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.

• MeSH
• Lymphocyte Activation immunology   MeSH
• RNA, Antisense * genetics   immunology   MeSH
• CD8-Positive T-Lymphocytes * immunology   MeSH
• Leukocytes, Mononuclear immunology   MeSH
• Humans MeSH
• Histocompatibility Antigens Class I * immunology   genetics   MeSH
• Peptides * immunology   genetics   MeSH
• Antigen Presentation MeSH
• Protein Biosynthesis * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Playing an important role in the etiology of substance use disorder (SUD), dopamine (DA) neurons are subject to various regulations but transcriptional regulations are largely understudied. For the first time, we report here that the Human Immunodeficiency Virus Type I Enhancer Binding Protein 2 (HIVEP2) is a dopaminergic transcriptional regulator. HIVEP2 is expressed in both the cytoplasm and nuclei of DA neurons. Therein, HIVEP2 can target the intronic sequence GTGGCTTTCT of SLC6A3 and thereby activate the gene. In naive rats from the bi-directional selectively bred substance-preferring P vs -nonpreferring NP rat model of substance abuse vulnerability, increased gene activity in males was associated with the vulnerability, whereas decreased gene activity in the females was associated with the same vulnerability. In clinical subjects, extensive and significant HIVEP2-SLC6A3 interactions were observed for SUD. Collectively, HIVEP2-mediated transcriptional mechanisms are implicated in dopaminergic pathophysiology of SUD.

• MeSH
• Cell Nucleus metabolism   MeSH
• DNA-Binding Proteins genetics   metabolism   MeSH
• Dopaminergic Neurons metabolism   MeSH
• Rats MeSH
• Humans MeSH
• Mice, Inbred C57BL MeSH
• Substance-Related Disorders etiology   genetics   MeSH
• Dopamine Plasma Membrane Transport Proteins genetics   metabolism   MeSH
• Gene Expression Regulation MeSH
• Sex Factors MeSH
• Transcription Factors genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Humans MeSH
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

Dinucleoside polyphosphates (NpnNs) were discovered 50 years ago in all cells. They are often called alarmones, even though the molecular target of the alarm has not yet been identified. Recently, we showed that they serve as noncanonical initiating nucleotides (NCINs) and fulfill the role of 5' RNA caps in Escherichia coli. Here, we present molecular insight into their ability to be used as NCINs by T7 RNA polymerase in the initiation phase of transcription. In general, we observed NpnNs to be equally good substrates as canonical nucleotides for T7 RNA polymerase. Surprisingly, the incorporation of ApnGs boosts the production of RNA 10-fold. This behavior is due to the pairing ability of both purine moieties with the -1 and +1 positions of the antisense DNA strand. Molecular dynamic simulations revealed noncanonical pairing of adenosine with the thymine of the DNA.

• MeSH
• Bacteriophage T7 enzymology   MeSH
• Dinucleoside Phosphates genetics   metabolism   MeSH
• DNA-Directed RNA Polymerases genetics   metabolism   MeSH
• DNA metabolism   MeSH
• Transcription Initiation, Genetic * MeSH
• Base Pairing MeSH
• RNA Caps genetics   MeSH
• RNA genetics   metabolism   MeSH
• Molecular Dynamics Simulation MeSH
• Protein Binding MeSH
• Viral Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

It has been more than 50 years since the discovery of dinucleoside polyphosphates (NpnNs) and yet their roles and mechanisms of action remain unclear. Here, we show that both methylated and non-methylated NpnNs serve as RNA caps in Escherichia coli. NpnNs are excellent substrates for T7 and E. coli RNA polymerases (RNAPs) and efficiently initiate transcription. We demonstrate, that the E. coli enzymes RNA 5'-pyrophosphohydrolase (RppH) and bis(5'-nucleosyl)-tetraphosphatase (ApaH) are able to remove the NpnN-caps from RNA. ApaH is able to cleave all NpnN-caps, while RppH is unable to cleave the methylated forms suggesting that the methylation adds an additional layer to RNA stability regulation. Our work introduces a different perspective on the chemical structure of RNA in prokaryotes and on the role of RNA caps. We bring evidence that small molecules, such as NpnNs are incorporated into RNA and may thus influence the cellular metabolism and RNA turnover.

• MeSH
• RNA, Bacterial genetics   MeSH
• Dinucleoside Phosphates genetics   MeSH
• DNA-Directed RNA Polymerases genetics   MeSH
• Escherichia coli genetics   MeSH
• Acid Anhydride Hydrolases metabolism   MeSH
• Nucleic Acid Conformation MeSH
• Methylation MeSH
• Escherichia coli Proteins metabolism   MeSH
• RNA Caps genetics   MeSH
• RNA Stability MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Systemin (SYS), an octadecapeptide hormone processed from a 200-amino-acid precursor (prosystemin, PS), plays a central role in the systemic activation of defense genes in tomato in response to herbivore and pathogen attacks. However, whether PS mRNA is transferable and its role in systemic defense responses remain unknown. We created the transgenic tomato PS gene tagged with the green fluorescent protein (PS-GFP) using a shoot- or root-specific promoter, and the constitutive 35S promoter in Arabidopsis. Subcellular localization of PS-/SYS-GFP was observed using confocal laser scanning microscopy and gene transcripts were determined using quantitative real-time PCR. In Arabidopsis, PS protein can be processed and SYS is secreted. Shoot-/root-specific expression of PS-GFP in Arabidopsis, and grafting experiments, revealed that the PS mRNA moves in a bi-directional manner. We also found that ectopic expression of PS improves Arabidopsis resistance to the necrotrophic fungus Botrytis cinerea, consistent with substantial upregulation of the transcript levels of specific pathogen-responsive genes. Our results provide novel insights into the multifaceted mechanism of SYS signaling transport and its potential application in genetic engineering for increasing pathogen resistance across diverse plant families.

