consequences of such variation remain largely unexplored. We developed a computational, structure-based approach to evaluate TF variants for their impact on DNA binding activity and used universal protein-binding microarrays to assay sequence-specific DNA binding activity across 41 reference and 117 variant alleles found in individuals of diverse ancestries and families with Mendelian diseases. We found 77 variants in 28 genes that affect DNA binding affinity or specificity and identified thousands of rare alleles likely to alter the DNA binding activity of human sequence-specific TFs. Our results suggest that most individuals have unique repertoires of TF DNA binding activities, which may contribute to phenotypic variation. PMID:27013732

  9. A riboswitch-regulated antisense RNA in Listeria monocytogenes.

    PubMed

    Mellin, J R; Tiensuu, Teresa; Bécavin, Christophe; Gouin, Edith; Johansson, Jörgen; Cossart, Pascale

    2013-08-01

    Riboswitches are ligand-binding elements located in 5' untranslated regions of messenger RNAs, which regulate expression of downstream genes. In Listeria monocytogenes, a vitamin B12-binding (B12) riboswitch was identified, not upstream of a gene but downstream, and antisense to the adjacent gene, pocR, suggesting it might regulate pocR in a nonclassical manner. In Salmonella enterica, PocR is a transcription factor that is activated by 1,2-propanediol, and subsequently activates expression of the pdu genes. The pdu genes mediate propanediol catabolism and are implicated in pathogenesis. As enzymes involved in propanediol catabolism require B12 as a cofactor, we hypothesized that the Listeria B12 riboswitch might be involved in pocR regulation. Here we demonstrate that the B12 riboswitch is transcribed as part of a noncoding antisense RNA, herein named AspocR. In the presence of B12, the riboswitch induces transcriptional termination, causing aspocR to be transcribed as a short transcript. In contrast, in the absence of B12, aspocR is transcribed as a long antisense RNA, which inhibits pocR expression. Regulation by AspocR ensures that pocR, and consequently the pdu genes, are maximally expressed only when both propanediol and B12 are present. Strikingly, AspocR can inhibit pocR expression in trans, suggesting it acts through a direct interaction with pocR mRNA. Together, this study demonstrates how pocR and the pdu genes can be regulated by B12 in bacteria and extends the classical definition of riboswitches from elements governing solely the expression of mRNAs to a wider role in controlling transcription of noncoding RNAs. PMID:23878253

  10. A novel antisense long noncoding RNA regulates the expression of MDC1 in bladder cancer

    PubMed Central

    Hua, Qiuhan; Chu, Haiyan; Tong, Na; Yuan, Lin; Qin, Chao; Yin, Changjun; Zhang, Zhengdong; Wang, Meilin

    2015-01-01

    Antisense long noncoding RNAs (lncRNAs) play important roles in regulating the expression of coding genes in post-transcriptional level. However, detailed expression profile of lncRNAs and functions of antisense lncRNAs in bladder cancer remains unclear. To investigate regulation of lncRNAs in bladder cancer and demonstrate their functions, we performed lncRNAs microarray analysis in 3 paired bladder cancer tissues. Further molecular assays were conducted to determine the potential role of identified antisense lncRNA MDC1-AS. As a result, a series of lncRNAs were differentially expressed in bladder cancer tissues in microarray screen. In a larger size of samples validation, we found that the expression levels of MDC1-AS and MDC1 was down-regulated in bladder cancer. After over-expression of MDC1-AS, increased levels of MDC1 were observed in bladder cancer cells. We also found a remarkably inhibitory role of antisense lncRNA MDC1-AS on malignant cell behaviors in bladder cancer cells EJ and T24. Subsequently, knockdown of MDC1 revealed that suppressing role of MDC1-AS was attributed to up-regulation of MDC1. In summary, we have identified a novel antisense lncRNA MDC1-AS, which may participate in bladder cancer through up-regulation of its antisense tumor-suppressing gene MDC1. Further studies should be conducted to demonstrate detailed mechanism of our findings. PMID:25514464

  11. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

    PubMed

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe

  12. Antisense oligonucleotide induction of progerin in human myogenic cells.

    PubMed

    Luo, Yue-Bei; Mitrpant, Chalermchai; Adams, Abbie M; Johnsen, Russell D; Fletcher, Sue; Mastaglia, Frank L; Wilton, Steve D

    2014-01-01

    We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA) transcript in human myogenic cells. The progerin transcript (LMNA Δ150) lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS). HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model to investigate

  13. The role of antisense long noncoding RNA in small RNA-triggered gene activation

    PubMed Central

    Zhang, Xizhe; Li, Haitang; Rossi, John J.

    2014-01-01

    Long noncoding RNAs (lncRNAs) are known to regulate neighboring protein-coding genes by directing chromatin remodeling complexes, imprinting, and X-chromosome inactivation. In this study, we explore the function of lncRNAs in small RNA-triggered transcriptional gene activation (TGA), a process in which microRNAs (miRNAs) or small interfering RNAs (siRNAs) associated with Argonaute (Ago) proteins induce chromatin remodeling and gene activation at promoters with sequence complementarity. We designed a model system with different lncRNA and chromatin environments to elucidate the molecular mechanisms required for mammalian TGA. Using RNA-fluorescence in situ hybridization (FISH) and rapid amplification of cDNA ends (RACE)-PCR, we demonstrated that small RNA-triggered TGA occurs at sites where antisense lncRNAs are transcribed through the reporter gene and promoter. Small RNA-induced TGA coincided with the enrichment of Ago2 at the promoter region, but Ago2-mediated cleavage of antisense lncRNAs was not observed. Moreover, we examined the allele-specific effects of lncRNAs through a Cre-induced inversion of a poly(A) sequence that was designed to block the transcription of antisense lncRNAs through the reporter gene region in an inducible and reversible manner. Termination of nascent antisense lncRNAs abrogated gene activation triggered by small RNAs, and only allele-specific cis-acting antisense lncRNAs, but not trans-acting lncRNAs, were capable of rescuing TGA. Hence, this model revealed that antisense lncRNAs can mediate TGA in cis and not in trans, serving as a molecular scaffold for a small RNA–Ago2 complex and chromatin remodeling. PMID:25344398

  14. Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum

    PubMed Central

    Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron

    2015-01-01

    The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression. PMID:25691743

  15. Antisense-mediated exon inclusion

    PubMed Central

    Hua, Yimin; Krainer, Adrian R

    2012-01-01

    Exon skipping induced by gene mutations is a common mechanism responsible for many genetic diseases. A practical approach to correct the aberrant splicing of defective genes is to use antisense oligonucleotides (ASOs). The recognition of splice sites and the regulation of splicing involve multiple positive or negative cis-acting elements and trans-acting factors. Base-pairing of ASOs to a negative element in a targeted pre-mRNA blocks the binding of splicing repressors to this cis-element and/or disrupts an unfavorable secondary structure; as a result, the ASO restores exon inclusion. For example, we have recently shown that appropriate 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified ASOs can efficiently correct survival motor neuron 2 (SMN2) exon 7 splicing in a cell-free splicing assay, in cultured human cells—including patient fibroblasts—and in both peripheral tissues and the CNS of SMA mouse models. These ASOs are promising drug leads for SMA therapy. PMID:22454070

  16. A new antisense tRNA construct for the genetic treatment of human immunodeficiency virus type 1 infection.

    PubMed Central

    Biasolo, M A; Radaelli, A; Del Pup, L; Franchin, E; De Giuli-Morghen, C; Palu, G

    1996-01-01

    Different strategies proposed in the literature to attempt gene therapy of AIDS are based mainly on the intracellular production of RNA and protein therapeutics. This report describes the construction and the anti-human immunodeficiency virus type 1 (HIV-1) activity of a new type of antisense tRNA directed against a nucleotide region in the first coding exon of HIV-1 tat (nucleotides 5924 to 5943; Los Alamos data bank) which is conserved among many HIV-1 clones. The anti-tat antisense sequence was inserted into a tRNA(Pro) backbone by replacement of the anticodon loop, without altering the tRNA canonic tetraloop structure. The antisense tRNA was able to interact effectively with its target in vitro. Jurkat cells that constitutively expressed the anti-tat tRNA following retroviral vector transduction exhibited significant resistance to HIV-1 de novo infection. Resistance seemed to correlate with the level of antisense expression. This is the first time that such a tRNA antisense strategy has been shown to be effective as a genetic treatment of HIV-1 infection in tissue culture. The construct design proposed in this report has some intrinsic advantages: the transcript is driven by a polymerase III promoter, the short length of the RNA minimizes effects of intramolecular base pairing that may impair target recognition, and the antisense RNA has the stability and intracellular fate of a native tRNA molecule. PMID:8642637

  17. An Xist-activating antisense RNA required for X-chromosome inactivation

    PubMed Central

    Sarkar, Mrinal K.; Gayen, Srimonta; Kumar, Surinder; Maclary, Emily; Buttigieg, Emily; Hinten, Michael; Kumari, Archana; Harris, Clair; Sado, Takashi; Kalantry, Sundeep

    2015-01-01

    The transcriptional imbalance due to the difference in the number of X chromosomes between male and female mammals is remedied through X-chromosome inactivation, the epigenetic transcriptional silencing of one of the two X chromosomes in females. The X-linked Xist long non-coding RNA functions as an X inactivation master regulator; Xist is selectively upregulated from the prospective inactive X chromosome and is required in cis for X inactivation. Here we discover an Xist antisense long non-coding RNA, XistAR (Xist Activating RNA), which is encoded within exon 1 of the mouse Xist gene and is transcribed only from the inactive X chromosome. Selective truncation of XistAR, while sparing the overlapping Xist RNA, leads to a deficiency in Xist RNA expression in cis during the initiation of X inactivation. Thus, the Xist gene carries within its coding sequence an antisense RNA that drives Xist expression. PMID:26477563

  18. Functionalization of an Antisense Small RNA

    PubMed Central

    Rodrigo, Guillermo; Prakash, Satya; Cordero, Teresa; Kushwaha, Manish; Jaramillo, Alfonso

    2016-01-01

    In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5′ untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology. PMID:26756967

  19. Simultaneous characterization of sense and antisense genomic processes by the double-stranded hidden Markov model.

    PubMed

    Glas, Julia; Dümcke, Sebastian; Zacher, Benedikt; Poron, Don; Gagneur, Julien; Tresch, Achim

    2016-03-18

    Hidden Markov models (HMMs) have been extensively used to dissect the genome into functionally distinct regions using data such as RNA expression or DNA binding measurements. It is a challenge to disentangle processes occurring on complementary strands of the same genomic region. We present the double-stranded HMM (dsHMM), a model for the strand-specific analysis of genomic processes. We applied dsHMM to yeast using strand specific transcription data, nucleosome data, and protein binding data for a set of 11 factors associated with the regulation of transcription.The resulting annotation recovers the mRNA transcription cycle (initiation, elongation, termination) while correctly predicting strand-specificity and directionality of the transcription process. We find that pre-initiation complex formation is an essentially undirected process, giving rise to a large number of bidirectional promoters and to pervasive antisense transcription. Notably, 12% of all transcriptionally active positions showed simultaneous activity on both strands. Furthermore, dsHMM reveals that antisense transcription is specifically suppressed by Nrd1, a yeast termination factor. PMID:26578558

  20. Potent Antibacterial Antisense Peptide–Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    PubMed Central

    Ghosal, Anubrata

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce antisense peptide–peptide nucleic acid (PNA) conjugates as antibacterial agents against P. aeruginosa. We have designed and optimized antisense peptide–PNA conjugates targeting the translation initiation region of the ftsZ gene (an essential bacterial gene involved in cell division) or the acpP gene (an essential bacterial gene involved in fatty acid synthesis) of P. aeruginosa (PA01) and characterized these compounds according to their antimicrobial activity and mode of action. Four antisense PNA oligomers conjugated to the H-(R-Ahx-R)4-Ahx-βala or the H-(R-Ahx)6-βala peptide exhibited complete growth inhibition of P. aeruginosa strains PA01, PA14, and LESB58 at 1–2 μM concentrations without any indication of bacterial membrane disruption (even at 20 μM), and resulted in specific reduction of the targeted mRNA levels. One of the four compounds showed clear bactericidal activity while the other significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections. PMID:23030590

  1. Antisense RNA suppression of peroxidase gene expression

    SciTech Connect

    Lagrimini, L.M.; Bradford, S.; De Leon, F.D. )

    1989-04-01

    The 5{prime} half the anionic peroxidase cDNA of tobacco was inserted into a CaMV 35S promoter/terminator expression cassette in the antisense configuration. This was inserted into the Agrobacterium-mediated plant transformation vector pCIBIO which includes kanamycin selection, transformed into two species of tobacco (N. tabacum and M. sylvestris), and plants were subsequently regenerated on kanamycin. Transgenic plants were analyzed for peroxidase expression and found to have 3-5 fold lower levels of peroxidase than wild-type plants. Isoelectric focusing demonstrated that the antisense RNA only suppressed the anionic peroxidase. Wound-induced peroxidase expression was found not to be affected by the antisense RNA. Northern blots show a greater than 5 fold suppression of anionic peroxidase mRNA in leaf tissue, and the antisense RNA was expressed at a level 2 fold over the endogenous mRNA. Plants were self-pollinated and F1 plants showed normal segregation. N. sylvestris transgenic plants with the lowest level of peroxidase are epinastic, and preliminary results indicate elevated auxin levels. Excised pith tissue from both species of transgenic plants rapidly collapse when exposed to air, while pith tissue from wild-type plants showed little change when exposed to air. Further characterization of these phenotypes is currently being made.

  2. Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

    SciTech Connect

    Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

    2008-01-15

    Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen

  3. Antisense oligonucleotide therapy for the treatment of C9ORF72 ALS/FTD diseases.

    PubMed

    Riboldi, Giulietta; Zanetta, Chiara; Ranieri, Michela; Nizzardo, Monica; Simone, Chiara; Magri, Francesca; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania

    2014-12-01

    Motor neuron disorders, and particularly amyotrophic lateral sclerosis (ALS), are fatal diseases that are due to the loss of motor neurons in the brain and spinal cord, with progressive paralysis and premature death. It has been recently shown that the most frequent genetic cause of ALS, frontotemporal dementia (FTD), and other neurological diseases is the expansion of a hexanucleotide repeat (GGGGCC) in the non-coding region of the C9ORF72 gene. The pathogenic mechanisms that produce cell death in the presence of this expansion are still unclear. One of the most likely hypotheses seems to be the gain-of-function that is achieved through the production of toxic RNA (able to sequester RNA-binding protein) and/or toxic proteins. In recent works, different authors have reported that antisense oligonucleotides complementary to the C9ORF72 RNA transcript sequence were able to significantly reduce RNA foci generated by the expanded RNA, in affected cells. Here, we summarize the recent findings that support the idea that the buildup of "toxic" RNA containing the GGGGCC repeat contributes to the death of motor neurons in ALS and also suggest that the use of antisense oligonucleotides targeting this transcript is a promising strategy for treating ALS/frontotemporal lobe dementia (FTLD) patients with the C9ORF72 repeat expansion. These data are particularly important, given the state of the art antisense technology, and they allow researchers to believe that a clinical application of these discoveries will be possible soon. PMID:24809691

  4. Reduction of EGF receptor levels in human tumor cells transfected with an antisense RNA expression vector

    SciTech Connect

    Yamada, Hirotomo; Koizumi, Shinji; Kimura, Masami ); Shimizu, Nobuyoshi )

    1989-09-01

    An expression vector was constructed from part of pSV2neo with the 3{prime}-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction cell-surface EGF receptor in response to ZnSO{sub 4}. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

  5. Transcriptional Silencing by Hairpin RNAs Complementary to a Gene Promoter

    PubMed Central

    Chu, Yongjun; Kalantari, Roya; Dodd, David W.

