Genome-wide view and characterization of natural antisense transcripts in Cannabis Sativa L.

Natural Antisense Transcripts (NATs) are a kind of complex regulatory RNAs that play crucial roles in gene expression and regulation. However, the NATs in Cannabis Sativa L., a widely economic and medicinal plant rich in cannabinoids remain unknown. In this study, we comprehensively predicted C. sativa NATs genome-wide using strand-specific RNA sequencing (ssRNA-Seq) data, and validated the expression profiles by strand-specific quantitative reverse transcription PCR (ssRT-qPCR). Consequently, a total of 307 NATs were predicted in C. sativa, including 104 cis- and 203 trans- NATs. Functional enrichment analysis demonstrated the potential involvement of the C. sativa NATs in DNA polymerase activity, RNA-DNA hybrid ribonuclease activity, and nucleic acid binding. Finally, 18 cis- and 376 trans- NAT-ST pairs were predicted to produce 621 cis- and 5,679 trans- small interfering RNA (nat-siRNAs), respectively. These nat-siRNAs were potentially involved in the biosynthesis of cannabinoids and cellulose. All these results will shed light on the regulation of NATs and nat-siRNAs in C. sativa.

Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi.

Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise while others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned.

Identification of transcribed sequences in Arabidopsis thaliana by using high-resolution genome tiling arrays.

Using a maskless photolithography method, we produced DNA oligonucleotide microarrays with probe sequences tiled throughout the genome of the plant Arabidopsis thaliana. RNA expression was determined for the complete nuclear, mitochondrial, and chloroplast genomes by tiling 5 million 36-mer probes. These probes were hybridized to labeled mRNA isolated from liquid grown T87 cells, an undifferentiated Arabidopsis cell culture line. Transcripts were detected from at least 60% of the nearly 26,330 annotated genes, which included 151 predicted genes that were not identified previously by a similar genome-wide hybridization study on four different cell lines. In comparison with previously published results with 25-mer tiling arrays produced by chromium masking-based photolithography technique, 36-mer oligonucleotide probes were found to be more useful in identifying intron-exon boundaries. Using two-dimensional HPLC tandem mass spectrometry, a small-scale proteomic analysis was performed with the same cells. A large amount of strongly hybridizing RNA was found in regions "antisense" to known genes. Similarity of antisense activities between the 25-mer and 36-mer data sets suggests that it is a reproducible and inherent property of the experiments. Transcription activities were also detected for many of the intergenic regions and the small RNAs, including tRNA, small nuclear RNA, small nucleolar RNA, and microRNA. Expression of tRNAs correlates with genome-wide amino acid usage.

A new reporter gene system suited for cell-free protein synthesis and high-throughput screening in small reaction volumes.

The properties of M-hirudin as a new reporter gene system were examined using rabbit reticulocyte lysate for cell-free protein expression. In contrast to the luciferase gene, in vitro translation of M-hirudin is highly robust against changes in concentrations of K+ (and Rb+). In addition, M-hirudin can be detected very sensitively using a reasonably priced fluorimetric thrombin assay. To show that the new reporter gene system is well suited for (u)HTS-applications, cell-free synthesis as well as the fluorimetric assay of M-hirudin were carried out in nanotiter and microtiter plates, respectively.

Differential distribution of the G protein gamma3 subunit in the developing zebrafish nervous system.

G proteins play an essential role in the transduction and propagation of extracellular signals across the plasma membrane. It was once thought that the G protein alpha subunit was the sole regulator of intracellular molecules. The G protein betagamma complex is now recognized as participating in many signaling events. While screening a zebrafish cDNA library to identify members of the protein 4.1 superfamily (Kelly, G.M., Reversade, B., Biochem. Cell Biol. 75 (1997), 623), we fortuitously identified a clone that encodes a zebrafish G protein gamma subunit. The 666 nucleotides of the zebrafish G protein gamma subunit cDNA encodes a polypeptide of 75 amino acids with high degree of homology to human, bovine, rat and mouse gamma subunits. BLAST search analysis of GenBank revealed that the zebrafish gamma subunit is 93% identical and 97% similar to the mammalian gamma3 subunit. The gamma3 gene was mapped to the zebrafish linkage group 21, approximately 10.76 cRays from bf, a gene with sequence homology to the human properdin factor gene. RT-PCR and in situ hybridization analyses first detected gamma3 mRNA during late somitogenesis, where it was expressed preferentially in the Vth cranial nerve, the forebrain and in ventrolateral regions of the mid- and hindbrain including the spinal cord. The ability of the zebrafish gamma3 subunit to form a signaling heterodimeric complex with a beta subunit was tested using a human beta2 subunit. The gamma3 formed a heterodimer with beta2 and the complex was capable of binding calmodulin in a calcium-dependent manner. Overexpression of the beta2gamma3 complex in zebrafish embryos lead to the loss of dorsoanterior structures and heart defects, possibly owing to an up-regulation of mitogen-activated protein kinase activity and/or decline in protein kinase A signaling. Together, these data imply that a betagamma heterodimer plays a role in signal transduction events involving G protein coupled receptors and that these events occur in specific regions in the nervous system of the developing zebrafish.

