Insertion sequences (IS) represent mobile genetic elements that have been shown to be associated with bacterial evolution and adaptation due to their effects on genome plasticity. In Bordetella pertussis, the causative agent of whooping cough, the numerous IS elements induce genomic rearrangements and contribute to the diversity of the global B. pertussis population. Previously, we have shown that the majority of IS-specific endogenous promoters induce the synthesis of alternative transcripts and thereby affect the transcriptional landscape of B. pertussis. Here, we describe the regulatory RNA Rfi2, which is transcribed from the Pout promoter of the IS481 gene BP1118 antisense to the adjacent fim2 gene encoding the major serotype 2 fimbrial subunit of B. pertussis. Among the classical bordetellae, Rfi2 is unique to B. pertussis, suggesting its specific role in virulence. We show that Rfi2 RNA attenuates fim2 transcription and, consequently, the production of the Fim2 protein. Interestingly, the mutant that does not produce Rfi2 displayed significantly increased cytotoxicity towards human macrophages compared to the parental strain. This observation suggests that the Rfi2-mediated reduction in cytotoxicity represents an evolutionary adaptation of B. pertussis that fine-tunes its interaction with the human host. Given the immunogenicity of Fim2, we further hypothesize that Rfi2-mediated modulation of Fim2 production contributes to immune evasion. To our knowledge, Rfi2 represents the first functionally characterized IS element-driven antisense RNA that modulates the expression of a virulence gene.

• Klíčová slova
• Bordetella pertussis, antisense RNA, cytotoxicity towards macrophages, fimbriae serotype 2, insertion sequence, modulation of virulence,
• MeSH
• antigeny bakteriální MeSH
• antisense RNA * genetika   metabolismus   MeSH
• bakteriální fimbrie * genetika   metabolismus   MeSH
• Bordetella pertussis * genetika   patogenita   metabolismus   MeSH
• faktory virulence rodu Bordetella genetika   MeSH
• lidé MeSH
• makrofágy mikrobiologie   MeSH
• pertuse mikrobiologie   MeSH
• promotorové oblasti (genetika) MeSH
• proteiny fimbrií * genetika   metabolismus   MeSH
• regulace genové exprese u bakterií * MeSH
• séroskupina MeSH
• transpozibilní elementy DNA * MeSH
• virulence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny bakteriální MeSH
• antisense RNA * MeSH
• faktory virulence rodu Bordetella MeSH
• fim2 protein, Bordetella MeSH Prohlížeč
• proteiny fimbrií * MeSH
• transpozibilní elementy DNA * MeSH

Antisense transcripts play an important role in generating regulatory non-coding RNAs but whether these transcripts are also translated to generate functional peptides remains poorly understood. In this study, RNA sequencing and six-frame database generation were combined with mass spectrometry analysis of peptides isolated from polysomes to identify Nascent Pioneer Translation Products (Na-PTPs) originating from alternative reading frames of bi-directional transcripts. Two Na-PTP originating peptides derived from antisense strands stimulated CD8+ T cell proliferation when presented to peripheral blood mononuclear cells (PBMCs) from nine healthy donors. Importantly, an antigenic peptide derived from the reverse strand of two cDNA constructs was presented on MHC-I molecules and induced CD8+ T cell activation. The results demonstrate that three-frame translation of bi-directional transcripts generates antigenic peptide substrates for the immune system. This discovery holds significance for understanding the origin of self-discriminating peptide substrates for the major histocompatibility class I (MHC-I) pathway and for enhancing immune-based therapies against infected or transformed cells.

• Klíčová slova
• MHC-I epitope, Pioneer Translation Products, bi-directional transcripts, bi-directional translation, reverse strand antigenic peptides,
• MeSH
• aktivace lymfocytů imunologie   MeSH
• antisense RNA * genetika   imunologie   MeSH
• CD8-pozitivní T-lymfocyty * imunologie   MeSH
• leukocyty mononukleární imunologie   MeSH
• lidé MeSH
• MHC antigeny I. třídy * imunologie   genetika   MeSH
• peptidy * imunologie   genetika   MeSH
• prezentace antigenu MeSH
• proteosyntéza * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antisense RNA * MeSH
• MHC antigeny I. třídy * MeSH
• peptidy * MeSH

Transient plasmid transfection is a common approach in studies in cultured mammalian cells. To examine behavior of transfected plasmids, we analyzed their transcriptional landscape by deep sequencing. We have found that the entire plasmid sequence is transcribed at different levels. Spurious transcription may have undesirable effects as some plasmids, when co-transfected, inhibited expression of luciferase reporters in a dose-dependent manner. In one case, we attributed this effect to a Kan/Neo resistance cassette, which generated a unique population of edited sense and antisense small RNAs. The unexpected complexity of expression from transiently transfected plasmids underscores the importance of appropriate experimental controls.