    2012-01-01

    Double-stranded RNAs can target gene promoters and inhibit transcription. To date, most research has focused on synthetic RNA duplexes. Transcriptional silencing by hairpin RNAs would facilitate a better understanding of endogenous RNA-mediated regulation of transcription within cells. Here we examine transcriptional silencing of progesterone receptor (PR) expression by hairpin RNAs. We identify the guide strand as the strand complementary to an antisense transcript at the PR promoter and that hairpin RNAs are active transcriptional silencing agents. The sequence of the hairpin loop affects activity, with the highest activity achieved when the loop has the potential for full complementarity to the antisense transcript target. Introduction of centrally mismatched bases relative to the target transcript does not prevent transcriptional silencing unless the mismatches are present on both the guide and passenger strands. These data demonstrate that hairpin RNAs can cause transcriptional silencing and offer insights into the mechanism of gene modulation by RNAs that target gene promoters. PMID:22703280

  6. Extremely High Expression of Antisense RNA for Wilms' Tumor 1 in Active Osteoclasts: Suppression of Wilms' Tumor 1 Protein Expression during Osteoclastogenesis.

    PubMed

    Li, Yin-Ji; Kukita, Akiko; Kyumoto-Nakamura, Yukari; Kukita, Toshio

    2016-09-01

    Wilms' tumor 1 (WT1), a zinc-finger transcription regulator of the early growth response family, identified as the product of a tumor suppressor gene of Wilms' tumors, bears potential ability to induce macrophage differentiation in blood cell differentiation. Herein, we examined the involvement of WT1 in the regulation of osteoclastogenesis. We detected a high level of WT1 protein expression in osteoclast precursors; however, WT1 expression was markedly suppressed during osteoclastogenesis. We examined expression of WT1 transcripts in bone tissue by RNA in situ hybridization. We found a high level of antisense transcripts in osteoclasts actively resorbing bone in mandible of newborn rats. Expression of antisense WT1 RNA in mandible was also confirmed by Northern blot analysis and strand-specific RT-PCR. Overexpression of antisense WT1 RNA in RAW-D cells, an osteoclast precursor cell line, resulted in a marked enhancement of osteoclastogenesis, suggesting that antisense WT1 RNA functions to suppress expression of WT1 protein in osteoclastogenesis. High level expression of antisense WT1 RNA may contribute to commitment to osteoclastogenesis, and may allow osteoclasts to maintain or stabilize their differentiation state. PMID:27393793

  7. Optimal antisense target reducing INS intron 1 retention is adjacent to a parallel G quadruplex

    PubMed Central

    Kralovicova, Jana; Lages, Ana; Patel, Alpa; Dhir, Ashish; Buratti, Emanuele; Searle, Mark; Vorechovsky, Igor

    2014-01-01

    Splice-switching oligonucleotides (SSOs) have been widely used to inhibit exon usage but antisense strategies that promote removal of entire introns to increase splicing-mediated gene expression have not been developed. Here we show reduction of INS intron 1 retention by SSOs that bind transcripts derived from a human haplotype expressing low levels of proinsulin. This haplotype is tagged by a polypyrimidine tract variant rs689 that decreases the efficiency of intron 1 splicing and increases the relative abundance of mRNAs with extended 5' untranslated region (5' UTR), which curtails translation. Co-expression of haplotype-specific reporter constructs with SSOs bound to splicing regulatory motifs and decoy splice sites in primary transcripts revealed a motif that significantly reduced intron 1-containing mRNAs. Using an antisense microwalk at a single nucleotide resolution, the optimal target was mapped to a splicing silencer containing two pseudoacceptor sites sandwiched between predicted RNA guanine (G) quadruplex structures. Circular dichroism spectroscopy and nuclear magnetic resonance of synthetic G-rich oligoribonucleotide tracts derived from this region showed formation of a stable parallel 2-quartet G-quadruplex on the 3' side of the antisense retention target and an equilibrium between quadruplexes and stable hairpin-loop structures bound by optimal SSOs. This region interacts with heterogeneous nuclear ribonucleoproteins F and H that may interfere with conformational transitions involving the antisense target. The SSO-assisted promotion of weak intron removal from the 5' UTR through competing noncanonical and canonical RNA structures may facilitate development of novel strategies to enhance gene expression. PMID:24944197

  8. Splice-switching antisense oligonucleotides as therapeutic drugs.

    PubMed

    Havens, Mallory A; Hastings, Michelle L

    2016-08-19

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

  9. Small regulatory RNAs in lambdoid bacteriophages and phage-derived plasmids: Not only antisense.

    PubMed

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Licznerska, Katarzyna; Felczykowska, Agnieszka; Dydecka, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-03-01

    Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs. PMID:25111672

  10. Overexpression of HOX genes is prevalent in Ewing sarcoma and is associated with altered epigenetic regulation of developmental transcription programs

    PubMed Central

    Svoboda, Laurie K; Harris, Ashley; Bailey, Natashay J; Schwentner, Raphaela; Tomazou, Eleni; von Levetzow, Cornelia; Magnuson, Brian; Ljungman, Mats; Kovar, Heinrich; Lawlor, Elizabeth R

    2014-01-01

    The polycomb proteins BMI-1 and EZH2 are highly overexpressed by Ewing sarcoma (ES), a tumor of stem cell origin that is driven by EWS-ETS fusion oncogenes, most commonly EWS-FLI1. In the current study we analyzed expression of transcription programs that are controlled by polycomb proteins during embryonic development to determine if they are abnormal in ES. Our results show that polycomb target gene expression in ES deviates from normal tissues and stem cells and that, as expected, most targets are relatively repressed. However, we also discovered a paradoxical up regulation of numerous polycomb targets and these were highly enriched for homeobox (HOX) genes. Comparison of HOX profiles between malignant and non-malignant tissues revealed a distinctive HOX profile in ES, which was characterized by overexpression of posterior HOXD genes. In addition, ectopic expression of EWS-FLI1 during stem cell differentiation led to aberrant up regulation of posterior HOXD genes. Mechanistically, this up regulation was associated with altered epigenetic regulation. Specifically, ES and EWS-FLI1+ stem cells displayed a relative loss of polycomb-dependent H3K27me3 and gain of trithorax-dependent H3K4me3 at the promoters of posterior HOXD genes and also at the HOXD11.12 polycomb response element. In addition, a striking correlation was evident between HOXD13 and other genes whose regulation is coordinately regulated during embryonic development by distal enhancer elements. Together, these studies demonstrate that epigenetic regulation of polycomb target genes, in particular HOXD genes, is altered in ES and that these changes are mediated downstream of EWS-FLI1. PMID:25625846

  11. Expression of TGMV antisense RNA in transgenic tobacco inhibits replication of BCTV but not ACMV geminiviruses.

    PubMed

    Bejarano, E R; Lichtenstein, C P

    1994-01-01

    Transgenic tobacco plants expressing an antisense RNA targeted against tomato golden mosaic virus (TGMV) show reduced/no symptoms and viral DNA accumulation upon TGMV infection [5]. The targeted region includes the AL1 gene, encoding an essential viral replication protein. This DNA sequence is conserved in various other geminiviruses, suggesting they too might show inhibition of replication in these plants. We infected leaf material with African cassava mosaic virus (ACMV) and beet curly top virus (BTCV) and saw a 4-fold reduction of BCTV, but not ACMV, DNA accumulation, compared to controls. The equivalent regions of BCTV and ACMV show similar overall homology to the TGMV target (63% and 64% respectively), but within this, BCTV displays a 280 nucleotide region of high homology (82%). In contrast, for ACMV, the homology is more dispersed. This indicates that a critical stretch of good complementarity is needed to block expression of the target mRNA, that is effective even within along antisense transcript. These studies indicate the potential for developing a multifunctional antisense cassette. PMID:8111023

  12. Modulation of lipoprotein metabolism by antisense technology: preclinical drug discovery methodology.

    PubMed

    Crooke, Rosanne M; Graham, Mark J

    2013-01-01

    Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs. PMID:23912993

  13. Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5

    PubMed Central

    Bahal, Raman; McNeer, Nicole Ali; Ly, Danith H.; Saltzman, W. Mark; Glazer, Peter M.

    2013-01-01

    The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of Chemokine Receptor 5 (CCR5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism. PMID:23954968

  14. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  15. Natural antisense RNA promotes 3′ end processing and maturation of MALAT1 lncRNA

    PubMed Central

    Zong, Xinying; Nakagawa, Shinichi; Freier, Susan M.; Fei, Jingyi; Ha, Taekjip; Prasanth, Supriya G.; Prasanth, Kannanganattu V.

    2016-01-01

    The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3′ end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3′ end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3′ end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans. Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3′ end processing. PMID:26826711

  16. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    PubMed

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing. PMID:26553470

  17. Sense and Antisense DMPK RNA Foci Accumulate in DM1 Tissues during Development

    PubMed Central

    Michel, Lise; Huguet-Lachon, Aline; Gourdon, Geneviève

    2015-01-01

    Myotonic dystrophy type 1 (DM1) is caused by an unstable expanded CTG repeat located within the DMPK gene 3’UTR. The nature, severity and age at onset of DM1 symptoms are very variable in patients. Different forms of the disease are described, among which the congenital form (CDM) is the most severe. Molecular mechanisms of DM1 are well characterized for the adult form and involve accumulation of mutant DMPK RNA forming foci in the nucleus. These RNA foci sequester proteins from the MBNL family and deregulate CELF proteins. These proteins are involved in many cellular mechanisms such as alternative splicing, transcriptional, translational and post-translational regulation miRNA regulation as well as mRNA polyadenylation and localization. All these mechanisms can be impaired in DM1 because of the deregulation of CELF and MBNL functions. The mechanisms involved in CDM are not clearly described. In order to get insight into the mechanisms underlying CDM, we investigated if expanded RNA nuclear foci, one of the molecular hallmarks of DM1, could be detected in human DM1 fetal tissues, as well as in embryonic and neonatal tissues from transgenic mice carrying the human DMPK gene with an expanded CTG repeat. We observed very abundant RNA foci formed by sense DMPK RNA and, to a lesser extent, antisense DMPK RNA foci. Sense DMPK RNA foci clearly co-localized with MBNL1 and MBNL2 proteins. In addition, we studied DMPK sense and antisense expression during development in the transgenic mice. We found that DMPK sense and antisense transcripts are expressed from embryonic and fetal stages in heart, muscle and brain and are regulated during development. These results suggest that mechanisms underlying DM1 and CDM involved common players including toxic expanded RNA forming numerous nuclear foci at early stages during development. PMID:26339785

  18. Transgenic male-sterile plant induced by an unedited atp9 gene is restored to fertility by inhibiting its expression with antisense RNA.

    PubMed Central

    Zabaleta, E; Mouras, A; Hernould, M; Suharsono; Araya, A

    1996-01-01

    We have previously shown that the expression of an unedited atp9 chimeric gene correlated with male-sterile phenotype in transgenic tobacco plant. To study the relationship between the expression of chimeric gene and the male-sterile trait, hemizygous and homozygous transgenic tobacco lines expressing the antisense atp9 RNA were constructed. The antisense producing plants were crossed with a homozygous male-sterile line, and the F1 progeny was analyzed. The offspring from crosses between homozygous lines produced only male-fertile plants, suggesting that the expression antisense atp9 RNA abolishes the effect of the unedited chimeric gene. In fact, the plants restored to male fertility showed a dramatic reduction of the unedited atp9 transcript levels, resulting in normal flower development and seed production. These results support our previous observation that the expression of unedited atp9 gene can induce male sterility. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855343

  19. Undetected antisense tRNAs in mitochondrial genomes?

    PubMed Central

    2010-01-01

    Background The hypothesis that both mitochondrial (mt) complementary DNA strands of tRNA genes code for tRNAs (sense-antisense coding) is explored. This could explain why mt tRNA mutations are 6.5 times more frequently pathogenic than in other mt sequences. Antisense tRNA expression is plausible because tRNA punctuation signals mt sense RNA maturation: both sense and antisense tRNAs form secondary structures potentially signalling processing. Sense RNA maturation processes by default 11 antisense tRNAs neighbouring sense genes. If antisense tRNAs are expressed, processed antisense tRNAs should have adapted more for translational activity than unprocessed ones. Four tRNA properties are examined: antisense tRNA 5' and 3' end processing by sense RNA maturation and its accuracy, cloverleaf stability and misacylation potential. Results Processed antisense tRNAs align better with standard tRNA sequences with the same cognate than unprocessed antisense tRNAs, suggesting less misacylations. Misacylation increases with cloverleaf fragility and processing inaccuracy. Cloverleaf fragility, misacylation and processing accuracy of antisense tRNAs decrease with genome-wide usage of their predicted cognate amino acid. Conclusions These properties correlate as if they adaptively coevolved for translational activity by some antisense tRNAs, and to avoid such activity by other antisense tRNAs. Analyses also suggest previously unsuspected particularities of aminoacylation specificity in mt tRNAs: combinations of competition between tRNAs on tRNA synthetases with competition between tRNA synthetases on tRNAs determine specificities of tRNA amino acylations. The latter analyses show that alignment methods used to detect tRNA cognates yield relatively robust results, even when they apparently fail to detect the tRNA's cognate amino acid and indicate high misacylation potential. Reviewers This article was reviewed by Dr Juergen Brosius, Dr Anthony M Poole and Dr Andrei S Rodin (nominated

  20. Diversity of transcripts and transcript processing forms in plastids of the dinoflagellate alga Karenia mikimotoi.

    PubMed

    Dorrell, Richard G; Hinksman, George A; Howe, Christopher J

    2016-02-01

    Plastids produce a vast diversity of transcripts. These include mature transcripts containing coding sequences, and their processing precursors, as well as transcripts that lack direct coding functions, such as antisense transcripts. Although plastid transcriptomes have been characterised for many plant species, less is known about the transcripts produced in other plastid lineages. We characterised the transcripts produced in the fucoxanthin-containing plastids of the dinoflagellate alga Karenia mikimotoi. This plastid lineage, acquired through tertiary endosymbiosis, utilises transcript processing pathways that are very different from those found in plants and green algae, including 3' poly(U) tail addition, and extensive substitutional editing of transcript sequences. We have sequenced the plastid transcriptome of K. mikimotoi, and have detected evidence for divergent evolution of fucoxanthin plastid genomes. We have additionally characterised polycistronic and monocistronic transcripts from two plastid loci, psbD-tRNA (Met)-ycf4 and rpl36-rps13-rps11. We find evidence for a range of transcripts produced from each locus that differ in terms of editing state, 5' end cleavage position, and poly(U) tail addition. Finally, we identify antisense transcripts in K. mikimotoi, which appear to undergo different processing events from the corresponding sense transcripts. Overall, our study provides insights into the diversity of transcripts and processing intermediates found in plastid lineages across the eukaryotes. PMID:26768263

  1. [Treatment with antisense oligonucleotides in Duchenne's disease].

    PubMed

    Pascual-Pascual, Samuel I

    2012-05-21

    In this paper I review the results of the treatments directed to modify the mRNA of dystrophin with the goal of converting the severe Duchenne type to the milder Becker muscular dystrophy. Antisense oligomers potential to modify Duchenne muscular dystrophy (DMD) gene expression and therapeutic strategies to induce ribosomal read-through of nonsense mutations (PTC124) are described. They are an important advance in the treatment of DMD, so far unspecific. Significant expression of new dystrophin is observed in biopsies of peripheral muscle, although the functional improvement is not so encouraging. New modification of chemistries are expected to improve the liberation, broad distribution in muscles, as well as their efficacy and safety enough to allow a positive chronic treatment of DMD. PMID:22605630

  2. Identification of Multiple Forms of RNA Transcripts Associated with Human-Specific Retrotransposed Gene Copies.

    PubMed

    Mori, Saori; Hayashi, Masaaki; Inagaki, Shun; Oshima, Takuji; Tateishi, Ken; Fujii, Hiroshi; Suzuki, Shunsuke

    2016-01-01

    The human genome contains thousands of retrocopies, mostly as processed pseudogenes, which were recently shown to be prevalently transcribed. In particular, those specifically acquired in the human lineage are able to modulate gene expression in a manner that contributed to the evolution of human-specific traits. Therefore, knowledge of the human-specific retrocopies that are transcribed or their full-length transcript structure contributes to better understand human genome evolution. In this study, we identified 16 human-specific retrocopies that harbor 5' CpG islands by in silico analysis and showed that 12 were transcribed in normal tissues and cancer cell lines with a variety of expression patterns, including cancer-specific expression. Determination of the structure of the transcripts associated with the retrocopies revealed that none were transcribed from their 5' CpG islands, but rather, from inside the 3' UTR and the nearby 5' flanking region of the retrocopies as well as the promoter of neighboring genes. The multiple forms of the transcripts, such as chimeric and individual transcripts in both the sense and antisense orientation, might have introduced novel post-transcriptional regulation into the genome during human evolution. These results shed light on the potential role of human-specific retrocopies in the evolution of gene regulation and genomic disorders. PMID:27389689

  3. Identification of Multiple Forms of RNA Transcripts Associated with Human-Specific Retrotransposed Gene Copies

    PubMed Central

    Mori, Saori; Hayashi, Masaaki; Inagaki, Shun; Oshima, Takuji; Tateishi, Ken; Fujii, Hiroshi; Suzuki, Shunsuke

    2016-01-01

    The human genome contains thousands of retrocopies, mostly as processed pseudogenes, which were recently shown to be prevalently transcribed. In particular, those specifically acquired in the human lineage are able to modulate gene expression in a manner that contributed to the evolution of human-specific traits. Therefore, knowledge of the human-specific retrocopies that are transcribed or their full-length transcript structure contributes to better understand human genome evolution. In this study, we identified 16 human-specific retrocopies that harbor 5′ CpG islands by in silico analysis and showed that 12 were transcribed in normal tissues and cancer cell lines with a variety of expression patterns, including cancer-specific expression. Determination of the structure of the transcripts associated with the retrocopies revealed that none were transcribed from their 5′ CpG islands, but rather, from inside the 3′ UTR and the nearby 5′ flanking region of the retrocopies as well as the promoter of neighboring genes. The multiple forms of the transcripts, such as chimeric and individual transcripts in both the sense and antisense orientation, might have introduced novel post-transcriptional regulation into the genome during human evolution. These results shed light on the potential role of human-specific retrocopies in the evolution of gene regulation and genomic disorders. PMID:27389689

  4. Construction of a directed hammerhead ribozyme library: towards the identification of optimal target sites for antisense-mediated gene inhibition.