Inhibitory effect of antisense epidermal growth factor receptor RNA on the proliferation of rat C6 glioma cells in vitro and in vivo.

OBJECT: The goal of this study was to evaluate the effect of antisense epidermal growth factor receptor (EGFR) RNA on the growth of rat glioma cells in vitro and in vivo and to determine the feasibility of targeting the EGFR gene for gene therapy in gliomas. METHODS: Antisense EGFR complementary (c)DNA was transfected into C6 glioma cells by using lipofectamine. In vitro studies, Southern and Northern blot analyses, in situ hybridization, and immunohistochemical staining were designed to examine the integration and expression of antisense EGFR constructs. The 3'(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay and the average number of argyrophilic nuclear organizer regions (Ag-NORs) were used to evaluate cell proliferation, whereas the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method and microscopy were used to observe cell apoptosis. As part of the in vivo studies, parental C6 cells and C6 cells transfected with EGFR antisense cDNA were implanted stereotactically into the right caudate nucleus of Wistar rats (C6-injected animals and transfected C6-injected animals). Rats with well-established cerebral C6 glioma foci were treated intratumorally with either antisense EGFR cDNA or empty-vector DNA by using lipofectamine (treated-C6 and control treated group). The general behavior and survival of the rats, findings on magnetic resonance images of their brains, histopathological changes, proliferation activity, and apoptosis of the cerebral gliomas in each group of rats were examined. Exogenous antisense EGFR cDNA was integrated into the genome of C6 cells and expressed. In clones with a high expression of the antisense construct, there was a dramatic decrease in endogenous EGFR messenger RNA and protein levels, reduced proliferation activity, and induction of apoptosis in vitro. The mean survival time of rats injected with C6 cells was 17.3 days. The mean survival time of rats injected with C6 cells followed by treatment with empty vector in lipofectamine was 15.4 days. Survival time was significantly prolonged in 100% of the rats injected with antisense-transfected C6 cells and in two thirds of the rats injected with C6 cells followed by antisense EGFR cDNA. Magnetic resonance imaging revealed distinct cerebral tumor foci in C6-injected rats and in control rats of the treated group, but none were found in the rats injected with transfected C6 cells. Furthermore, tumor foci disappeared completely in C6-injected rats treated with antisense EGFR cDNA. The cerebral gliomas of the rats treated by injection of antisense EGFR RNA were characterized by reduced proliferation activity and the induction of apoptosis. CONCLUSIONS: The results of this study indicate that EGFR plays an important role in the genesis of malignant gliomas. It may, therefore, be an effective target of antisense gene therapy in patients with gliomas.

Antisense expression of the human pro-melanin-concentrating hormone genes.

Expression of transcripts for human pro-melanin concentrating hormone (pMCH) were studied in the hypothalamus, the primary location for pMCH producing cells in the mammalian CNS. Human hypothalamic tissue was extracted for total RNA and the cDNA generated with reverse transcriptase (RT). PCR amplification with primers spanning exons 2 and 3 of the pMCH human-variant genes (pMCHL), yielded an unspliced product, confirming prior work [T.B. Campbell, C.K. McDonald, M. Hagen, The effect of structure in a long target RNA on ribozyme cleavage efficiency, Nucleic Acids Res. 25 (1997) 4985-4993]. In addition, this product was shown to be exclusively antisense, and to be derived from the 5p (pMCHL1), not the 5q (pMCHL2) locus. Thus, there is no evidence that the MCH peptide-precursor molecule is produced in the brain by the human-variant pMCHL loci. In contrast, corresponding RT-PCR for pMCH RNA generated by the locus on 12q, demonstrated the presence of both sense and antisense spliced RNA. Partial sequencing of the spliced product confirmed that production of at least the two C-terminal peptides would occur from the 12q pMCH locus. The significance of the findings for pMCH and pMCHL1 are discussed relative to what is known about the function of endogenous antisense RNA.