RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum

The recent identification of antisense RNA in the transcriptomes of many eukaryotes has generated enormous interest. The presence of antisense RNA in Plasmodium falciparum, the causative agent of severe malaria, remains controversial. Elucidation of the mechanism of antisense RNA in P. falciparum synthesis is critical in order to demonstrate the origin and function of these transcripts. Therefore, a systematic analysis of antisense and sense RNA synthesis was performed using direct labeling experiments. Nuclear run on experiments with single-stranded DNA probes demonstrated that antisense RNA is synthesized in the nucleus at several genomic loci. Antisense RNA synthesis is sensitive to the potent RNA polymerase II inhibitor α-amanitin. Antisense and sense transcription was also detected in nuclei isolated from synchronized parasites, suggesting concurrent synthesis. In summary, our experiments directly demonstrate that antisense RNA synthesis is a common transcriptional phenomenon in P. falciparum, and is catalyzed by RNA polymerase II. Copyright © 2005 RNA Society

Translation of mRNA injected into Xenopus oocytes is specifically inhibited by antisense RNA.

The bacteriophage SP6 promoter and RNA polymerase were used to synthesize sense and antisense RNAs coding for the enzymes thymidine kinase (TK) and chloramphenicol acetyl transferase (CAT). Injection of antisense CAT RNA into frog oocytes inhibited expression of sense CAT mRNA. Similarly, antisense TK RNA inhibited expression of sense TK mRNA. Antisense RNAs were stable in oocytes and had no detectable effect on either the expression of endogenous proteins or on the expression of nonhomologous RNA transcripts. CAT activity expressed from a plasmid transcribed in the oocyte nucleus was also inhibited by antisense RNA injected into the oocyte cytoplasm. The data suggest that antisense RNA will be useful in identifying the function of specific mRNA sequences during early development of the frog

Cell type-specific expression of endogenous cardiac Troponin I antisense RNA in the neonatal rat heart

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 gen

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.Peer reviewe

SEQUENCING ANTISENSE TRANSCRIPTOME OF POPULUS THROUGH ANTISENSE/SENSE TRANSCRIPT PAIR ENRICHMENT

Past transcriptome research on plants focused primarily on protein coding genes, and only recently researchers began looking into the non-protein coding regions that may play significant roles in gene regulation. Antisense RNA transcripts that are found naturally in the cell share complementary sequence with sense transcripts and have been shown to regulate expression of their sense counterparts. Since antisense RNA has been largely under-studied and difficult to sequence because of their low relative abundance, new methods are needed to target antisense RNA for efficient genome-wide profiling. To address this gap in methods to efficiently and cost effectively enrich antisense RNA transcripts for sequencing, we developed methods that allow for the enrichment of antisense RNA through chemically guided annealing of sense/antisense transcript pairs of RNA and cDNA, and the separation of double stranded structures for library formation. These methods utilize guanidinium thiocyanate (GuSCN) to enrich antisense transcripts through the annealing of sense/antisense transcript pairs. Annealed RNA can then be separated through J2 monoclonal anti-dsRNA antibody binding, mRNA purification of enriched transcript pairs, and the custom tailing of sense/antisense transcript pair enriched libraries for Nanopore direct RNA sequencing. We also developed a novel method for the separation of annealed double stranded first strand cDNA using hydroxyapatite powder (HAP) chromatography to form antisense enriched Illumina libraries. The long-term goal of this project is for these methods to be used to form targeted antisense enriched libraries for the genome wide characterization of the antisense RNA response to drought stress in Populus

Antisense RNA transcripts in the blood may be novel diagnostic markers for colorectal cancer

Numerous genetic studies have been conducted regarding the occurrence of colorectal cancer (CRC) and the prognosis using microarrays. However, adequate investigations into the diagnostic application of microarrays have yet to be performed. The simplicity and accuracy of diagnosis and prognosis tracking are important requirements for its processes, and the use of blood cells for diagnosis is considered to be suitable to meet these requirements. The patients involved in the study were 28 preoperative patients with CRC and 6 healthy individuals who served as controls. RNA was extracted from the blood cells of the patients and analyzed using a sense/antisense RNA custom microarray. In the patients with CRC, the expression levels of 20 sense RNA and 20 antisense RNA species were identified as being significantly altered compared with that of the healthy volunteers (P2.0). Cluster analysis of these RNA species revealed that the top 10 antisense RNAs significantly clustered patients with cancer and healthy individuals separately. Patients with stage I or II CRC exhibited significant changes in the expression levels of 33 sense and 39 antisense RNA species, as compared with healthy volunteers (P2.0). Cluster analysis demonstrated that patients with stage I or II CRC and healthy volunteers formed separate clusters only among the top 20 antisense RNA species. A tracking study of expression levels of haloacid dehalogenase‑like hydrolase domain‑containing 1 (HDHD1) antisense RNA was performed and a significant difference was identified between the CRC and healthy groups revealing that the levels at one week and three months following surgical removal of the cancerous tissue, decreased to almost same levels of the healthy individuals. The results of the current study indicate that HDHD1 antisense RNA may serve as a potential biomarker for the prognosis of CRC