    PubMed Central

    Pierce, M L; Ruffner, D E

    1998-01-01

    Antisense-mediated gene inhibition uses short complementary DNA or RNA oligonucleotides to block expression of any mRNA of interest. A key parameter in the success or failure of an antisense therapy is the identification of a suitable target site on the chosen mRNA. Ultimately, the accessibility of the target to the antisense agent determines target suitability. Since accessibility is a function of many complex factors, it is currently beyond our ability to predict. Consequently, identification of the most effective target(s) requires examination of every site. Towards this goal, we describe a method to construct directed ribozyme libraries against any chosen mRNA. The library contains nearly equal amounts of ribozymes targeting every site on the chosen transcript and the library only contains ribozymes capable of binding to that transcript. Expression of the ribozyme library in cultured cells should allow identification of optimal target sites under natural conditions, subject to the complexities of a fully functional cell. Optimal target sites identified in this manner should be the most effective sites for therapeutic intervention. PMID:9801305

  5. Expression analysis of the long non-coding RNA antisense to Uchl1 (AS Uchl1) during dopaminergic cells' differentiation in vitro and in neurochemical models of Parkinson's disease

    PubMed Central

    Carrieri, Claudia; Forrest, Alistair R. R.; Santoro, Claudio; Persichetti, Francesca; Carninci, Piero; Zucchelli, Silvia; Gustincich, Stefano

    2015-01-01

    Antisense (AS) transcripts are RNA molecules that are transcribed from the opposite strand to sense (S) genes forming S/AS pairs. The most prominent configuration is when a lncRNA is antisense to a protein coding gene. Increasing evidences prove that antisense transcription may control sense gene expression acting at distinct regulatory levels. However, its contribution to brain function and neurodegenerative diseases remains unclear. We have recently identified AS Uchl1 as an antisense to the mouse Ubiquitin carboxy-terminal hydrolase L1 (Uchl1) gene (AS Uchl1), the synthenic locus of UCHL1/PARK5. This is mutated in rare cases of early-onset familial Parkinson's Disease (PD) and loss of UCHL1 activity has been reported in many neurodegenerative diseases. Importantly, manipulation of UchL1 expression has been proposed as tool for therapeutic intervention. AS Uchl1 induces UchL1 expression by increasing its translation. It is the representative member of SINEUPs (SINEB2 sequence to UP-regulate translation), a new functional class of natural antisense lncRNAs that activate translation of their sense genes. Here we take advantage of FANTOM5 dataset to identify the transcription start sites associated to S/AS pair at Uchl1 locus. We show that AS Uchl1 expression is under the regulation of Nurr1, a major transcription factor involved in dopaminergic cells' differentiation and maintenance. Furthermore, AS Uch1 RNA levels are strongly down-regulated in neurochemical models of PD in vitro and in vivo. This work positions AS Uchl1 RNA as a component of Nurr1-dependent gene network and target of cellular stress extending our understanding on the role of antisense transcription in the brain. PMID:25883552

  6. A novel dysferlin mutant pseudoexon bypassed with antisense oligonucleotides

    PubMed Central

    Dominov, Janice A; Uyan, Özgün; Sapp, Peter C; McKenna-Yasek, Diane; Nallamilli, Babi R R; Hegde, Madhuri; Brown, Robert H

    2014-01-01

    Objective Mutations in dysferlin (DYSF), a Ca2+-sensitive ferlin family protein important for membrane repair, vesicle trafficking, and T-tubule function, cause Miyoshi myopathy, limb-girdle muscular dystrophy type 2B, and distal myopathy. More than 330 pathogenic DYSF mutations have been identified within exons or near exon–intron junctions. In ~17% of patients who lack normal DYSF, only a single disease-causing mutation has been identified. We studied one family with one known mutant allele to identify both the second underlying genetic defect and potential therapeutic approaches. Methods We sequenced the full DYSF cDNA and investigated antisense oligonucleotides (AONs) as a tool to modify splicing of the mRNA transcripts in order to process out mutant sequences. Results We identified a novel pseudoexon between exons 44 and 45, (pseudoexon 44.1, PE44.1), which inserts an additional 177 nucleotides into the mRNA and 59 amino acids within the conserved C2F domain of the DYSF protein. Two unrelated dysferlinopathy patients were also found to carry this mutation. Using AONs targeting PE44.1, we blocked the abnormal splicing event, yielding normal, full-length DYSF mRNA, and increased DYSF protein expression. Interpretation This is the first report of a deep intronic mutation in DYSF that alters mRNA splicing to include a mutant peptide fragment within a key DYSF domain. We report that AON-mediated exon-skipping restores production of normal, full-length DYSF in patients’ cells in vitro, offering hope that this approach will be therapeutic in this genetic context, and providing a foundation for AON therapeutics targeting other pathogenic DYSF alleles. PMID:25493284

  7. Antisense approach to inflammatory bowel disease: prospects and challenges.

    PubMed

    Marafini, Irene; Di Fusco, Davide; Calabrese, Emma; Sedda, Silvia; Pallone, Francesco; Monteleone, Giovanni

    2015-05-01

    Despite the great success of anti-tumour necrosis factor-based therapies, the treatment of Crohn's disease (CD) and ulcerative colitis (UC) still remains a challenge for clinicians, as these drugs are not effective in all patients, their efficacy may wane with time, and their use can increase the risk of adverse events and be associated with the development of new immune-mediated diseases. Therefore, new therapeutic targets are currently being investigated both in pre-clinical studies and in clinical trials. Among the technologies used to build new therapeutic compounds, the antisense oligonucleotide (ASO) approach is slowly gaining space in the field of inflammatory bowel diseases (IBDs), and three ASOs have been investigated in clinical trials. Systemic administration of alicaforsen targeting intercellular adhesion molecule-1, a protein involved in the recruitment of leukocytes to inflamed intestine, was not effective in CD, even though the same compound was of benefit when given as an enema to UC patients. DIMS0150, targeting nuclear factor (NF) κB-p65, a transcription factor that promotes pro-inflammatory responses, was very promising in pre-clinical studies and is currently being tested in clinical trials. Oral mongersen, targeting Smad7, an intracellular protein that inhibits transforming growth factor (TGF)-β1 activity, was safe and well tolerated by CD patients, and the results of a phase II clinical trial showed the efficacy of the drug in inducing clinical remission in patients with active disease. In this leading article, we review the rationale and the clinical data available regarding these three agents, and we discuss the challenge of using ASOs in IBD. PMID:25911184

  8. RNA therapeutics: RNAi and antisense mechanisms and clinical applications

    PubMed Central

    Chery, Jessica; Näär, Anders

    2016-01-01

    RNA therapeutics refers to the use of oligonucleotides to target primarily ribonucleic acids (RNA) for therapeutic efforts or in research studies to elucidate functions of genes. Oligonucleotides are distinct from other pharmacological modalities, such as small molecules and antibodies that target mainly proteins, due to their mechanisms of action and chemical properties. Nucleic acids come in two forms: deoxyribonucleic acids (DNA) and ribonucleic acids (RNA). Although DNA is more stable, RNA offers more structural variety ranging from messenger RNA (mRNA) that codes for protein to non-coding RNAs, microRNA (miRNA), transfer RNA (tRNA), short interfering RNAs (siRNAs), ribosomal RNA (rRNA), and long-noncoding RNAs (lncRNAs). As our understanding of the wide variety of RNAs deepens, researchers have sought to target RNA since >80% of the genome is estimated to be transcribed. These transcripts include non-coding RNAs such as miRNAs and siRNAs that function in gene regulation by playing key roles in the transfer of genetic information from DNA to protein, the final product of the central dogma in biology1. Currently there are two main approaches used to target RNA: double stranded RNA-mediated interference (RNAi) and antisense oligonucleotides (ASO). Both approaches are currently in clinical trials for targeting of RNAs involved in various diseases, such as cancer and neurodegeneration. In fact, ASOs targeting spinal muscular atrophy and amyotrophic lateral sclerosis have shown positive results in clinical trials2. Advantages of ASOs include higher affinity due to the development of chemical modifications that increase affinity, selectivity while decreasing toxicity due to off-target effects. This review will highlight the major therapeutic approaches of RNA medicine currently being applied with a focus on RNAi and ASOs. PMID:27570789

  9. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    PubMed

    Gerard, Xavier; Garanto, Alejandro; Rozet, Jean-Michel; Collin, Rob W J

    2016-01-01

    Inherited retinal dystrophies (IRDs) are an extremely heterogeneous group of genetic diseases for which currently no effective treatment strategies exist. Over the last decade, significant progress has been made utilizing gene augmentation therapy for a few genetic subtypes of IRD, although several technical challenges so far prevent a broad clinical application of this approach for other forms of IRD. Many of the mutations leading to these retinal diseases affect pre-mRNA splicing of the mutated genes . Antisense oligonucleotide (AON)-mediated splice modulation appears to be a powerful approach to correct the consequences of such mutations at the pre-mRNA level , as demonstrated by promising results in clinical trials for several inherited disorders like Duchenne muscular dystrophy, hypercholesterolemia and various types of cancer. In this mini-review, we summarize ongoing pre-clinical research on AON-based therapy for a few genetic subtypes of IRD , speculate on other potential therapeutic targets, and discuss the opportunities and challenges that lie ahead to translate splice modulation therapy for retinal disorders to the clinic. PMID:26427454

  10. Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape.

    PubMed

    Cecere, Germano; Hoersch, Sebastian; O'Keeffe, Sean; Sachidanandam, Ravi; Grishok, Alla

    2014-04-01

    Argonaute proteins and their small RNA cofactors short interfering RNAs are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) that are antisense to germline transcripts. However, its role in gene expression regulation remains controversial. Here we used genome-wide profiling of nascent RNA transcripts and found that the CSR-1 RNA interference pathway promoted sense-oriented RNA polymerase II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. On the basis of these findings, we propose that the CSR-1 pathway helps maintain the directionality of active transcription, thereby propagating the distinction between transcriptionally active and silent genomic regions. PMID:24681887

  11. NERF encodes a RING E3 ligase important for drought resistance and enhances the expression of its antisense gene NFYA5 in Arabidopsis

    PubMed Central

    Gao, Wei; Liu, Wenwen; Zhao, Meng; Li, Wen-Xue

    2015-01-01

    NFYA5 is an important drought-stress inducible transcription factor gene that is targeted by miR169 in Arabidopsis. We show here that the cis-natural antisense transcript gene of NFYA5, NFYA5 Enhancing RING FINGER (NERF), can produce siRNAs from their overlapping region (OR) and affect NFYA5 transcripts by functioning together with miR169. The NERF protein functions as an E3 ligase for ubiquitination. Overexpression of NERF or OR cDNA leads to siRNANERF accumulation, miR169 repression, and NFYA5 transcript enhancement; knock-down of NERF transcripts by an artificial miRNA enhances miR169 abundance and reduces NFYA5 transcripts. Overexpression of NFYA5 does not affect the NERF mRNA level. Deep sequencing of the small RNA library from 35S::OR plants identifies 960 sequences representing 323 unique siRNAs that originate from OR; the sequences of some siRNANERF are similar/complementary to those of miR169. Overexpression of the 195- to 280-bp OR cDNA-containing siRNAs similar/complementary to miR169 also leads to the accumulation of NFYA5 transcripts. Analysis of NERF knock-down plants and NERF overexpression lines showed that, like NFYA5, NERF is important for controlling stomatal aperture and drought resistance. This regulatory model might apply to other natural antisense transcripts with positively correlated expression patterns. PMID:25514924

  12. Antisense-Based Progerin Downregulation in HGPS-Like Patients' Cells.

    PubMed

    Harhouri, Karim; Navarro, Claire; Baquerre, Camille; Da Silva, Nathalie; Bartoli, Catherine; Casey, Frank; Mawuse, Guedenon Koffi; Doubaj, Yassamine; Lévy, Nicolas; De Sandre-Giovannoli, Annachiara

    2016-01-01

    Progeroid laminopathies, including Hutchinson-Gilford Progeria Syndrome (HGPS, OMIM #176670), are premature and accelerated aging diseases caused by defects in nuclear A-type Lamins. Most HGPS patients carry a de novo point mutation within exon 11 of the LMNA gene encoding A-type Lamins. This mutation activates a cryptic splice site leading to the deletion of 50 amino acids at its carboxy-terminal domain, resulting in a truncated and permanently farnesylated Prelamin A called Prelamin A Δ50 or Progerin. Some patients carry other LMNA mutations affecting exon 11 splicing and are named "HGPS-like" patients. They also produce Progerin and/or other truncated Prelamin A isoforms (Δ35 and Δ90) at the transcriptional and/or protein level. The results we present show that morpholino antisense oligonucleotides (AON) prevent pathogenic LMNA splicing, markedly reducing the accumulation of Progerin and/or other truncated Prelamin A isoforms (Prelamin A Δ35, Prelamin A Δ90) in HGPS-like patients' cells. Finally, a patient affected with Mandibuloacral Dysplasia type B (MAD-B, carrying a homozygous mutation in ZMPSTE24, encoding an enzyme involved in Prelamin A maturation, leading to accumulation of wild type farnesylated Prelamin A), was also included in this study. These results provide preclinical proof of principle for the use of a personalized antisense approach in HGPS-like and MAD-B patients, who may therefore be eligible for inclusion in a therapeutic trial based on this approach, together with classical HGPS patients. PMID:27409638

  13. Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

    PubMed Central

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel

    2014-01-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns—multisystemic, hepatic, or in central nervous system (CNS)—and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  14. Antisense mediated splicing modulation for inherited metabolic diseases: challenges for delivery.

    PubMed

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R

    2014-02-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  15. Nanoparticle Delivery of Antisense Oligonucleotides and Their Application in the Exon Skipping Strategy for Duchenne Muscular Dystrophy

    PubMed Central

    Falzarano, Maria Sofia; Passarelli, Chiara

    2014-01-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy. PMID:24506782

  16. Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA.

    PubMed

    Hemenway, C; Fang, R X; Kaniewski, W K; Chua, N H; Tumer, N E

    1988-05-01

    Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. Plants expressing antisense RNA were protected only at low inoculum concentrations. The extent of this protection was even lower than that observed in plants expressing low levels of CP. In contrast to previous reports for plants expressing tobacco mosaic virus or alfalfa mosaic virus CP, inoculation of plants expressing high levels of PVX CP with PVX RNA did not overcome the protection. Specifically, lesion numbers on inoculated leaves and PVX levels on inoculated and systemtic leaves of the CP+ plants were reduced to a similar extent in both virus and RNA inoculated plants. Although these results do not rule out that CP-mediated protection involves inhibition of uncoating of the challenge virus, they suggest that PVX CP (or its RNA) can moderate early events in RNA infection by a different mechanism. PMID:16453840

  17. On primordial sense-antisense coding

    PubMed Central

    Rodin, Andrei S.; Rodin, Sergei N.; Carter, Charles W.

