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E. Gerhart H. Wagner - One of the best experts on this subject based on the ideXlab platform.
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Kill the messenger: bacterial Antisense RNA promotes mRNA decay
Nature structural & molecular biology, 2009Co-Authors: E. Gerhart H. WagnerAbstract:Bacterial Antisense RNAs target translation initiation regions (TIR s) to compete with ribosome binding, thus repressing translation and—secondarily—causing degradation of the naked mRNA. A new study reports on an Antisense RNA that directly accelerates mRNA decay by targeting a sequence deep within the coding region, far downstream of the TIR.
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An Antisense RNA inhibits translation by competing with standby ribosomes.
Molecular cell, 2007Co-Authors: Fabien Darfeuille, Cecilia Unoson, Jörg Vogel, E. Gerhart H. WagnerAbstract:Most Antisense RNAs in bacteria inhibit translation by competing with ribosomes for translation initiation regions (TIRs) on nascent mRNA. We propose a mechanism by which an Antisense RNA inhibits ...
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An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions in Escherichia coli
Journal of bacteriology, 2002Co-Authors: Sabine Brantl, E. Gerhart H. WagnerAbstract:Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the Antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate Antisense RNA, as expected for this mechanism. Similarly, both Antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate Antisense RNAs had no effect. Thus, this report shows that Antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed. Regulation of gene expression in bacteria is carried out by a number of different mechanisms. Transcriptional control works primarily through repressor or activator proteins as in regulation of metabolic processes (25). Posttranscriptional control is exerted at the mRNA level, either by proteins or RNAs. Many accessory genetic elements use Antisense RNAs for regulation. Antisense RNAs act on their complementary
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Antisense RNA-mediated transcriptional attenuation: an in vitro study of plasmid pT181.
Molecular microbiology, 2002Co-Authors: Sabine Brantl, E. Gerhart H. WagnerAbstract:Summary Antisense RNAs regulate plasmid replication by several different mechanisms. One of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pT181, and later for the streptococcal plasmids pIP501 and pAMb1. Previously, we performed detailed in vitro and in vivo analyses of the pIP501 system. Here, we present an in vitro analysis of the Antisense system of plasmid pT181. The secondary structures of Antisense and sense RNA species of different lengths were determined. Binding rate constants for sense/Antisense RNA pairs were measured, and functional segments required for complex formation were determined. A single-round transcription assay was used for in vitro analysis of transcriptional attenuation. A comparison between pT181 and pIP501 revealed several differences; whereas a truncated derivative of pIP501 Antisense RNA was sufficient for stable complex formation, both stem‐loop structures of pT181-RNAI were required. In contrast to the sense RNA of pIP501, which showed an intrinsic propensity to terminate (30‐50% in the absence of Antisense RNA), the sense RNA of pT181 required Antisense RNA for induced termination. Rate constants of formation of pT181 sense‐Antisense RNA complexes were similar to inhibition rate constants, in striking contrast to pIP501, in which inhibition occurred at least 10-fold faster than stable binding.
Sabine Brantl - One of the best experts on this subject based on the ideXlab platform.
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Antisense RNA mediated transcriptional attenuation in plasmid pip501 the simultaneous interaction between two complementary loop pairs is required for efficient inhibition by the Antisense RNA
Microbiology, 2007Co-Authors: Nadja Heidrich, Sabine BrantlAbstract:Streptococcal plasmid pIP501 uses Antisense RNA-mediated transcriptional attenuation to regulate its replication. Previous in vitro assays suggested that binding intermediates between RNAII (sense RNA) and RNAIII (Antisense RNA) are sufficient for inhibition, and a U-turn structure on RNAII loop L1 was found to be crucial for the interaction with RNAIII. Here, sequence and structural requirements for an efficient RNAII–RNAIII interaction were investigated. A detailed probing of RNA secondary structure combined with in vitro single-round transcription assays indicated that complex formation between the two molecules progresses into the lower stems of both loop pairs of the sense and Antisense RNAs, but that the complex between RNAII and RNAIII is not a full duplex. Stem–loops L3 and L4 were required to be linked to one other for efficient contact with the complementary loops L2 and L1 of the sense RNA, indicating a simultaneous interaction between these two loop pairs. Thereby, the sequence and length of the spacer connecting L3 and L4 were shown not to be important for inhibition.