Antisense RNA based down-regulation of RNaseE in E.coli

BACKGROUND: Messenger RNA decay is an important mechanism for controlling gene expression in all organisms. The rate of the mRNA degradation directly affects the steady state concentration of mRNAs and therefore influences the protein synthesis. RNaseE has a key importance for the general mRNA decay in E.coli. While RNaseE initiates the degradation of most mRNAs in E.coli, it is likely that the enzyme is also responsible for the degradation of recombinant RNAs. As RNaseE is essential for cell viability and knockout mutants cannot be cultured, we investigated the possibility for a down-regulation of the intracellular level of RNaseE by antisense RNAs. During this study, an antisense RNA based approach could be established which revealed a strong reduction of the intracellular level of RNaseE in E.coli. RESULTS: Despite the autoregulation of rne mRNA by its gene product, significant antisense downregulation of RNaseE is possible. The expression of antisense RNAs did not effect the cell growth negatively. The amount of antisense RNA was monitored quantitatively by a fluorescence based sandwich hybridisation assay. Induction by anhydrotetracycline was followed by a 25-fold increase of the detectable antisense RNA molecules per cell. The antisense RNA level was maintained above 400 molecules per cell until the stationary phase, which caused the level of expressed antisense RNAs to decrease markedly. Western blot experiments revealed the strongest reduction in the RNaseE protein level 90 min after antisense RNA induction. The cellular level of RNaseE could be decreased to 35% of the wild type level. When the growth entered the stationary phase, the RNaseE level was maintained still at 50 to 60% of the wild type level. CONCLUSION: In difference to eukaryotic cells, where the RNAi technology is widely used, this technology is rather unexplored in bacteria, although different natural systems use antisense RNA-based silencing, and a few studies have earlier indicated the potential of this technology also in prokaryotes. Our results show that even complicated self-regulatory systems such as RNaseE may be controlled by antisense RNA technology, indicating that systems based on antisense RNA expression may have a potential for controlling detrimental factors with plasmid-based constructs in arbitrary strains while maintaining their beneficial characteristics. The study also proved that the RNA sandwich hybridisation technique is directly applicable to quantify small RNA molecules in crude cell extracts, which may have a broader application potential as a monitoring tool in RNA inhibition applications

An antisense RNA expression vector for Neurospora crassa

The artificial expression of antisense RNA is commonly used in eucaryotes, especially higher plants, to reduce the level of specific proteins (van der Krol et al. 1988 Nature 333:866-869). Here we report the use of antisense RNA to inhibit the translation of a subunit of the mitochondrial NADH:ubiquinone oxidoreductase, the respiratory complex I in N. crass

Novel applications of shotgun phage display

In a shotgun phage display library, theoretically, the entire proteome of a bacterium is represented. Phages displaying specific polypeptides can be isolated by affinity selection, while the corresponding gene remains physically linked to the gene product. The overall objective of the study in this thesis was to explore the shotgun phage display technique in new areas. Initially, it was used to study interactions between Staphylococcus aureus and an in vivo coated biomaterial. It was shown to be well suited for the identification of bacterial proteins that bind to ex vivo central venous catheters. Several known interactions were detected, but it was also found that β2-glycoprotein I (β2-GPI) is deposited on this type of biomaterial – a finding that is of interest both for the adherence of S. aureus, but perhaps also in view of the occurrence of autoantibodies in certain autoimmune diseases. Further, it is of interest to identify the subset of extracellular proteins in a bacterium since they are involved in important functions like pathogenesis and symbiosis. A method that allows for the rapid and general isolation of extracellular proteins is desirable, and may prove particularly useful when applied to bacteria for which the genome sequences are not known. For this purpose, a specialised phage display method was developed to isolate extracellular proteins by virtue of the presence of signal peptides (SS phage display). It was successfully applied to S. aureus and, on a larger scale, to the symbiotic bacterium Bradyrhizobium japonicum. In elaboration of the SS phage display method, an inducible antisense RNA system was incorporated to enable gene silencing of the isolated genes. A tetracycline-regulated promoter was inserted in such a way, that an antisense RNA covering the cloned gene could be expressed. The new element was shown to be compatible with the properties of SS phage display, and to promote gene expression upon induction on both the transcriptional and translational level. However, screening for clones affected by the induction of antisense RNA transcription was unsuccessful, and further developments of the system are required to improve the efficiency of this attractive application