    2010-01-01

    The genetic code is implemented by aminoacyl-tRNA synthetases (aaRS). These twenty enzymes are divided into two classes that, despite performing same functions, have nothing common in structure. The mystery of this striking partition of aaRSs might have been concealed in their sterically complementary modes of tRNA recognition that, as we have found recently, protect the tRNAs with complementary anticodons from confusion in translation. This finding implies that, in the beginning, life increased its coding repertoire by the pairs of complementary codons (rather than one-by-one) and used both complementary strands of genes as templates for translation. The class I and class II aaRSs may represent one of the most important examples of such primordial sence-antisence (SAS) coding (Rodin and Ohno, 1995). In this report, we address the issue of SAS coding in a wider scope. We suggest a variety of advantages that such coding would have had in exploring a wider sequence space before translation became highly specific. In particular, we confirm that in Achylia klebsiana a single gene might have originally coded for an HSP70 chaperonin (class II aaRS homolog) and an NAD-specific GDH-like enzyme (class I aaRS homolog) via its sense and antisense strands. Thus, in contrast to the conclusions in (Williams et al., 2009), this could indeed be a “Rosetta stone” (eroded somewhat, though) gene for the SAS origin of the two aaRS classes (Carter and Duax, 2002). PMID:19956936

  18. Inhibition of Human Immunodeficiency Virus Replication by Antisense Oligodeoxynucleotides

    NASA Astrophysics Data System (ADS)

    Goodchild, John; Agrawal, Sudhir; Civeira, Maria P.; Sarin, Prem S.; Sun, Daisy; Zamecnik, Paul C.

    1988-08-01

    Twenty different target sites within human immunodeficiency virus (HIV) RNA were selected for studies of inhibition of HIV replication by antisense oligonucleotides. Target sites were selected based on their potential capacity to block recognition functions during viral replication. Antisense oligomers complementary to sites within or near the sequence repeated at the ends of retrovirus RNA (R region) and to certain splice sites were most effective. The effect of antisense oligomer length on inhibiting virus replication was also investigated, and preliminary toxicity studies in mice show that these compounds are toxic only at high levels. The results indicate potential usefulness for these oligomers in the treatment of patients with acquired immunodeficiency syndrome (AIDS) and AIDS-related complex either alone or in combination with other drugs.

  19. Inhibition of dengue virus by novel, modified antisense oligonucleotides.

    PubMed Central

    Raviprakash, K; Liu, K; Matteucci, M; Wagner, R; Riffenburgh, R; Carl, M

    1995-01-01

    Five different target regions along the length of the dengue virus type 2 genome were compared for inhibition of the virus following intracellular injection of the cognate antisense oligonucleotides and their analogs. Unmodified phosphodiester oligonucleotides as well as the corresponding phosphorothioate oligonucleotides were ineffective in bringing about a significant inhibition of the virus. Novel modified phosphorothioate oligonucleotides in which the C-5 atoms of uridines and cytidines were replaced by propynyl groups caused a significant inhibition of the virus. Antisense oligonucleotide directed against the target region near the translation initiation site of dengue virus RNA was the most effective, followed by antisense oligonucleotide directed against a target in the 3' untranslated region of the virus RNA. It is suggested that the inhibitory effect of these novel modified oligonucleotides is due to their increased affinity for the target sequences and that they probably function via an RNase H cleavage of the oligonucleotide:RNA heteroduplex. PMID:7983769

  20. Antisense downregulation of polyphenol oxidase results in enhanced disease susceptibility.

    PubMed

    Thipyapong, Piyada; Hunt, Michelle D; Steffens, John C

    2004-11-01

    Polyphenol oxidases (PPOs; EC 1.14.18.1 or EC 1.10.3.2) catalyze the oxidation of phenolics to quinones, highly reactive intermediates whose secondary reactions are responsible for much of the oxidative browning that accompanies plant senescence, wounding, and responses to pathogens. To assess the impact of PPO expression on resistance to Pseudomonas syringae pv. tomato we introduced a chimeric antisense potato PPO cDNA into tomato (Lycopersicon esculentum L.). Oxidation of caffeic acid, the dominant o-diphenolic aglycone of tomato foliage, was decreased ca. 40-fold by antisense expression of PPO. All members of the PPO gene family were downregulated: neither immunoreactive PPO nor PPO-specific mRNA were detectable in the transgenic plants. In addition, the antisense PPO construct suppressed inducible increases in PPO activity. Downregulation of PPO in antisense plants did not affect growth, development, or reproduction of greenhouse-grown plants. However, antisense PPO expression dramatically increased susceptibility to P. syringae expressing the avirulence gene avrPto in both Pto and pto backgrounds. In a compatible (pto) interaction, plants constitutively expressing an antisense PPO construct exhibited a 55-fold increase in bacterial growth, three times larger lesion area, and ten times more lesions cm(-2) than nontransformed plants. In an incompatible (Pto) interaction, antisense PPO plants exhibited 100-fold increases in bacterial growth and ten times more lesions cm(-2) than nontransformed plants. Although it is not clear whether hypersusceptibility of antisense plants is due to low constitutive PPO levels or failure to induce PPO upon infection, these findings suggest a critical role for PPO-catalyzed phenolic oxidation in limiting disease development. As a preliminary effort to understand the role of induced PPO in limiting disease development, we also examined the response of PPO promoter::beta-glucuronidase constructs when plants are challenged with P

  1. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD).

    PubMed

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA). PMID:21686247

  2. Diversity of Antisense and Other Non-Coding RNAs in Archaea Revealed by Comparative Small RNA Sequencing in Four Pyrobaculum Species

    PubMed Central

    Bernick, David L.; Dennis, Patrick P.; Lui, Lauren M.; Lowe, Todd M.

    2012-01-01

    A great diversity of small, non-coding RNA (ncRNA) molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs (sRNA) in archaea is limited. We employed RNA-seq to identify novel sRNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense sRNAs encoded opposite to key regulatory (ferric uptake regulator), metabolic (triose-phosphate isomerase), and core transcriptional apparatus genes (transcription factor B). We also found a large increase in the number of conserved C/D box sRNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these sRNAs indicates they are relatively recent, stable adaptations. PMID:22783241

  3. Transcription mediated insulation and interference direct gene cluster expression switches

    PubMed Central

    Nguyen, Tania; Brown, David; Murray, Struan C; Haenni, Simon; Halstead, James M; O'Connor, Leigh; Shipkovenska, Gergana; Steinmetz, Lars M; Mellor, Jane

    2014-01-01

    In yeast, many tandemly arranged genes show peak expression in different phases of the metabolic cycle (YMC) or in different carbon sources, indicative of regulation by a bi-modal switch, but it is not clear how these switches are controlled. Using native elongating transcript analysis (NET-seq), we show that transcription itself is a component of bi-modal switches, facilitating reciprocal expression in gene clusters. HMS2, encoding a growth-regulated transcription factor, switches between sense- or antisense-dominant states that also coordinate up- and down-regulation of transcription at neighbouring genes. Engineering HMS2 reveals alternative mono-, di- or tri-cistronic and antisense transcription units (TUs), using different promoter and terminator combinations, that underlie state-switching. Promoters or terminators are excluded from functional TUs by read-through transcriptional interference, while antisense TUs insulate downstream genes from interference. We propose that the balance of transcriptional insulation and interference at gene clusters facilitates gene expression switches during intracellular and extracellular environmental change. DOI: http://dx.doi.org/10.7554/eLife.03635.001 PMID:25407679

  4. Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.

    PubMed

    Bruin, G J; Börnsen, K O; Hüsken, D; Gassmann, E; Widmer, H M; Paulus, A

    1995-08-11

    The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented. PMID:7581844

  5. Short antisense-locked nucleic acids (all-LNAs) correct alternative splicing abnormalities in myotonic dystrophy

    PubMed Central

    Wojtkowiak-Szlachcic, Agnieszka; Taylor, Katarzyna; Stepniak-Konieczna, Ewa; Sznajder, Lukasz J.; Mykowska, Agnieszka; Sroka, Joanna; Thornton, Charles A.; Sobczak, Krzysztof

    2015-01-01

    Myotonic dystrophy type 1 (DM1) is an autosomal dominant multisystemic disorder caused by expansion of CTG triplet repeats in 3′-untranslated region of DMPK gene. The pathomechanism of DM1 is driven by accumulation of toxic transcripts containing expanded CUG repeats (CUGexp) in nuclear foci which sequester several factors regulating RNA metabolism, such as Muscleblind-like proteins (MBNLs). In this work, we utilized very short chemically modified antisense oligonucleotides composed exclusively of locked nucleic acids (all-LNAs) complementary to CUG repeats, as potential therapeutic agents against DM1. Our in vitro data demonstrated that very short, 8- or 10-unit all-LNAs effectively bound the CUG repeat RNA and prevented the formation of CUGexp/MBNL complexes. In proliferating DM1 cells as well as in skeletal muscles of DM1 mouse model the all-LNAs induced the reduction of the number and size of CUGexp foci and corrected MBNL-sensitive alternative splicing defects with high efficacy and specificity. The all-LNAs had low impact on the cellular level of CUGexp-containing transcripts and did not affect the expression of other transcripts with short CUG repeats. Our data strongly indicate that short all-LNAs complementary to CUG repeats are a promising therapeutic tool against DM1. PMID:25753670

  6. Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

    PubMed

    Marsollier, Anne-Charlotte; Ciszewski, Lukasz; Mariot, Virginie; Popplewell, Linda; Voit, Thomas; Dickson, George; Dumonceaux, Julie

    2016-04-15

    Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy. PMID:26787513

  7. Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle.

    PubMed

    Pandorf, Clay E; Jiang, Weihua; Qin, Anqi X; Bodell, Paul W; Baldwin, Kenneth M; Haddad, Fadia

    2012-04-01

    Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development. PMID:22262309

  8. Boosting transcription by transcription: enhancer-associated transcripts.

    PubMed

    Darrow, Emily M; Chadwick, Brian P

    2013-12-01

    Enhancers are traditionally viewed as DNA sequences located some distance from a promoter that act in cis and in an orientation-independent fashion to increase utilization of specific promoters and thereby regulate gene expression. Much progress has been made over the last decade toward understanding how these distant elements interact with target promoters, but how transcription is enhanced remains an object of active inquiry. Recent reports convey the prevalence and diversity of enhancer transcription and transcripts and support both as key factors with mechanistically distinct, but not mutually exclusive roles in enhancer function. Decoupling the causes and effects of transcription on the local chromatin landscape and understanding the role of enhancer transcripts in the context of long-range interactions are challenges that require additional attention. In this review, we focus on the possible functions of enhancer transcription by highlighting several recent enhancer RNA papers and, within the context of other enhancer studies, speculate on the role of enhancer transcription in regulating differential gene expression. PMID:24178450

  9. Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

    PubMed Central

    Lorenz, Peter; Baker, Brenda F.; Bennett, C. Frank; Spector, David L.

    1998-01-01

    Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides. PMID:9571236

  10. Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization.

    PubMed

    Csorba, Tibor; Questa, Julia I; Sun, Qianwen; Dean, Caroline

    2014-11-11

    Long noncoding RNAs (lncRNAs) have been proposed to play important roles in gene regulation. However, their importance in epigenetic silencing and how specificity is determined remain controversial. We have investigated the cold-induced epigenetic switching mechanism involved in the silencing of Arabidopsis thaliana Flowering Locus C (FLC), which occurs during vernalization. Antisense transcripts, collectively named COOLAIR, are induced by prolonged cold before the major accumulation of histone 3 lysine 27 trimethylation (H3K27me3), characteristic of Polycomb silencing. We have found that COOLAIR is physically associated with the FLC locus and accelerates transcriptional shutdown of FLC during cold exposure. Removal of COOLAIR disrupted the synchronized replacement of H3K36 methylation with H3K27me3 at the intragenic FLC nucleation site during the cold. Consistently, genetic analysis showed COOLAIR and Polycomb complexes work independently in the cold-dependent silencing of FLC. Our data reveal a role for lncRNA in the coordinated switching of chromatin states that occurs during epigenetic regulation. PMID:25349421

  11. Antisense COOLAIR mediates the coordinated switching of chromatin states at FLC during vernalization

    PubMed Central

    Csorba, Tibor; Questa, Julia I.; Sun, Qianwen; Dean, Caroline

    2014-01-01

    Long noncoding RNAs (lncRNAs) have been proposed to play important roles in gene regulation. However, their importance in epigenetic silencing and how specificity is determined remain controversial. We have investigated the cold-induced epigenetic switching mechanism involved in the silencing of Arabidopsis thaliana FLOWERING LOCUS C (FLC), which occurs during vernalization. Antisense transcripts, collectively named COOLAIR, are induced by prolonged cold before the major accumulation of histone 3 lysine 27 trimethylation (H3K27me3), characteristic of Polycomb silencing. We have found that COOLAIR is physically associated with the FLC locus and accelerates transcriptional shutdown of FLC during cold exposure. Removal of COOLAIR disrupted the synchronized replacement of H3K36 methylation with H3K27me3 at the intragenic FLC nucleation site during the cold. Consistently, genetic analysis showed COOLAIR and Polycomb complexes work independently in the cold-dependent silencing of FLC. Our data reveal a role for lncRNA in the coordinated switching of chromatin states that occurs during epigenetic regulation. PMID:25349421

  12. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli

    PubMed Central

    Guruge, Keerthi S.; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I.; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  13. Source-Related Effects of Wastewater on Transcription Factor (AhR, CAR and PXR)-Mediated Induction of Gene Expression in Cultured Rat Hepatocytes and Their Association with the Prevalence of Antimicrobial-Resistant Escherichia coli.

    PubMed

    Guruge, Keerthi S; Yamanaka, Noriko; Sonobe, Miyuki; Fujizono, Wataru; Yoshioka, Miyako; Akiba, Masato; Yamamoto, Takehisa; Joshua, Derrick I; Balakrishna, Keshava; Yamashita, Nobuyoshi; Kannan, Kurunthachalam; Tsutsui, Toshiyuki

    2015-01-01

    Extracts of wastewater collected from 4 sewage treatment plants (STPs) receiving effluents from different sources in South India were investigated for their levels of transcription factor-mediated gene induction in primary cultured rat hepatocytes. In addition, the relation between gene induction levels and the prevalence of antimicrobial-resistant Escherichia coli (E. coli) in wastewater was examined. STP-3, which treats only hospital wastewater, exhibited significantly greater induction potency of all 6 drug metabolizing cytochrome P450 (CYP) genes examined, CYP1A1, 1A2, 1B1, 2B15, 3A1, and 3A2, whereas the wastewater at STP-1, which exclusively receives domestic sewage, showed significantly diminished levels of induction of 3 CYP genes when compared to the levels of CYP induction at STP-2, which receives mixed wastewater. Samples collected during the monsoon season showed a significantly altered gene induction capacity compared to that of samples from the pre-monsoon period. The data suggest that the toxicity of wastewater in STPs was not significantly diminished during the treatment process. The chemical-gene interaction data predicted that a vast number of chemicals present in the wastewater would stimulate the genes studied in the rat hepatocytes. The multivariable logistic regression analysis demonstrated that the prevalence of isolates resistant to cefotaxime, imipenem and streptomycin was significantly correlated with the levels of induction of at least three CYP-isozymes in STP wastewater. In addition, the resistance of isolates in treatment plants was not altered by the treatment steps, whereas the sampling season did have an impact on the resistance to specific antimicrobials. The identification of receptor-mediated gene regulation capacities offers important data not limited to the (synergistic) physiological role of chemicals in biological systems but may provide new insight into the link between the effects of known/unknown drugs and prevalence of

  14. SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells

    PubMed Central

    Zucchelli, Silvia; Fasolo, Francesca; Russo, Roberta; Cimatti, Laura; Patrucco, Laura; Takahashi, Hazuki; Jones, Michael H.; Santoro, Claudio; Sblattero, Daniele; Cotella, Diego; Persichetti, Francesca; Carninci, Piero; Gustincich, Stefano

    2015-01-01

    Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies. PMID:26029048

  15. Gene Silencing by Gold Nanoshell-Mediated Delivery and Laser-Triggered Release of Antisense Oligonucleotide and siRNA

    PubMed Central

    Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A.; Ji, Lin; Halas, Naomi J.