• MeSH
• antisense RNA genetika   MeSH
• buněčné linie MeSH
• lidé MeSH
• plazmidy genetika   MeSH
• průtoková cytometrie MeSH
• vysoce účinné nukleotidové sekvenování metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense RNA MeSH

BACKGROUND: Tight regulation of testicular gene expression is a prerequisite for male reproductive success, while differentiation of gene activity in spermatogenesis is important during speciation. Thus, comparison of testicular transcriptomes between closely related species can reveal unique regulatory patterns and shed light on evolutionary constraints separating the species. METHODOLOGY/PRINCIPAL FINDINGS: Here, we compared testicular transcriptomes of two closely related mouse species, Mus musculus and Mus spretus, which diverged more than one million years ago. We analyzed testicular expression using tiling arrays overlapping Chromosomes 2, X, Y and mitochondrial genome. An excess of differentially regulated non-coding RNAs was found on Chromosome 2 including the intronic antisense RNAs, intergenic RNAs and premature forms of Piwi-interacting RNAs (piRNAs). Moreover, striking difference was found in the expression of X-linked G6pdx gene, the parental gene of the autosomal retrogene G6pd2. CONCLUSIONS/SIGNIFICANCE: The prevalence of non-coding RNAs among differentially expressed transcripts indicates their role in species-specific regulation of spermatogenesis. The postmeiotic expression of G6pdx in Mus spretus points towards the continuous evolution of X-chromosome silencing and provides an example of expression change accompanying the out-of-the X-chromosomal retroposition.

• MeSH
• antisense RNA genetika   MeSH
• chromozom X genetika   MeSH
• druhová specificita MeSH
• glukosa-6-fosfátdehydrogenasa genetika   MeSH
• malá interferující RNA genetika   MeSH
• meióza genetika   MeSH
• molekulární evoluce * MeSH
• myši MeSH
• nekódující RNA genetika   MeSH
• retroelementy genetika   MeSH
• testis cytologie   metabolismus   MeSH
• transkriptom * MeSH
• umlčování genů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense RNA MeSH
• glukosa-6-fosfátdehydrogenasa MeSH
• malá interferující RNA MeSH
• nekódující RNA MeSH
• retroelementy MeSH

The first observation of chromosomally encoded small antisense RNA in Corynebacterium glutamicum is reported. Transcription oriented in the reverse direction to the transcription of the genes cg1934 and cg1935 was demonstrated within the chromosomal cg1934-cg1935 intergenic region. The transcription was found to be increased after heat shock. The transcriptional start point of this RNA designated ArnA was localized 21 bp upstream of the cg1935 translational start point by primer extension analysis, when the total RNA was isolated from cells grown at 30 degrees C. After heat shock, the transcriptional start point of an additional species of ArnA RNA was detected 19 bp upstream of the cg1935 translational start point. The stress-response sigma factor SigH was found to be involved in the synthesis of ArnA RNAs. The 3' end of the ArnA RNAs was identified using the 3'-rapid amplification of cDNA ends technique. The length of the two ArnA RNA species was thus determined to be 129 and 131 nt, respectively. The ArnA RNAs were found to overlap the 5'-untranslated region of the transcript of the cg1935 gene coding for a transcriptional regulator of the GntR family. These results suggest that the noncoding ArnA RNAs have a regulatory function.

• MeSH
• 5' nepřekládaná oblast genetika   MeSH
• antisense RNA genetika   MeSH
• bakteriální chromozomy MeSH
• bakteriální geny MeSH
• bakteriální proteiny metabolismus   MeSH
• bakteriální RNA genetika   MeSH
• Corynebacterium glutamicum genetika   MeSH
• genetická transkripce MeSH
• intergenová DNA MeSH
• konformace nukleové kyseliny MeSH
• molekulární sekvence - údaje MeSH
• počátek transkripce MeSH
• regulace genové exprese u bakterií MeSH
• sekvence nukleotidů MeSH
• sigma faktor metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• antisense RNA MeSH
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• intergenová DNA MeSH
• SigH protein, bacteria MeSH Prohlížeč
• sigma faktor MeSH