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An Antisense RNA-Mediated Transcriptional Attenuation Mechanism Functions in Escherichia coli
Journal of bacteriology, 2002Co-Authors: Sabine Brantl, E. Gerhart H. WagnerAbstract:Antisense RNA-mediated transcriptional attenuation is a regulatory mechanism operating in the replication control of two groups of plasmids in gram-positive bacteria, the pT181 group and the inc18 family, represented by pIP501. In contrast, this control mechanism has so far not been identified in gram-negative bacteria or their plasmids. In this work we asked whether such a mechanism can be supported by Escherichia coli. The core replication control regions of plasmids pT181 and pIP501 were transferred into this heterologous host. In vivo lacZ reporter gene assays showed that the Antisense RNAs of these plasmids can inhibit lacZ expression and that most of this effect can be accounted for by reduced mRNA readthrough. Northern analyses confirmed that the ratio of attenuated to readthrough target RNA was increased in the presence of the cognate Antisense RNA, as expected for this mechanism. Similarly, both Antisense RNAs induced premature termination of their cognate target RNAs in an E. coli in vitro transcription system, whereas the noncognate Antisense RNAs had no effect. Thus, this report shows that Antisense RNA-mediated transcriptional attenuation is supported by at least one gram-negative host, although the data indicate that inhibitory efficiencies are lower than those for, e.g., Bacillus subtilis. Possible explanations for the apparent absence of this control mode in plasmids of gram-negative bacteria are discussed. Regulation of gene expression in bacteria is carried out by a number of different mechanisms. Transcriptional control works primarily through repressor or activator proteins as in regulation of metabolic processes (25). Posttranscriptional control is exerted at the mRNA level, either by proteins or RNAs. Many accessory genetic elements use Antisense RNAs for regulation. Antisense RNAs act on their complementary
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Antisense-RNA regulation and RNA interference.
Biochimica et biophysica acta, 2002Co-Authors: Sabine BrantlAbstract:For a long time, RNA has been merely regarded as a molecule that can either function as a messenger (mRNA) or as part of the translational machinery (tRNA, rRNA). Meanwhile, it became clear that RNAs are versatile molecules that do not only play key roles in many important biological processes like splicing, editing, protein export and others, but can also--like enzymes--act catalytically. Two important aspects of RNA function--Antisense-RNA control and RNA interference (RNAi)--are emphasized in this review. Antisense-RNA control functions in all three kingdoms of life--although the majority of examples are known from bacteria. In contrast, RNAi, gene silencing triggered by double-stranded RNA, the oldest and most ubiquitous antiviral system, is exclusively found in eukaryotes. Our current knowledge about occurrence, biological roles and mechanisms of action of Antisense RNAs as well as the recent findings about involved genes/enzymes and the putative mechanism of RNAi are summarized. An interesting intersection between both regulatory mechanisms is briefly discussed.
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Antisense RNA-mediated transcriptional attenuation: an in vitro study of plasmid pT181.
Molecular microbiology, 2002Co-Authors: Sabine Brantl, E. Gerhart H. WagnerAbstract:Summary Antisense RNAs regulate plasmid replication by several different mechanisms. One of these mechanisms, transcriptional attenuation, was first described for the staphylococcal plasmid pT181, and later for the streptococcal plasmids pIP501 and pAMb1. Previously, we performed detailed in vitro and in vivo analyses of the pIP501 system. Here, we present an in vitro analysis of the Antisense system of plasmid pT181. The secondary structures of Antisense and sense RNA species of different lengths were determined. Binding rate constants for sense/Antisense RNA pairs were measured, and functional segments required for complex formation were determined. A single-round transcription assay was used for in vitro analysis of transcriptional attenuation. A comparison between pT181 and pIP501 revealed several differences; whereas a truncated derivative of pIP501 Antisense RNA was sufficient for stable complex formation, both stem‐loop structures of pT181-RNAI were required. In contrast to the sense RNA of pIP501, which showed an intrinsic propensity to terminate (30‐50% in the absence of Antisense RNA), the sense RNA of pT181 required Antisense RNA for induced termination. Rate constants of formation of pT181 sense‐Antisense RNA complexes were similar to inhibition rate constants, in striking contrast to pIP501, in which inhibition occurred at least 10-fold faster than stable binding.
Hans Peter Luther - One of the best experts on this subject based on the ideXlab platform.
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Cell type-specific expression of endogenous cardiac Troponin I Antisense RNA in the neonatal rat heart
Molecular and Cellular Biochemistry, 2009Co-Authors: Stefanie Voigtsberger, Holger Bartsch, Gert Baumann, Hans Peter LutherAbstract:Since the number of detected natural Antisense RNA is growing, investigations upon the expression pattern of the Antisense RNA become more important. As we focused our work on natural occurring Antisense transcripts in human and rat heart tissues, we were interested in the question, whether the expression pattern of Antisense and sense RNA can vary in different cell types of the same tissue. In our previous analysis of total neonatal rat heart tissue, we demonstrated the co-expression of both cTnI RNA species in this tissue. Now we investigated the expression of Antisense and sense RNA quantitatively in neonatal cardiomyocytes (NCMs) and neonatal cardiac fibroblasts (NCFs). Performing northern blot as well as RT-PCR, we could detect natural Antisense and sense RNA transcripts of cTnI in NCM and NCF implying that these transcripts are co-expressed in both cell types. The absolute amounts of the RNA transcripts were higher in NCM. Both RNA species showed identical sizes in the northern blot. Quantification by real-time PCR revealed a higher relative level of natural Antisense RNA in NCF compared to NCM which points out to a cell type-specific expression of sense and Antisense RNA. Our observations suggest that Antisense RNA transcription may contribute to a cell type-specific regulation of the cTnI gene.