    2013-01-01

    The approach of RNA interference (RNAi)- using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein- is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly(L)lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ∼ 47% and ∼49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

  16. Gene silencing by gold nanoshell-mediated delivery and laser-triggered release of antisense oligonucleotide and siRNA.

    PubMed

    Huschka, Ryan; Barhoumi, Aoune; Liu, Qing; Roth, Jack A; Ji, Lin; Halas, Naomi J

    2012-09-25

    RNA interference (RNAi)--using antisense DNA or RNA oligonucleotides to silence activity of a specific pathogenic gene transcript and reduce expression of the encoded protein--is very useful in dissecting genetic function and holds significant promise as a molecular therapeutic. A major obstacle in achieving gene silencing with RNAi technology is the systemic delivery of therapeutic oligonucleotides. Here we demonstrate an engineered gold nanoshell (NS)-based therapeutic oligonucleotide delivery vehicle, designed to release its cargo on demand upon illumination with a near-infrared (NIR) laser. A poly-L-lysine peptide (PLL) epilayer covalently attached to the NS surface (NS-PLL) is used to capture intact, single-stranded antisense DNA oligonucleotides, or alternatively, double-stranded short-interfering RNA (siRNA) molecules. Controlled release of the captured therapeutic oligonucleotides in each case is accomplished by continuous wave NIR laser irradiation at 800 nm, near the resonance wavelength of the nanoshell. Fluorescently tagged oligonucleotides were used to monitor the time-dependent release process and light-triggered endosomal release. A green fluorescent protein (GFP)-expressing human lung cancer H1299 cell line was used to determine cellular uptake and gene silencing mediated by the NS-PLL carrying GFP gene-specific single-stranded DNA antisense oligonucleotide (AON-GFP), or a double-stranded siRNA (siRNA-GFP), in vitro. Light-triggered delivery resulted in ~47% and ~49% downregulation of the targeted GFP expression by AON-GFP and siRNA-GFP, respectively. Cytotoxicity induced by both the NS-PLL delivery vector and by laser irradiation is minimal, as demonstrated by a XTT cell proliferation assay. PMID:22862291

  17. SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation

    PubMed Central

    Zucchelli, S; Cotella, D; Takahashi, H; Carrieri, C; Cimatti, L; Fasolo, F; Jones, MH; Sblattero, D; Sanges, R; Santoro, C; Persichetti, F; Carninci, P; Gustincich, S

    2015-01-01

    Over the past 10 years, it has emerged that pervasive transcription in mammalian genomes has a tremendous impact on several biological functions. Most of transcribed RNAs are lncRNAs and repetitive elements. In this review, we will detail the discovery of a new functional class of natural and synthetic antisense lncRNAs that stimulate translation of sense mRNAs. These molecules have been named SINEUPs since their function requires the activity of an embedded inverted SINEB2 sequence to UP-regulate translation. Natural SINEUPs suggest that embedded Transposable Elements may represent functional domains in long non-coding RNAs. Synthetic SINEUPs may be designed by targeting the antisense sequence to the mRNA of choice representing the first scalable tool to increase protein synthesis of potentially any gene of interest. We will discuss potential applications of SINEUP technology in the field of molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies. PMID:26259533

  18. Avoidance of antisense, antiterminator tRNA anticodons in vertebrate mitochondria.

    PubMed

    Seligmann, Hervé

    2010-07-01

    Protein synthesis (translation) stops at stop codons, codons not complemented by tRNA anticodons. tRNAs matching stops, antitermination (Ter) tRNAs, prevent translational termination, producing dysfunctional proteins. Genomes avoid tRNAs with anticodons whose complement (the anticodon of the 'antisense' tRNA) matches stops. This suggests that antisense tRNAs, which also form cloverleaves, are occasionally expressed. Mitochondrial antisense tRNA expression is plausible, because both DNA strands are transcribed as single RNAs, and tRNA structures signal RNA maturation. Results describe potential antisense Ter tRNAs in mammalian mitochondrial genomes detected by tRNAscan-SE, and evidence for adaptations preventing translational antitermination: genomes possessing Ter tRNAs use less corresponding stop codons; antisense Ter tRNAs form weaker cloverleaves than homologuous non-Ter antisense tRNAs; and genomic stop codon usages decrease with stabilities of codon-anticodon interactions and of Ter tRNA cloverleaves. This suggests that antisense tRNAs frequently function in translation. Results suggest that opposite strand coding is exceptional in modern genes, yet might be frequent for mitochondrial tRNAs. This adds antisense tRNA templating to other mitochondrial tRNA functions: sense tRNA templating, formation and regulation of secondary (light strand DNA) replication origins. Antitermination probably affects mitochondrial degenerative diseases and ageing: pathogenic mutations are twice as frequent in tRNAs with antisense Ter anticodons than in other tRNAs, and species lacking mitochondrial antisense Ter tRNAs have longer mean maximal lifespans than those possessing antisense Ter tRNAs. PMID:20399828

  19. A Universal Positive-Negative Selection System for Gene Targeting in Plants Combining an Antibiotic Resistance Gene and Its Antisense RNA.

    PubMed

    Nishizawa-Yokoi, Ayako; Nonaka, Satoko; Osakabe, Keishi; Saika, Hiroaki; Toki, Seiichi

    2015-09-01

    Gene targeting (GT) is a useful technology for accurate genome engineering in plants. A reproducible approach based on a positive-negative selection system using hygromycin resistance and the diphtheria toxin A subunit gene as positive and negative selection markers, respectively, is now available. However, to date, this selection system has been applied exclusively in rice (Oryza sativa). To establish a universally applicable positive-negative GT system in plants, we designed a selection system using a combination of neomycin phosphotransferaseII (nptII) and an antisense nptII construct. The concomitant transcription of both sense and antisense nptII suppresses significantly the level of expression of the sense nptII gene, and transgenic calli and plants become sensitive to the antibiotic geneticin. In addition, we were able to utilize the sense nptII gene as a positive selection marker and the antisense nptII construct as a negative selection marker for knockout of the endogenous rice genes Waxy and 33-kD globulin through GT, although negative selection with this system is relatively less efficient compared with diphtheria toxin A subunit. The approach developed here, with some additional improvements, could be applied as a universal selection system for the enrichment of GT cells in several plant species. PMID:26143254

  20. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    PubMed

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity. PMID:26643897

  1. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    PubMed

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation. PMID:26407519

  2. 3'-modified antisense oligodeoxyribonucleotides complementary to calmodulin mRNA alter behavioral responses in Paramecium.

    PubMed Central

    Hinrichsen, R D; Fraga, D; Reed, M W

    1992-01-01

    The calcium-binding protein calmodulin has been shown to modulate the Ca(2+)-dependent ion channels of Paramecium tetraurelia. Mutations in the calmodulin gene of Paramecium result in an altered pattern of behavioral responses. Antisense oligodeoxyribonucleotides (ODNs), complementary to calmodulin mRNA in Paramecium, were synthesized from a modified solid support that introduced a 3'-hydroxyhexyl phosphate. These 3'-modified ODNs were tested for their ability to alter the behavioral response of Paramecium. The microinjection of antisense ODNs temporarily reduced the backward swimming behavior of the cells in test solutions containing Na+. The injection of sense and random 3'-modified ODNs, or unmodified antisense ODNs, had no effect. The antisense ODN-induced effect was reversed by the injection of calmodulin protein. The pattern of response of the injected cells in various behavioral test solutions indicated that the calmodulin antisense ODNs reduce the Ca(2+)-dependent Na+ current. Antisense ODNs, complementary either to the 5' start site or to an internal sequence of the calmodulin mRNA, were similarly effective in altering behavior. These results show that antisense ODNs may be utilized in ciliated protozoa as a tool for reducing the expression of specific gene products. In addition, Paramecium represents a powerful model system with which to study and develop antisense ODN technology. PMID:1528867

  3. Intrinsic disorder in transcription factors†

    PubMed Central

    Liu, Jiangang; Perumal, Narayanan B.; Oldfield, Christopher J.; Su, Eric W.; Uversky, Vladimir N.; Dunker, A. Keith

    2008-01-01

    Intrinsic disorder (ID) is highly abundant in eukaryotes, which reflect the greater need for disorder-associated signaling and transcriptional regulation in nucleated cells. Although several well-characterized examples of intrinsically disordered proteins in transcriptional regulation have been reported, no systematic analysis has been reported so far. To test for a general prevalence of intrinsic disorder in transcriptional regulation, we used the Predictor Of Natural Disorder Regions (PONDR) to analyze the abundance of intrinsic disorder in three transcription factor datasets and two control sets. This analysis revealed that from 94.13% to 82.63% of transcription factors posses extended regions of intrinsic disorder, relative to 54.51% and 18.64% of the proteins in two control datasets, which indicates the significant prevalence of intrinsic disorder in transcription factors. This propensity of transcription factors for intrinsic disorder was confirmed by cumulative distribution function analysis and charge-hydropathy plots. The amino acid composition analysis showed that all three transcription factor datasets were substantially depleted in order-promoting residues, and significantly enriched in disorder-promoting residues. Our analysis of the distribution of disorder within the transcription factor datasets revealed that: (a) The AT-hooks and basic regions of transcription factor DNA-binding domains are highly disordered; (b) The degree of disorder in transcription factor activation regions is much higher than that in DNA-binding domains; (c) The degree of disorder is significantly higher in eukaryotic transcription factors than in prokaryotic transcription factors; (d) The level of α-MoRFs (molecular recognition feature) prediction is much higher in transcription factors. Overall, our data reflected the fact that the eukaryotes with well-developed gene transcription machinery require transcription factor flexibility to be more efficient. PMID:16734424

  4. PRO-051, an antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy.

    PubMed

    Hammond, Suzan M; Wood, Matthew Ja

    2010-08-01

    PRO-051 (GSK-2402968), being developed by GlaxoSmithKline plc, under license from Leiden University Medical Center and Prosensa Therapeutics BV, is a 2'-O-methyl phosphorothioate antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy (DMD). The PRO-051 oligonucleotide sequence induces skipping of exon 51 of the dystrophin gene by binding to a sequence within the dystrophin pre-mRNA and masking the exon inclusion signals that are used for splicing. Removal of exon 51 from an exon 45 to 50, 47 to 50, 48 to 50, 49 to 50, 50, 52 or 52 to 63 deleted transcript allows restoration of the open reading frame and synthesis of an internally truncated, semi-functional dystrophin protein. By targeting exon 51, approximately 13% of patients with DMD could be treated, the largest proportion of patients that could benefit from targeting a single dystrophin exon. A proof-of-concept clinical trial of PRO-051 in patients with DMD demonstrated that a single intramuscular administration of PRO-051 induced exon skipping within muscle fibers adjacent to the injection site, while biopsies revealed dystrophin expression in treated but not control muscle fibers. At the time of publication, a phase I/IIa trial to evaluate subcutaneous delivery of PRO-051 had been completed, although full results were yet to be published. PMID:20677099

  5. Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness.

    PubMed

    Lentz, Jennifer J; Jodelka, Francine M; Hinrich, Anthony J; McCaffrey, Kate E; Farris, Hamilton E; Spalitta, Matthew J; Bazan, Nicolas G; Duelli, Dominik M; Rigo, Frank; Hastings, Michelle L

    2013-03-01

    Hearing impairment is the most common sensory disorder, with congenital hearing impairment present in approximately 1 in 1,000 newborns. Hereditary deafness is often mediated by the improper development or degeneration of cochlear hair cells. Until now, it was not known whether such congenital failures could be mitigated by therapeutic intervention. Here we show that hearing and vestibular function can be rescued in a mouse model of human hereditary deafness. An antisense oligonucleotide (ASO) was used to correct defective pre-mRNA splicing of transcripts from the USH1C gene with the c.216G>A mutation, which causes human Usher syndrome, the leading genetic cause of combined deafness and blindness. Treatment of neonatal mice with a single systemic dose of ASO partially corrects Ush1c c.216G>A splicing, increases protein expression, improves stereocilia organization in the cochlea, and rescues cochlear hair cells, vestibular function and low-frequency hearing in mice. These effects were sustained for several months, providing evidence that congenital deafness can be effectively overcome by treatment early in development to correct gene expression and demonstrating the therapeutic potential of ASOs in the treatment of deafness. PMID:23380860

  6. Antitumor Effects of EGFR Antisense Guanidine-Based Peptide Nucleic Acids in Cancer Models

    PubMed Central

    Thomas, Sufi M.; Sahu, Bichismita; Rapireddy, Srinivas; Bahal, Raman; Wheeler, Sarah E.; Procopio, Eva M.; Kim, Joseph; Joyce, Sonali C.; Contrucci, Sarah; Wang, Yun; Chiosea, Simion I.; Lathrop, Kira L.; Watkins, Simon; Grandis, Jennifer R.; Armitage, Bruce A.; Ly, Danith H.

    2013-01-01

    Peptide nucleic acids have emerged over the past two decades as a promising class of nucleic acid mimics because of their strong binding affinity and sequence selectivity toward DNA and RNA, and resistance to enzymatic degradation by proteases and nucleases. While they have been shown to be effective in regulation of gene expression in vitro, and to a small extent in vivo, their full potential for molecular therapy has not yet been fully realized due to poor cellular uptake. Herein, we report the development of cell-permeable, guanidine-based peptide nucleic acids targeting the epidermal growth factor receptor (EGFR) in preclinical models as therapeutic modality for head and neck squamous cell carcinoma (HNSCC) and nonsmall cell lung cancer (NSCLC). A GPNA oligomer, 16 nucleotides in length, designed to bind to EGFR gene transcript elicited potent antisense effects in HNSCC and NSCLC cells in preclinical models. When administered intraperitoneally in mice, EGFRAS-GPNA was taken-up by several tissues including the xenograft tumor. Systemic administration of EGFRAS-GPNA induced antitumor effects in HNSCC xenografts, with similar efficacies as the FDA-approved EGFR inhibitors: cetuximab and erlotinib. In addition to targeting wild-type EGFR, EGFRAS-GPNA is effective against the constitutively active EGFR vIII mutant implicated in cetuximab resistance. Our data reveals that GPNA is just as effective as a molecular platform for treating cetuximab resistant cells, demonstrating its utility in the treatment of cancer. PMID:23113581

  7. Antisense-induced messenger depletion corrects a COL6A2 dominant mutation in Ullrich myopathy.

    PubMed

    Gualandi, Francesca; Manzati, Elisa; Sabatelli, Patrizia; Passarelli, Chiara; Bovolenta, Matteo; Pellegrini, Camilla; Perrone, Daniela; Squarzoni, Stefano; Pegoraro, Elena; Bonaldo, Paolo; Ferlini, Alessandra

    2012-12-01

    Collagen VI gene mutations cause Ullrich and Bethlem muscular dystrophies. Pathogenic mutations frequently have a dominant negative effect, with defects in collagen VI chain secretion and assembly. It is agreed that, conversely, collagen VI haploinsufficiency has no pathological consequences. Thus, RNA-targeting approaches aimed at preferentially inactivating the mutated COL6 messenger may represent a promising therapeutic strategy. By in vitro studies we obtained the preferential depletion of the mutated COL6A2 messenger, by targeting a common single-nucleotide polymorphism (SNP), cistronic with a dominant COL6A2 mutation. We used a 2'-O-methyl phosphorothioate (2'OMePS) antisense oligonucleotide covering the SNP within exon 3, which is out of frame. Exon 3 skipping has the effect of depleting the mutated transcript via RNA nonsense-mediated decay, recovering the correct collagen VI secretion and restoring the ability to form an interconnected microfilament network into the extracellular matrix. This novel RNA modulation approach to correcting dominant mutations may represent a therapeutic strategy potentially applicable to a great variety of mutations and diseases. PMID:22992134

  8. Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy

    PubMed Central

    Gramlich, Michael; Pane, Luna Simona; Zhou, Qifeng; Chen, Zhifen; Murgia, Marta; Schötterl, Sonja; Goedel, Alexander; Metzger, Katja; Brade, Thomas; Parrotta, Elvira; Schaller, Martin; Gerull, Brenda; Thierfelder, Ludwig; Aartsma-Rus, Annemieke; Labeit, Siegfried; Atherton, John J; McGaughran, Julie; Harvey, Richard P; Sinnecker, Daniel; Mann, Matthias; Laugwitz, Karl-Ludwig; Gawaz, Meinrad Paul; Moretti, Alessandra

    2015-01-01

    Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM. PMID:25759365

  9. Antisense Reduction of Tau in Adult Mice Protects against Seizures

    PubMed Central

    DeVos, Sarah L.; Goncharoff, Dustin K.; Chen, Guo; Kebodeaux, Carey S.; Yamada, Kaoru; Stewart, Floy R.; Schuler, Dorothy R.; Maloney, Susan E.; Wozniak, David F.; Rigo, Frank; Bennett, C. Frank; Cirrito, John R.; Holtzman, David M.