SMAD5, a transducer of TGF-beta/BMP inhibitory signals and a tumor suppressor candidate, localizes to the region of invariant loss in human myeloid neoplasms, on chromosome 5q31.1. Recent evidence indicates a gene-dosage effect along the TGF-beta/BMP signaling pathways. We have identified a novel transcript designated DAMS, whose 3' exonic sequences contain in part an alternate 5' exon of SMAD5, in the antisense orientation. Expressed sequenced tags (ESTs) for DAMS are found in fetal tissues (heart, adrenal glands, and total fetus) and pancreatic tumor cDNA libraries. In contrast to SMAD5, DAMS expression is not readily detectable in adult and fetal tissues. Semiquantitative PCR suggests that the stoichiometry between SMAD5 and DAMS transcripts ranges between 15 and 120 in normal and malignant hematopoietic cells. The findings raise the possibility that DAMS may be a fail-safe mechanism for precise regulation of SMAD5 transcript levels that may be critical in maintaining normal homeostasis.

• MeSH
• 5' nepřekládaná oblast genetika   MeSH
• antisense RNA genetika   MeSH
• DNA vazebné proteiny genetika   MeSH
• fosfoproteiny genetika   MeSH
• genetická transkripce genetika   MeSH
• hematopoéza genetika   MeSH
• lidé MeSH
• lidské chromozomy, pár 5 genetika   MeSH
• messenger RNA metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• myeloidní leukemie genetika   MeSH
• nádorové buňky kultivované MeSH
• polymerázová řetězová reakce MeSH
• protein Smad5 MeSH
• regulace genové exprese u nádorů genetika   MeSH
• RNA dlouhá nekódující MeSH
• RNA nádorová genetika   MeSH
• trans-aktivátory genetika   MeSH
• transkripční faktory genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• 5' nepřekládaná oblast MeSH
• antisense RNA MeSH
• DNA vazebné proteiny MeSH
• fosfoproteiny MeSH
• messenger RNA MeSH
• protein Smad5 MeSH
• RNA dlouhá nekódující MeSH
• RNA nádorová MeSH
• SMAD5 protein, human MeSH Prohlížeč
• SMAD5-AS1 lncRNA, human MeSH Prohlížeč
• trans-aktivátory MeSH
• transkripční faktory MeSH

Complex formation between different antisense RNAs directed against either plus-strand or minus-strand sequences of the potato spindle tuber viroid (PSTVd) was studied using temperature-gradient gel electrophoresis and immunochemical detection with an antibody specific for double-stranded RNA. Short minus-strand sequences were directed against the upper central conserved region (UCCR) of plus-strand viroid replication intermediates, a plus-strand corresponding to the left half of the rod-like secondary structure (VL+) against minus-strand replication intermediates. It was shown that antisense RNA forms complexes with the corresponding target RNA only with low yield during incubation at low (physiological) temperatures but with high yield during in vitro transcription of the target RNA when the antisense RNA is already present in the solution. The antisense RNA sequences were integrated into Solanum tuberosum L. by Agrobacterium tumefaciens transformation. Antisense RNA expression in vivo was analyzed by Northern analysis. Infection tests were performed using the transgenic potato lines in order to evaluate their degree of resistance against PSTVd infection. Although some lines showed a significant inhibition of viroid accumulation, a high variability of viroid infection in different transgenic potato lines was obtained. Since strongly infected plants were observed in all transgenic lines 6 to 8 weeks post inoculation, a threshold concentration of viroid, overcoming the antisense effect has to be assumed. When the rate of viroid accumulation was tested using agroinfection assays on leaf discs, a stronger antisense effect could be achieved.

• MeSH
• Agrobacterium tumefaciens genetika   metabolismus   MeSH
• antisense RNA biosyntéza   genetika   metabolismus   MeSH
• dvouvláknová RNA analýza   chemie   metabolismus   MeSH
• genetická transkripce MeSH
• geneticky modifikované rostliny genetika   MeSH
• molekulární sekvence - údaje MeSH
• monoklonální protilátky imunologie   MeSH
• RNA virová antagonisté a inhibitory   metabolismus   MeSH
• sekvence nukleotidů MeSH
• Solanum tuberosum genetika   virologie   MeSH
• transformace genetická MeSH
• viroidy genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antisense RNA MeSH
• dvouvláknová RNA MeSH
• monoklonální protilátky MeSH
• RNA virová MeSH