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Role of endogenous Antisense RNA in cardiac gene regulation
Journal of Molecular Medicine, 2005Co-Authors: Hans Peter LutherAbstract:Endogenous Antisense RNA has been detected for a range of eukaryotic genes and now appears to be a common phenomenon in mammalian cells. Its abundance compared to levels of its complementary sense mRNA indicates that Antisense RNA may be involved in posttrancriptional regulation of a gene. In general a downregulating effect on gene expression has been demonstrated or suggested. Due to the heterogeneity in origin and character of different Antisense transcripts alteRNAtive functions such as stabilizing the corresponding sense transcript and being part of gene recombination must be considered. Regulation by endogenous Antisense RNA has been shown for a plethora of genes, including cardiac genes, such as myosin heavy chainMHC, atrial light chain, and troponin I. There is now growing evidence that Antisense transcription is involved in human disease, and it is reasonable to consider Antisense as a target for intervention procedures. Here we review the progress in our understanding of as well as the controversies arising from investigating the regulatory mechanisms of Antisense RNA, with special focus on cardiac genes. Finally, links between Antisense transcription and heart disease and the possible use of Antisense as a target of cardiac intervention procedures are discussed.
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Detection of a novel sense-Antisense RNA-hybrid structure by RACE experiments on endogenous troponin I Antisense RNA.
RNA (New York N.Y.), 2004Co-Authors: Holger Bartsch, Stefanie Voigtsberger, Gert Baumann, Ingo Morano, Hans Peter LutherAbstract:Conformational changes in the troponin/tropomyosin complex significantly alter the mechanical properties of cardiac muscle. Phosphorylation of cardiac troponin I, part of the troponin/tropomyosin complex, reduces calcium affinity, which leads to increased relaxation of cardiac muscle. Because cardiac troponin I plays a central role in tuning the heart to different work demands, detailed knowledge of troponin I protein regulation is required. Our group previously detected naturally occurring Antisense RNA for troponin I in human and rat hearts, and here, attempt to unravel the structure of rat cardiac troponin I Antisense RNA. We performed rapid amplification of cDNA ends (RACE) experiments and discovered Antisense sequences identical to a copy of the sense mRNA, which led us to conclude that the Antisense RNA must be transcribed from troponin I mRNA in the cytoplasm. Moreover, we isolated RNA structures comprising sense and Antisense sequences in one continuous molecule. As we found no homolog structures described in the literature, we called this "hybrid RNA." Because a duplex formation was demonstrated previously we concluded that hybrid RNA is a consequence of a tight interaction between sense and Antisense troponin I RNA in vivo, which we discuss in the article.
G. H. Rank - One of the best experts on this subject based on the ideXlab platform.
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Antisense RNA regulation of the ILV2 gene in yeast : a correction
Current genetics, 1994Co-Authors: Greg M. Arndt, Wei Xiao, G. H. RankAbstract:A previous report of the use of Antisense RNA to regulate gene expression in yeast is incorrect.
Yuzhi Wang - One of the best experts on this subject based on the ideXlab platform.
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Inhibition Growth of Multidrug Resistant KBV200Cells by MDR1 Antisense RNA
Biochemical and biophysical research communications, 1997Co-Authors: Yuzhi WangAbstract:Abstract Acquisition of resistance to multiple drugs of tumor cell caused by overexpression of the MDR1 gene is one of major obstacles in cancer chemotherapy. We have attempted to reverse the multidrug resistance (MDR) phenotype by treating vincristine (VCR) and adriamycin (ADM) resistant KBV200cells with MDR1 Antisense RNA. Retroviral vector expressing the Antisense RNA was transfected into KBV200. In the transfected cells, a stable expression of Antisense RNA and a reduction of cellular MDR1 mRNA could be detected by RT-PCR, and a reduction of MDR1 specific P-glycoprotein (P-gp) was also detected by Western blot, whereas an increase of the drug concentration in the cells was detected by FACS. The IC50of transfected cells to VCR and ADM was reduced by 65 and 47%. This study demonstrates that Antisense RNA can increase the sensitivity of tumor cells to anticancer drug by decreasing the expression of the MDR1 gene. This strategy may be applicable to cure cancer patients with P-gp mediated MDR phenotype.