    2013-01-01

    Tau, a microtubule-associated protein, is implicated in the pathogenesis of Alzheimer's Disease (AD) in regard to both neurofibrillary tangle formation and neuronal network hyperexcitability. The genetic ablation of tau substantially reduces hyperexcitability in AD mouse lines, induced seizure models, and genetic in vivo models of epilepsy. These data demonstrate that tau is an important regulator of network excitability. However, developmental compensation in the genetic tau knock-out line may account for the protective effect against seizures. To test the efficacy of a tau reducing therapy for disorders with a detrimental hyperexcitability profile in adult animals, we identified antisense oligonucleotides that selectively decrease endogenous tau expression throughout the entire mouse CNS—brain and spinal cord tissue, interstitial fluid, and CSF—while having no effect on baseline motor or cognitive behavior. In two chemically induced seizure models, mice with reduced tau protein had less severe seizures than control mice. Total tau protein levels and seizure severity were highly correlated, such that those mice with the most severe seizures also had the highest levels of tau. Our results demonstrate that endogenous tau is integral for regulating neuronal hyperexcitability in adult animals and suggest that an antisense oligonucleotide reduction of tau could benefit those with epilepsy and perhaps other disorders associated with tau-mediated neuronal hyperexcitability. PMID:23904623

  10. Does Active Learning through an Antisense Jigsaw Make Sense?

    NASA Astrophysics Data System (ADS)

    Seetharaman, Mahadevan; Musier-Forsyth, Karin

    2003-12-01

    Three journal articles on nucleic acid antisense modification strategies were assigned to 12 students as part of an active learning "jigsaw" exercise for a graduate-level chemistry course on nucleic acids. Each student was required to read one of the three articles. This assignment was preceded by an hour-long lecture on the basic concepts in antisense antigene technology. On the day of the jigsaw, the students with the same article (three groups of four students) discussed their article briefly, and then formed four new groups where no one had read the same article. Each student spent about five minutes teaching his or her article to the other group members, using specific questions provided to guide the discussion. This exercise laid the foundation for bringing the discussion to the entire class, where most of the students actively participated. To test the students' comprehension of the reading materials, a problem set was designed that required not only an understanding of the three articles, but also application of the concepts learned. The effectiveness of this active learning strategy and its applicability to other topics are discussed in this article.

  11. Anxiolytic effect and memory improvement in rats by antisense oligodeoxynucleotide to 5-hydroxytryptamine-2A precursor protein.

    PubMed

    Cohen, Hagit

    2005-01-01

    Serotonergic (5-hydroxytryptamine; 5-HT) mechanisms have been implicated in a number of physiological and pathophysiological processes including mood, anxiety, and cognitive functioning. Among the many 5-HT receptor subtypes, the 5-HT2A receptors (5-HT2A-R) seem to be of particular importance in mediating these effects, and they are prime targets for a variety of psychoactive substances-from hallucinogenic drugs, through atypical antipsychotics, to anxiolytics and antidepressants. Various selective 5-HT2A-R ligands induce different behavioral responses. To determine whether receptor downregulation is an essential part of anxiolytic action, levels of 5-HT2A receptors were manipulated in rats using a nonpharmacological approach-by the administration of an antisense oligodeoxynucleotide (ASODN) to 5-HT2A-R. Each ASODN was injected icv between two and five times at 24-hr intervals. Control rats received injections of either a scrambled oligodeoxynucleotide (ScrODN) or the vehicle only. On Day 6, anxiety-related behavior was assessed in the elevated plus maze paradigm and performance of memory tasks in the Morris water maze. Gene transcripts were measured by quantitative reverse transcription polymerase chain reaction (PCR). The results show that compared to vehicle and ScrODN control animals, icv 5-HT2A-R-ASODN administrations for 4 consecutive days (but not less) significantly decreased anxietylike behavior and improved memory retention performance. The reduction in anxiety-related behavior in 5-HT2A-R-ASODN rats was accompanied by a decrease in 5-HT2A-R-mRNA expression in the frontal cortex and in the hippocampus. Receptor downregulation has been proposed as one of the central mechanisms for anxiolytic drug actions. Antisense-mediated downmanipulation of receptors in this study, especially of 5-HT2A, supports this theory. PMID:16149040

  12. Characterization of a Novel Antisense RNA in the Major Pilin Locus of Neisseria meningitidis Influencing Antigenic Variation

    PubMed Central

    Tan, Felicia Y. Y.; Wörmann, Mirka E.; Tang, Christoph M.

    2015-01-01

    ABSTRACT Expression of type four pili (Tfp) is essential for virulence in Neisseria meningitidis. Pili mediate adhesion, bacterial aggregation, and DNA uptake. In N. meningitidis, the major pilin subunit is encoded by the pilE gene. In some strains, PilE is subject to phase and antigenic variation, which can alter Tfp properties and together offer a possible mechanism of immune escape. Pilin expression and antigenic variation can be modulated in response to environmental cues; however, the precise mechanisms of such regulation remain unclear. We identified a promoter in the pilE locus, 3′ of the pilE coding sequence, on the antisense (AS) strand which is conserved in meningococci. We show that this promoter directs transcription of an AS RNA that is expressed during specific growth phases and in response to salt stress. Furthermore, we demonstrate that the transcript encompasses sequences complementary to the entire pilE coding sequence and 5′ untranslated region. AS RNAs can regulate the gene on the sense strand by altering transcript stability or translation. However, by using Northern blotting, quantitative reverse transcription-PCR (RT-PCR), and Western blotting, we found no significant AS RNA-dependent changes in pilE transcript or protein level. Instead, our data indicate that the AS RNA influences pilin antigenic variation. This work provides further insights into the complex regulation of pilin expression and variation in pathogenic Neisseria. IMPORTANCE Pathogenic Neisseria spp. express type four pili (Tfp) which are important for adhesion, aggregation and transformation. Some strains of N. meningitidis are able to vary the sequence of the major subunit (PilE) of the Tfp. The mechanisms underlying this variation are not fully defined, but the process requires several noncoding elements that are found adjacent to the pilE gene. In this work, we identified a cis-encoded RNA antisense to pilE in N. meningitidis. By using Northern blotting and RT

  13. Photosynthetic Performance and Fertility Are Repressed in GmAOX2b Antisense Soybean1[OA

    PubMed Central

    Chai, Tsun-Thai; Simmonds, Daina; Day, David A.; Colmer, Timothy D.; Finnegan, Patrick M.

    2010-01-01

    The alternative oxidase (AOX) is a cyanide-resistant oxidase that provides an alternative outlet for electrons from the respiratory electron transport chain embedded in the inner membrane of plant mitochondria. Examination of soybean (Glycine max) plants carrying a GmAOX2b antisense gene showed AOX to have a central role in reproductive development and fecundity. In three independently transformed antisense lines, seed set was reduced by 16% to 43%, whereas ovule abortion increased by 1.2- to 1.7-fold when compared with nontransgenic transformation control plants. Reduced fecundity was associated with reductions in whole leaf cyanide-resistant, salicylhydroxamic acid-sensitive respiration and net photosynthesis, but there was no change in total respiration in the dark. The frequency of potential fertilization events was reduced by at least one-third in the antisense plants as a likely consequence of prefertilization defects. Pistils of the antisense plants contained a higher proportion of immature-sized, nonfertile embryo sacs compared with nontransgenic control plants. Increased rates of pollen abortion in vivo and reduced rates of pollen germination in vitro suggested that the antisense gene compromised pollen development and function. Reciprocal crosses between antisense and nontransgenic plants revealed that pollen produced by antisense plants was less active in fertilization. Taken together, the results presented here indicate that AOX expression has an important role in determining normal gametophyte development and function. PMID:20097793

  14. Functional analysis of polyphenol oxidases by antisense/sense technology.

    PubMed

    Thipyapong, Piyada; Stout, Michael J; Attajarusit, Jutharat

    2007-01-01

    Polyphenol oxidases (PPOs) catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum) plants with modified PPO expression levels (suppressed PPO and overexpressing PPO). These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F.)), cotton bollworm (Helicoverpa armigera (Hübner)) and beet army worm (Spodoptera exigua (Hübner)), whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say)) feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants overexpressing PPO, suggesting

  15. Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA.

    PubMed Central

    Koppelhus, U; Zachar, V; Nielsen, P E; Liu, X; Eugen-Olsen, J; Ebbesen, P

    1997-01-01

    We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs) was investigated. We found that a bis-PNA (parallel antisense 10mer linked to antiparallel antisense 10mer) was superior to both the parallel antisense 10mer and antiparallel antisense 10mer in inhibiting reverse transcription of the gene, thus indicating triplex formation at the target sequence. A complete arrest of reverse transcription was obtained at approximately 6-fold molar excess of the bis-PNA with respect to the gag RNA. At this molar ratio we found no effect on in vitro translation of gag RNA. A 15mer duplex-forming PNA was also found to inhibit reverse transcription at very low molar ratios of PNA/ gag RNA. Specificity of the inhibition of reverse transcription by PNA was confirmed by RNA sequencing, which revealed that all tested RTs were stopped by the PNA/RNA complex at the predicted site. We propose that the effect of PNA is exclusively due to steric hindrance, as we found no signs of RNA degradation that would indicate PNA-mediated RNase H activation of the tested RTs. In conclusion, PNA appears to have a potential to become a specific and efficient inhibitor of reverse transcription in vivo , provided sufficient intracellular levels are achievable. PMID:9153317

  16. Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo

    NASA Astrophysics Data System (ADS)

    Simons, Michael; Edelman, Elazer R.; Dekeyser, Jean-Luc; Langer, Robert; Rosenberg, Robert D.

    1992-09-01

    SYNTHETIC antisense oligonucleotides have been used to dissect gene function in vitro. Technical difficulties prevented the use of this approach for investigating the effect of gene products in vivo. Here we report the use of local delivery of antisense c-myb oligonu-cleotide to suppress intimal accumulation of rat carotid arterial smooth muscle cells. Our results suggest that antisense oligonucleotides can be used to define the in vivo biological role of specific macromolecules in the blood vessel wall and could potentially serve as a new class of therapeutic agents for cardiovascular disorders.

  17. Antisense oligonucleotide for tissue factor inhibits hepatic ischemic reperfusion injury.

    PubMed

    Nakamura, Kenji; Kadotani, Yayoi; Ushigome, Hidetaka; Akioka, Kiyokazu; Okamoto, Masahiko; Ohmori, Yoshihiro; Yaoi, Takeshi; Fushiki, Shinji; Yoshimura, Rikio; Yoshimura, Norio

    2002-09-27

    Tissue factor (TF) is an initiation factor for blood coagulation and its expression is induced on endothelial cells during inflammatory or immune responses. We designed an antisense oligodeoxynucleotide (AS-1/TF) for rat TF and studied its effect on hepatic ischemic reperfusion injury. AS-1/TF was delivered intravenously to Lewis rats. After 10 h, hepatic artery and portal vein were partially clamped. Livers were reperfused after 180 min and harvested. TF expression was studied using immunohistochemical staining. One of 10 rats survived in a 5-day survival rate and TF was strongly stained on endothelial cells in non-treatment group. However, by treatment with AS-1/TF, six of seven survived and TF staining was significantly reduced. Furthermore, we observed that fluorescein-labeled AS-1/TF was absorbed into endothelial cells. These results suggest that AS-1/TF can strongly suppress the expression of TF and thereby inhibit ischemic reperfusion injury to the rat liver. PMID:12270110

  18. Genome wide transcription start sites analysis of Xanthomonas campestris pv. campestris B100 with insights into the gum gene cluster directing the biosynthesis of the exopolysaccharide xanthan.

    PubMed

    Alkhateeb, Rabeaa S; Vorhölter, Frank-Jörg; Rückert, Christian; Mentz, Almut; Wibberg, Daniel; Hublik, Gerd; Niehaus, Karsten; Pühler, Alfred

    2016-05-10

    Xanthomonas campestris pv. campestris (Xcc) is the major producer of the exopolysaccharide xanthan, the commercially most important natural polysaccharide of microbial origin. The current work provides deeper insights into the yet uncharacterized transcriptomic features of the xanthan producing strain Xcc-B100. Towards this goal, RNA sequencing of a library based on the selective enrichment of the 5' ends of native transcripts was performed. This approach resulted in the genome wide identification of 3067 transcription start sites (TSSs) that were further classified based on their genomic positions. Among them, 1545 mapped upstream of an actively transcribed CDS and 1363 were classified as novel TSSs representing antisense, internal, and TSSs belonging to previously unidentified genomic features. Analyzing the transcriptional strength of primary and antisense TSSs revealed that in some instances antisense transcription seemed to be initiated at a higher level than its sense counterpart. Mapping the exact positions of TSSs aided in the identification of promoter consensus motifs, ribosomal binding sites, and enhanced the genome annotation of 159 in silico predicted translational start (TLS) sites. The global view on length distribution of the 5' untranslated regions (5'-UTRs) deduced from the data pointed to the occurrence of leaderless transcripts and transcripts with unusually long 5'-UTRs, in addition to identifying seven putative riboswitch elements for Xcc-B100. Concerning the biosynthesis of xanthan, we focused on the transcriptional organization of the gum gene cluster. Under the conditions tested, we present evidence for a complex transcription pattern of the gum genes with multiple TSSs and an obvious considerable role of antisense transcription. The gene gumB, encoding an outer membrane xanthan exporter, is presented here as an example for genes that possessed a strong antisense TSS. PMID:26975844

  19. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    PubMed Central

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  20. Transcriptional landscape and essential genes of Neisseria gonorrhoeae

    PubMed Central

    Remmele, Christian W.; Xian, Yibo; Albrecht, Marco; Faulstich, Michaela; Fraunholz, Martin; Heinrichs, Elisabeth; Dittrich, Marcus T.; Müller, Tobias; Reinhardt, Richard; Rudel, Thomas

    2014-01-01

    The WHO has recently classified Neisseria gonorrhoeae as a super-bacterium due to the rapid spread of antibiotic resistant derivatives and an overall dramatic increase in infection incidences. Genome sequencing has identified potential genes, however, little is known about the transcriptional organization and the presence of non-coding RNAs in gonococci. We performed RNA sequencing to define the transcriptome and the transcriptional start sites of all gonococcal genes and operons. Numerous new transcripts including 253 potentially non-coding RNAs transcribed from intergenic regions or antisense to coding genes were identified. Strikingly, strong antisense transcription was detected for the phase-variable opa genes coding for a family of adhesins and invasins in pathogenic Neisseria, that may have regulatory functions. Based on the defined transcriptional start sites, promoter motifs were identified. We further generated and sequenced a high density Tn5 transposon library to predict a core of 827 gonococcal essential genes, 133 of which have no known function. Our combined RNA-Seq and Tn-Seq approach establishes a detailed map of gonococcal genes and defines the first core set of essential gonococcal genes. PMID:25143534

  1. A modular strategy for engineering orthogonal chimeric RNA transcription regulators

    PubMed Central

    Takahashi, Melissa K.; Lucks, Julius B.

    2013-01-01

    Antisense RNA transcription attenuators are a key component of the synthetic biology toolbox, with their ability to serve as building blocks for both signal integration logic circuits and transcriptional cascades. However, a central challenge to building more sophisticated RNA genetic circuitry is creating larger families of orthogonal attenuators that function independently of each other. Here, we overcome this challenge by developing a modular strategy to create chimeric fusions between the engineered transcriptional attenuator from plasmid pT181 and natural antisense RNA translational regulators. Using in vivo gene expression assays in Escherichia coli, we demonstrate our ability to create chimeric attenuators by fusing sequences from five different translational regulators. Mutagenesis of these functional attenuators allowed us to create a total of 11 new chimeric attenutaors. A comprehensive orthogonality test of these culminated in a 7 × 7 matrix of mutually orthogonal regulators. A comparison between all chimeras tested led to design principles that will facilitate further engineering of orthogonal RNA transcription regulators, and may help elucidate general principles of non-coding RNA regulation. We anticipate that our strategy will accelerate the development of even larger families of orthogonal RNA transcription regulators, and thus create breakthroughs in our ability to construct increasingly sophisticated RNA genetic circuitry. PMID:23761434

  2. Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes.

    PubMed

    Chiu, Shih-Jiuan; Liu, Shujun; Perrotti, Danilo; Marcucci, Guido; Lee, Robert J

    2006-05-15

    A novel transferrin receptor (TfR)-targeted liposomal formulation was synthesized and evaluated for the delivery of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) (G3139, oblimerson sodium, or Genasense) to Bcl-2 in K562 leukemia cells. Liposomes composed of DC-Chol/egg PC/PEG-DSPE (25:73.5:1.5, mol/mol/mol) were loaded with G3139 with high efficiency (70-80%). To prepare targeted liposomes, transferrin was first coupled to PEG-DSPE and then incorporated into the bilayer by post-insertion. The liposomes had a mean diameter of 100 to 150 nm and exhibited colloidal stability for up to 8 weeks. Uptake of Tf-conjugated G3139-containing liposomes in TfR positive K562 cells was found to be more efficient than that of the non-targeted control formulation and could be blocked by excess free Tf. Treatment with Tf-conjugated liposomes resulted in Bcl-2 protein downregulation in K562 cells that was approximately 2-fold greater than with non-targeted liposomes (p<0.05) and 10-fold greater than with free G3139. Treatment with 2 microM G3139 in Tf-conjugated liposomes resulted in >80% reduction in Bcl-2 transcript. In addition, Tf-conjugated liposomal G3139-sensitized K562 cells to daunorubicin, lowering IC50 from 1.8 microM to 0.18 microM. In conclusion, Tf-conjugated liposomes are effective delivery vehicles for G3139 antisense oligos in TfR positive K562 cells and warrant further investigation as an in vivo oligo delivery vehicle. PMID:16564596

  3. Role of CTCF protein in regulating FMR1 locus transcription.

    PubMed

    Lanni, Stella; Goracci, Martina; Borrelli, Loredana; Mancano, Giorgia; Chiurazzi, Pietro; Moscato, Umberto; Ferrè, Fabrizio; Helmer-Citterich, Manuela; Tabolacci, Elisabetta; Neri, Giovanni

    2013-01-01

    Fragile X syndrome (FXS), the leading cause of inherited intellectual disability, is caused by epigenetic silencing of the FMR1 gene, through expansion and methylation of a CGG triplet repeat (methylated full mutation). An antisense transcript (FMR1-AS1), starting from both promoter and intron 2 of the FMR1 gene, was demonstrated in transcriptionally active alleles, but not in silent FXS alleles. Moreover, a DNA methylation boundary, which is lost in FXS, was recently identified upstream of the FMR1 gene. Several nuclear proteins bind to this region, like the insulator protein CTCF. Here we demonstrate for the first time that rare unmethylated full mutation (UFM) alleles present the same boundary described in wild type (WT) alleles and that CTCF binds to this region, as well as to the FMR1 gene promoter, exon 1 and intron 2 binding sites. Contrariwise, DNA methylation prevents CTCF binding to FXS alleles. Drug-induced CpGs demethylation does not restore this binding. CTCF knock-down experiments clearly established that CTCF does not act as insulator at the active FMR1 locus, despite the presence of a CGG expansion. CTCF depletion induces heterochromatinic histone configuration of the FMR1 locus and results in reduction of FMR1 transcription, which however is not accompanied by spreading of DNA methylation towards the FMR1 promoter. CTCF depletion is also associated with FMR1-AS1 mRNA reduction. Antisense RNA, like sense transcript, is upregulated in UFM and absent in FXS cells and its splicing is correlated to that of the FMR1-mRNA. We conclude that CTCF has a complex role in regulating FMR1 expression, probably through the organization of chromatin loops between sense/antisense transcriptional regulatory regions, as suggested by bioinformatics analysis. PMID:23874213

  4. The reduction in small ribosomal subunit abundance in ethanol-stressed cells of Bacillus subtilis is mediated by a SigB-dependent antisense RNA.

    PubMed

    Mars, Ruben A T; Mendonça, Karoline; Denham, Emma L; van Dijl, Jan Maarten

    2015-10-01

    One of the best-characterized general stress responses in bacteria is the σB-mediated stress response of the Gram-positive soil bacterium Bacillus subtilis. The σB regulon contains approximately 200 protein-encoding genes and 136 putative regulatory RNAs. One of these σB-dependent RNAs, named S1136-S1134, was recently mapped as being transcribed from the S1136 promoter on the opposite strand of the essential rpsD gene, which encodes the ribosomal primary-binding protein S4. Accordingly, S1136-S1134 transcription results in an rpsD-overlapping antisense RNA (asRNA). Upon exposure of B. subtilis to ethanol, the S1136 promoter was found to be induced, while rpsD transcription was downregulated. By quantitative PCR, we show that the activation of transcription from the S1136 promoter is directly responsible for the downregulation of rpsD upon ethanol exposure. We also show that this downregulation of rpsD leads to a reduced level of the small (30S) ribosomal subunit upon ethanol stress. The activation of the S1136 promoter thus represents the first example of antisense transcription-mediated regulation in the general stress response of B. subtilis and implicates the reduction of ribosomal protein abundance as a new aspect in the σB-dependent stress response. We propose that the observed reduction in the level of the small ribosomal subunit, which contains the ribosome-decoding center, may protect B. subtilis cells against misreading and spurious translation of possibly toxic aberrant peptides under conditions of ethanol stress. PMID:26115952

  5. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    PubMed

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients. PMID:20030230

  6. Targeting TGF-β Signaling by Antisense Oligonucleotide-mediated Knockdown of TGF-β Type I Receptor

    PubMed Central

    Kemaladewi, Dwi U; Pasteuning, Svitlana; van der Meulen, Joke W; van Heiningen, Sandra H; van Ommen, Gert-Jan; ten Dijke, Peter; Aartsma-Rus, Annemieke; ‘t Hoen, Peter AC; Hoogaars, Willem M

    2014-01-01

    Duchenne muscular dystrophy (DMD) is caused by lack of functional dystrophin and results in progressive myofiber damage and degeneration. In addition, impaired muscle regeneration and fibrosis contribute to the progressive pathology of DMD. Importantly, transforming growth factor-β (TGF-β) is implicated in DMD pathology and is known to stimulate fibrosis and inhibit muscle regeneration. In this study, we present a new strategy to target TGF-β signaling cascades by specifically inhibiting the expression of TGF-β type I receptor TGFBR1 (ALK5). Antisense oligonucleotides (AONs) were designed to specifically induce exon skipping of mouse ALK5 transcripts. AON-induced exon skipping of ALK5 resulted in specific downregulation of full-length receptor transcripts in vitro in different cell types, repression of TGF-β activity, and enhanced C2C12 myoblast differentiation. To determine the effect of these AONs in dystrophic muscles, we performed intramuscular injections of ALK5 AONs in mdx mice, which resulted in a decrease in expression of fibrosis-related genes and upregulation of Myog expression compared to control AON-injected muscles. In summary, our study presents a novel method to target TGF-β signaling cascades with potential beneficial effects for DMD. PMID:24691207

  7. Activating the synthesis of progerin, the mutant prelamin A in Hutchinson-Gilford progeria syndrome, with antisense oligonucleotides.

    PubMed

    Fong, Loren G; Vickers, Timothy A; Farber, Emily A; Choi, Christine; Yun, Ui Jeong; Hu, Yan; Yang, Shao H; Coffinier, Catherine; Lee, Roger; Yin, Liya; Davies, Brandon S J; Andres, Douglas A; Spielmann, H Peter; Bennett, C Frank; Young, Stephen G

    2009-07-01

    Hutchinson-Gilford progeria syndrome (HGPS) is caused by point mutations that increase utilization of an alternate splice donor site in exon 11 of LMNA (the gene encoding lamin C and prelamin A). The alternate splicing reduces transcripts for wild-type prelamin A and increases transcripts for a truncated prelamin A (progerin). Here, we show that antisense oligonucleotides (ASOs) against exon 11 sequences downstream from the exon 11 splice donor site promote alternate splicing in both wild-type and HGPS fibroblasts, increasing the synthesis of progerin. Indeed, wild-type fibroblasts transfected with these ASOs exhibit progerin levels similar to (or greater than) those in fibroblasts from HGPS patients. This progerin was farnesylated, as judged by metabolic labeling studies. The synthesis of progerin in wild-type fibroblasts was accompanied by the same nuclear shape and gene-expression perturbations observed in HGPS fibroblasts. An ASO corresponding to the 5' portion of intron 11 also promoted alternate splicing. In contrast, an ASO against exon 11 sequences 5' to the alternate splice site reduced alternate splicing in HGPS cells and modestly lowered progerin levels. Thus, different ASOs can be used to increase or decrease 'HGPS splicing'. ASOs represent a new and powerful tool for recreating HGPS pathophysiology in wild-type cells. PMID:19376814

  8. Bioinformatic and functional optimization of antisense phosphorodiamidate morpholino oligomers (PMOs) for therapeutic modulation of RNA splicing in muscle.

    PubMed

    Popplewell, Linda J; Graham, Ian R; Malerba, Alberto; Dickson, George

    2011-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted, Becker muscular dystrophy (BMD)-like, but functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript so that gene expression is restored. AO-induced exon skipping producing functional truncated dystrophin exon has been demonstrated in animal models of DMD both in vitro and in vivo, and in DMD patient cells in vitro in culture, and in DMD muscle explants. More recently, AO-mediated exon skipping has been confirmed in DMD patients in Phase I clinical trials. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, and in particular phosphorodiamidate morpholino oligomers (PMOs), for the targeted skipping of specific exons on the DMD gene. PMID:21194027

  9. Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm

    PubMed Central

    Doran, Philip; Wilton, Steve D.; Fletcher, Sue; Ohlendieck, Kay

    2009-01-01

    The disintegration of the dystrophin-glycoprotein complex represents the initial pathobiochemical insult in Duchenne muscular dystrophy. However, secondary changes in signalling, energy metabolism and ion homeostasis are probably the main factors that eventually cause progressive muscle wasting. Thus, for the proper evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscles. Antisense oligomer-mediated exon skipping promises functional restoration of the primary deficiency in dystrophin. In this study, an established phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide was employed for the specific removal of exon 23 in the mutated mouse dystrophin gene transcript. Using DIGE analysis, we could show the reversal of secondary pathobiochemical abnormalities in the dystrophic diaphragm following exon-23 skipping. In analogy to the restoration of dystrophin, β-dystroglycan and neuronal nitric oxide synthase, the muscular dystrophy-associated differential expression of calsequestrin, adenylate kinase, aldolase, mitochondrial creatine kinase and cvHsp was reversed in treated muscle fibres. Hence, the re-establishment of Dp427 coded by the transcript missing exon 23 has counter-acted dystrophic alterations in Ca2+-handling, nucleotide metabolism, bioenergetic pathways and cellular stress response. This clearly establishes the exon-skipping approach as a realistic treatment strategy for diminishing diverse downstream alterations in dystrophinopathy. PMID:19132684

  10. Isolation and characterization of a cDNA encoding granule-bound starch synthase in cassava (Manihot esculenta Crantz) and its antisense expression in potato.

    PubMed

    Salehuzzaman, S N; Jacobsen, E; Visser, R G

    1993-12-01

    A tuber-specific cDNA library of cassava (Manihot esculenta Crantz) was constructed and a full-length cDNA for granule-bound starch synthase (GBSS, also known as waxy protein), the enzyme responsible for the synthesis of amylose in reserve starch, was cloned. Sequencing of the cloned cDNA showed that it has 74% identity with potato GBSS and 60-72% identity with GBSS from other plant species. The cDNA encodes a 608 amino acid protein of which 78 amino acids form a chloroplast/amyloplast transit peptide of 8.37 kDa. The mature protein has a predicted molecular mass of 58.61 kDa (530 amino acids). Comparison of the GBSS proteins of various plant species and glycogen synthase of bacteria showed extensive identity among the mature form of plant GBSS proteins, in which the monocots and dicots form two separate branches in the evolutionary tree. From analysis of the genomic DNA of allotetraploid cassava, it is shown that GBSS is a low-copy-number gene. GBSS transcript is synthesized in a number of different organs, but most abundantly in tubers. Potato plants were transformed with the cassava GBSS cDNA in antisense orientation fused between the potato GBSS promoter and the nopaline synthase terminator. The expression of the endogenous GBSS gene in these transgenic potato plants was partially or completely inhibited. Complete inhibition of GBSS activity by the cassava antisense gene resulted in absence of GBSS protein and amylose giving rise to almost complete amylose-free potato starch. This shows that also heterologous genes can be used to achieve antisense effects in other plant species. PMID:8260633

  11. Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime.

    PubMed

    Readman, John B; Dickson, George; Coldham, Nick G

    2016-01-01

    Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and -17 to -5, respectively) close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a bla CTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of bla CTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates. PMID:27047482

  12. Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime

    PubMed Central

    Readman, John B.; Dickson, George; Coldham, Nick G.

    2016-01-01

    Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson-Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25-mer phosphorodiamidate morpholino oligomer (PMO) and a 13-mer polyamide (peptide) nucleic acid (PNA) were designed to target mRNA (positions -4 to +21, and –17 to –5, respectively) close to the translational initiation site of the extended-spectrum β-lactamase resistance genes of CTX-M group 1. These antisense oligonucleotides were found to inhibit β-lactamase activity by up to 96% in a cell-free translation-transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up-regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime (CTX) in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0-40 nM). The PMO and PNA were covalently bound to the cell penetrating peptide (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates. PMID:27047482

  13. Oligodeoxynucleotide studies in primates: antisense and immune stimulatory indications.

    PubMed

    Farman, Cindy A; Kornbrust, Doug J

    2003-01-01

    Antisense oligodeoxynucleotide compounds (AS ODN) are being developed as therapeutics for various disease indications. Their safety and pharmacokinetics are most commonly evaluated in rodents and nonhuman primates. Traditional AS ODN are short, single strands of DNA, and they target specific mRNA sequences. Plasma clearance of AS ODN is rapid, broad tissue distribution occurs, and elimination is by nuclease metabolism. Structural modifications to AS ODN have been made to enhance their efficacy and improve their safety. A number of class effects are observed with AS ODN that are unrelated to the specific targeted mRNA sequence. Acute effects include activation of the alternative complement pathway and inhibition of the intrinsic coagulation pathway. In monkeys, rodents, and dogs given repeated doses of AS ODN, accumulation of AS ODN and/or metabolites occurs in the form of basophilic granules in various tissues, including the kidney, lymph nodes and liver. A new potential therapeutic application of ODN is that of immune stimulation. Immunostimulatory ODN (IS ODN) are being investigated for use in treating cancer, infectious disease, and allergy. For the development of both AS and IS ODN, primates will continue to be important for safety assessment. PMID:12597439

  14. Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

    PubMed Central

    Nishina, Tomoko; Numata, Junna; Nishina, Kazutaka; Yoshida-Tanaka, Kie; Nitta, Keiko; Piao, Wenying; Iwata, Rintaro; Ito, Shingo; Kuwahara, Hiroya; Wada, Takeshi; Mizusawa, Hidehiro; Yokota, Takanori

    2015-01-01

    We developed an efficient system for delivering short interfering RNA (siRNA) to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid)-DNA gapmer antisense oligonucleotide (ASO) was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA) as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS) molecules are bound to ASO with UNA sequences. PMID:25584900

  15. Annexin A2 facilitates endocytic trafficking of antisense oligonucleotides

    PubMed Central

    Wang, Shiyu; Sun, Hong; Tanowitz, Michael; Liang, Xue-hai; Crooke, Stanley T.

    2016-01-01

    Chemically modified antisense oligonucleotides (ASOs) designed to mediate site-specific cleavage of RNA by RNase H1 are used as research tools and as therapeutics. ASOs modified with phosphorothioate (PS) linkages enter cells via endocytotic pathways. The mechanisms by which PS-ASOs are released from membrane-enclosed endocytotic organelles to reach target RNAs remain largely unknown. We recently found that annexin A2 (ANXA2) co-localizes with PS-ASOs in late endosomes (LEs) and enhances ASO activity. Here, we show that co-localization of ANXA2 with PS-ASO is not dependent on their direct interactions or mediated by ANXA2 partner protein S100A10. Instead, ANXA2 accompanies the transport of PS-ASOs to LEs, as ANXA2/PS-ASO co-localization was observed inside LEs. Although ANXA2 appears not to affect levels of PS-ASO internalization, ANXA2 reduction caused significant accumulation of ASOs in early endosomes (EEs) and reduced localization in LEs and decreased PS-ASO activity. Importantly, the kinetics of PS-ASO activity upon free uptake show that target mRNA reduction occurs at least 4 hrs after PS-ASOs exit from EEs and is coincident with release from LEs. Taken together, our results indicate that ANXA2 facilitates PS-ASO trafficking from early to late endosomes where it may also contribute to PS-ASO release. PMID:27378781

  16. Altering behavioral responses and dopamine transporter protein with antisense peptide nucleic acids.

    PubMed

    Tyler-McMahon, B M; Stewart, J A; Jackson, J; Bitner, M D; Fauq, A; McCormick, D J; Richelson, E

    2001-10-01

    The dopamine transporter (DAT) plays a role in locomotion and is an obligatory target for amphetamines. We designed and synthesized an antisense peptide nucleic acid (PNA) to rat DAT to examine the effect of this antisense molecule on locomotion and on responsiveness to amphetamines. Rats were injected intraperitoneally daily for 9 days with either saline, an antisense DAT PNA, a scrambled DAT PNA, or a mismatch DAT PNA. On days 7 and 9 after initial motility measurements were taken, the animals were challenged with 10 mg/kg of amphetamine and scored for motility. On day 7, there was no significant difference between the baseline levels of activity of any of the groups or their responses to amphetamine. On day 9, the antisense PNA-treated rats showed a statistically significant increase in their resting motility (P < 0.01). When these rats were challenged with amphetamine, motility of the saline-, scrambled PNA-, and mismatch PNA-treated animals showed increases of 31-, 36-, and 20-fold, respectively, while the antisense PNA-treated animals showed increases of only 3.4-fold (P < 0.01). ELISA results revealed a 32% decrease in striatal DAT in antisense PNA-treated rats compared with the saline, scrambled PNA, and mismatch PNA controls (P < 0.001). These results extend our previous findings that brain proteins can be knocked down in a specific manner by antisense molecules administered extracranially. Additionally, these results suggest some novel approaches for the treatment of diseases dependent upon the function of the dopamine transporter. PMID:11543728

  17. Analysis of the Mechanism of Action of the Antisense RNA That Controls the Replication of the repABC Plasmid p42d ▿ †

    PubMed Central

    Cervantes-Rivera, Ramón; Romero-López, Cristina; Berzal-Herranz, Alfredo; Cevallos, Miguel A.

    2010-01-01

    Replication and segregation of the Rhizobium etli symbiotic plasmid (pRetCFN42d) depend on the presence of a repABC operon, which carries all the plasmid-encoded elements required for these functions. All repABC operons share three protein-encoding genes (repA, repB, and repC), an antisense RNA (ctRNA) coding gene, and at least one centromere-like region (parS). The products of repA and repB, in conjunction with the parS region, make up the segregation system, and they negatively regulate operon transcription. The last gene of the operon, repC, encodes the initiator protein. The ctRNA is a negative posttranscriptional regulator of repC. In this work, we analyzed the secondary structures of the ctRNA and its target and mapped the motifs involved in the complex formed between them. Essential residues for the effective interaction localize at the unpaired 5′ end of the antisense molecule and the loop of the target mRNA. In light of our results, we propose a model explaining the mechanism of action of this ctRNA in the regulation of plasmid replication in R. etli. PMID:20435728

  18. CREB antisense oligodeoxynucleotide administration into the dorsal hippocampal CA3 region impairs long- but not short-term spatial memory in mice

    PubMed Central

    Florian, Cédrick; Mons, Nicole; Roullet, Pascal

    2006-01-01

    The transcription factor cAMP response-element binding protein (CREB) has a pivotal role in hippocampal synaptic plasticity and hippocampus-dependent long-term memory. We recently demonstrated that the dorsal hippocampal CA3 region is involved in memory consolidation of spatial information tested on a Morris water maze in mice. To test whether activation of CREB in the CA3 region is required for memory consolidation of spatial information, bilaterally cannulated mice were infused 18 h before the beginning of the behavioral training with antisense or control sense CREB oligodeoxynucleotides (ODNs) or buffer. Mice were then subjected to massed training in a spatial version of the water maze and tested for retention 0 or 24 h after the last training session. We showed that CREB antisense ODN-infusion in the CA3 region impaired long-term memory when tested 24 h later but had no effect on spatial acquisition or short-term memory tested immediately after behavioral training. These findings provide evidence that the regionally restricted activation of CREB in the dorsal hippocampal CA3 region is critical for the long-term memory consolidation phase of spatial learning but not for short-term memory. PMID:16882863

  19. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown.

    PubMed

    Fakhoury, Johans J; Edwardson, Thomas G; Conway, Justin W; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S; Sleiman, Hanadi F

    2015-12-28

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into 'gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing. PMID:26597764

  20. Development of Antisense Therapeutic and Imaging Agents to Detect and Suppress Inducible Nitric Oxide Synthase (iNOS) Expression in Acute Lung Injury (ALI)

    NASA Astrophysics Data System (ADS)

    Shen, Yuefei

    This dissertation focuses on the development and investigation of antisense imaging and therapeutic agents, combined with nanotechnology, to detect and suppress inducible nitric oxide synthase (iNOS) expression for the diagnosis and treatment of acute lung injury (ALI). To achieve this goal, several efforts were made. The first effort was the identification and characterization of high binding affinity antisense peptide nucleic acids (PNAs) and shell-crosslinked knedel-like nanoparticle (SCK)-PNA conjugates to the iNOS mRNA. Antisense binding sites on the iNOS mRNA were first mapped by a procedure for rapidly generating a library of antisense accessible sites on native mRNAs (MASL) which involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific PCR. Antisense PNAs against the antisense accessible sites were accordingly synthesized and characterized. The second effort was the investigation of cationic shell crosslinked knedel-like nanoparticle (cSCK)-mediated siRNA delivery to suppress iNOS expression for the treatment of ALI. siRNA with its unique gene-specific properties could serve as a promising therapeutic agent, however success in this area has been challenged by a lack of efficient biocompatible transfection agents. cSCK with its nanometer size and positive charge previously showed efficient cellular delivery of phosphorothioate ODNs (oligodeoxynucleotides), plasmid DNA and PNA. Herein, cSCK showed good siRNA binding and facilitated efficient siRNA transfection in HeLa, a mouse macrophage cell line and other human cell lines. cSCK led to greater silencing efficiency than Lipofectamine 2000 in HeLa cells as determined by the viability following transfection with cytotoxic and non-cytotoxic siRNAs, as well in 293T and HEK cells, and was comparable in BEAS-2B and MCF10a cells. The third effort was the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabeled

  1. Use of Recombinase-Based In Vivo Expression Technology To Characterize Enterococcus faecalis Gene Expression during Infection Identifies In Vivo-Expressed Antisense RNAs and Implicates the Protease Eep in Pathogenesis

    PubMed Central

    Barnes, Aaron M. T.; Grindle, Suzanne M.; Manias, Dawn A.; Schlievert, Patrick M.; Dunny, Gary M.

    2012-01-01

    Enterococcus faecalis is a member of the mammalian gastrointestinal microflora that has become a leading cause of nosocomial infections over the past several decades. E. faecalis must be able to adapt its physiology based on its surroundings in order to thrive in a mammalian host as both a commensal and a pathogen. We employed recombinase-based in vivo expression technology (RIVET) to identify promoters on the E. faecalis OG1RF chromosome that were specifically activated during the course of infection in a rabbit subdermal abscess model. The RIVET screen identified 249 putative in vivo-activated loci, over one-third of which are predicted to generate antisense transcripts. Three predicted antisense transcripts were detected in in vitro- and in vivo-grown cells, providing the first evidence of in vivo-expressed antisense RNAs in E. faecalis. Deletions in the in vivo-activated genes that encode glutamate 5-kinase (proB [EF0038]), the transcriptional regulator EbrA (ebrA [EF1809]), and the membrane metalloprotease Eep (eep [EF2380]) did not hinder biofilm formation in in vitro assays. In a rabbit model of endocarditis, the ΔebrA strain was fully virulent, the ΔproB strain was slightly attenuated, and the Δeep strain was severely attenuated. The Δeep virulence defect could be complemented by the expression of the wild-type gene in trans. Microscopic analysis of early Δeep biofilms revealed an abundance of small cellular aggregates that were not observed in wild-type biofilms. This work illustrates the use of a RIVET screen to provide information about the temporal activation of genes during infection, resulting in the identification and confirmation of a new virulence determinant in an important pathogen. PMID:22144481

  2. Antisense overlapping open reading frames in genes from bacteria to humans.

    PubMed Central

    Merino, E; Balbás, P; Puente, J L; Bolívar, F

    1994-01-01

    Long Open Reading Frames (ORFs) in antisense DNA strands have been reported in the literature as being rare events. However, an extensive analysis of the GenBank database revealed that a substantial number of genes from several species contain an in-phase ORF in the antisense strand, that overlaps entirely the coding sequence of the sense strand, or even extends beyond. The findings described in this paper show that this is a frequent, non-random phenomenon, which is primarily dependent on codon usage, and to a lesser extent on gene size and GC content. Examination of the sequence database for several prokaryotic and eukaryotic organisms, demonstrates that coding sequences with in-phase, 100% overlapping antisense ORFs are present in every genome studied so far. PMID:8208617

  3. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  4. Gold nanoparticle mediated laser transfection for high-throughput antisense applications

    NASA Astrophysics Data System (ADS)

    Kalies, S.; Heinemann, D.; Schomaker, M.; Birr, T.; Ripken, T.; Meyer, H.

    2013-06-01

    The delivery of antisense structures, like siRNA, is beneficial for new therapeutic approaches in regenerative sciences. Optical transfection techniques enable high spatial control combined with minimal invasive treatment of cells due to the use of short laser pulses. However, single cell laser transfection by a tightly focused laser beam, for example femtosecond laser transfection, has the major drawback of low throughput. Compared to this, high-throughput in laser transfection is possible by applying gold nanoparticles irradiated by a weakly focused laser beam scanning over the cell sample. Herein, we show the delivery of antisense molecules and demonstrate the minimal cytotoxicity of a method called gold nanoparticle mediated (GNOME) laser transfection. A 532 nm microchip laser in conjugation with 200 nm gold nanoparticles at a concentration of 0.5 μg/cm2 is used. In addition to antisense molecules, the uptake of dextrans of several sizes is analyzed.

  5. Antisense inhibition of myoD expression in regenerating rat soleus muscle is followed by an increase in the mRNA levels of myoD, myf-5 and myogenin and by a retarded regeneration.

    PubMed

    Zádor, Erno; Bottka, Sándor; Wuytack, Frank

    2002-06-12

    It has been reported that muscles of myoD-/- mice present a lower potential to regenerate, but there are no reports on the effect of acute interference with myoD expression limited in space and time to only a particular regenerating muscle. Here we relied on antisense inhibition of this factor. Four different oligos were tested. The suppression of regeneration indices (the expression of desmin, the formation of myotubes and the initiation of endplates) was the most pronounced, with the oligomer targeting a region encompassing the translation start site of myoD. A mixed backbone phosphorothioate-phosphate diester oligo (200 microl at 20 microM) was still detectable in the muscles 1 h after its administration and reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the level of the targeted 5' end of the myoD mRNA was selectively decreased. The level of myoD protein was also lowered. Four hours after the antisense treatment, when the oligos were no longer detectable, the myoD mRNA level was restored and 24 h later it exceeded controls together with that of myf-5 and myogenin. After 4 weeks, the antisense-treated soleus muscles were similar to the control-treated and the untreated regenerated soleus with respect to fiber types and motor endplates, however, they contained smaller fibers which reflected the asynchronity of regeneration. This shows that successfully targeted simple antisense oligonucleotides can be used as selective tools for inhibition of individual factors in studying the process of muscle regeneration. PMID:12063168

  6. The 5′-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB

    PubMed Central

    López-Aguilar, Celeste; Romero-López, Cristina; Espinosa, Manuel; Berzal-Herranz, Alfredo; del Solar, Gloria

    2015-01-01

    Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5′-tail and an 8-nt-long 3′-terminal U-rich stretch. Here, the 3′ and 5′ regions of the 5′-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5′-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a “kissing” interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex. PMID:26175752

  7. A Compendium of Nucleosome and Transcript Profiles Reveals Determinants of Chromatin Architecture and Transcription

    PubMed Central

    van Bakel, Harm; Tsui, Kyle; Gebbia, Marinella; Mnaimneh, Sanie; Hughes, Timothy R.; Nislow, Corey

    2013-01-01

    Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology, and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors, including the CAF-1 complex, Spt10, and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning, and these data provide a valuable resource for future studies. PMID:23658529

  8. Cytoplasmic Control of Sense-Antisense mRNA Pairs.

    PubMed

    Sinturel, Flore; Navickas, Albertas; Wery, Maxime; Descrimes, Marc; Morillon, Antonin; Torchet, Claire; Benard, Lionel

    2015-09-22

    Transcriptome analyses have revealed that convergent gene transcription can produce many 3'-overlapping mRNAs in diverse organisms. Few studies have examined the fate of 3'-complementary mRNAs in double-stranded RNA-dependent nuclear phenomena, and nothing is known about the cytoplasmic destiny of 3'-overlapping messengers or their impact on gene expression. Here, we demonstrate that the complementary tails of 3'-overlapping mRNAs can interact in the cytoplasm and promote post-transcriptional regulatory events including no-go decay (NGD) in Saccharomyces cerevisiae. Genome-wide experiments confirm that these messenger-interacting mRNAs (mimRNAs) form RNA duplexes in wild-type cells and thus have potential roles in modulating the mRNA levels of their convergent gene pattern under different growth conditions. We show that the post-transcriptional fate of hundreds of mimRNAs is controlled by Xrn1, revealing the extent to which this conserved 5'-3' cytoplasmic exoribonuclease plays an unexpected but key role in the post-transcriptional control of convergent gene expression. PMID:26344770

  9. Nascent transcription affected by RNA polymerase IV in Zea mays.

    PubMed

    Erhard, Karl F; Talbot, Joy-El R B; Deans, Natalie C; McClish, Allison E; Hollick, Jay B

    2015-04-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3'-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306

  10. Nascent Transcription Affected by RNA Polymerase IV in Zea mays

    PubMed Central

    Erhard, Karl F.; Talbot, Joy-El R. B.; Deans, Natalie C.; McClish, Allison E.; Hollick, Jay B.

    2015-01-01

    All eukaryotes use three DNA-dependent RNA polymerases (RNAPs) to create cellular RNAs from DNA templates. Plants have additional RNAPs related to Pol II, but their evolutionary role(s) remain largely unknown. Zea mays (maize) RNA polymerase D1 (RPD1), the largest subunit of RNA polymerase IV (Pol IV), is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs), and transcriptional regulation of specific alleles. Here, we define the nascent transcriptomes of rpd1 mutant and wild-type (WT) seedlings using global run-on sequencing (GRO-seq) to identify the broader targets of RPD1-based regulation. Comparisons of WT and rpd1 mutant GRO-seq profiles indicate that Pol IV globally affects transcription at both transcriptional start sites and immediately downstream of polyadenylation addition sites. We found no evidence of divergent transcription from gene promoters as seen in mammalian GRO-seq profiles. Statistical comparisons identify genes and TEs whose transcription is affected by RPD1. Most examples of significant increases in genic antisense transcription appear to be initiated by 3ʹ-proximal long terminal repeat retrotransposons. These results indicate that maize Pol IV specifies Pol II-based transcriptional regulation for specific regions of the maize genome including genes having developmental significance. PMID:25653306