• MeSH
• Arabidopsis drug effects   genetics   microbiology   MeSH
• Botrytis drug effects   physiology   MeSH
• Fluorescence MeSH
• Plants, Genetically Modified MeSH
• Plant Roots drug effects   genetics   MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Plant Diseases genetics   microbiology   MeSH
• Disease Resistance drug effects   genetics   MeSH
• Peptides pharmacology   MeSH
• Proteolysis drug effects   MeSH
• Gene Expression Regulation, Plant drug effects   MeSH
• Plant Proteins genetics   metabolism   MeSH
• Seedlings drug effects   growth & development   physiology   MeSH
• Solanum lycopersicum microbiology   MeSH
• Subcellular Fractions metabolism   MeSH
• RNA Transport drug effects   genetics   MeSH
• Plant Shoots drug effects   genetics   MeSH
• Green Fluorescent Proteins metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The growth and development of oocyte affect the functional activities of the surrounding somatic cells. These cells are regulated by various types of hormones, proteins, metabolites, and regulatory molecules through gap communication, ultimately leading to the development and maturation of oocytes. The close association between somatic cells and oocytes, which together form the cumulus-oocyte complexes (COCs), and their bi-directional communication are crucial for the acquisition of developmental competences by the oocyte. In this study, oocytes were extracted from the ovaries obtained from crossbred landrace gilts and subjected to in vitro maturation. RNA isolated from those oocytes was used for the subsequent microarray analysis. The data obtained shows, for the first time, variable levels of gene expression (fold changes higher than |2| and adjusted p-value < 0.05) belonging to four ontological groups: regulation of cell proliferation (GO:0042127), regulation of cell migration (GO:0030334), and regulation of programmed cell death (GO:0043067) that can be used together as proliferation, migration or apoptosis markers. We have identified several genes of porcine oocytes (ID2, VEGFA, BTG2, ESR1, CCND2, EDNRA, ANGPTL4, TGFBR3, GJA1, LAMA2, KIT, TPM1, VCP, GRID2, MEF2C, RPS3A, PLD1, BTG3, CD47, MITF), whose expression after in vitro maturation (IVM) is downregulated with different degrees. Our results may be helpful in further elucidating the molecular basis and functional significance of a number of gene markers associated with the processes of migration, proliferation and angiogenesis occurring in COCs.

• MeSH
• Apoptosis genetics   MeSH
• Down-Regulation MeSH
• Gene Regulatory Networks MeSH
• In Vitro Oocyte Maturation Techniques MeSH
• Cumulus Cells metabolism   pathology   MeSH
• Oocytes growth & development   metabolism   pathology   MeSH
• Cell Movement genetics   MeSH
• Swine MeSH
• Cell Proliferation genetics   MeSH
• RNA genetics   metabolism   MeSH
• Oligonucleotide Array Sequence Analysis MeSH
• Gene Expression Profiling MeSH
• Transcriptome * MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Elevated summer temperature is reported to be the leading cause of stress in dairy and beef cows, which negatively affects various reproductive functions. Follicular cells respond to heat stress (HS) by activating the expression of heat shock family proteins (HSPs) and other antioxidants. HS is reported to negatively affect the bi-directional communication between the follicular cells and the oocyte, which is partly mediated by follicular fluid extracellular vesicles (EVs) released from surrounding cells. As carriers of bioactive molecules (DNA, RNA, protein, and lipids), the involvement of EVs in mediating the stress response in follicular cells is not fully understood. Here we used an in vitro model to decipher the cellular and EV-coupled miRNAs of bovine granulosa cells in response to HS. Moreover, the protective role of stress-related EVs against subsequent HS was assessed. For this, bovine granulosa cells from smaller follicles were cultured in vitro and after sub-confluency, cells were either kept at 37 °C or subjected to HS (42 °C). Results showed that granulosa cells exposed to HS increased the accumulation of ROS, total oxidized protein, apoptosis, and the expression of HSPs and antioxidants, while the viability of cells was reduced. Moreover, 14 and 6 miRNAs were differentially expressed in heat-stressed granulosa cells and the corresponding EVs, respectively. Supplementation of stress-related EVs in cultured granulosa cells has induced adaptive response to subsequent HS. However, this potential was not pronounced when the cells were kept under 37 °C. Taking together, EVs generated from granulosa cells exposed to HS has the potential to shuttle bioactive molecules to recipient cells and make them robust to subsequent HS.

• MeSH
• Apoptosis MeSH
• Extracellular Vesicles genetics   metabolism   pathology   MeSH
• Granulosa Cells metabolism   pathology   MeSH
• Cattle Diseases epidemiology   genetics   prevention & control   MeSH
• Ovarian Follicle metabolism   pathology   MeSH
• Heat Stress Disorders genetics   physiopathology   veterinary   MeSH
• Heat-Shock Response * MeSH
• Gene Expression Regulation MeSH
• Cattle MeSH
• Gene Expression Profiling MeSH
• Animals MeSH
• Check Tag
• Cattle MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

High-Risk neuroblastoma (NB) survival rate is still <50%, despite treatments being more and more aggressive. The biggest hurdle liable to cancer therapy failure is the drug resistance by tumor cells that is likely due to the intra-tumor heterogeneity (ITH). To investigate the link between ITH and therapy resistance in NB, we performed a single cell RNA sequencing (scRNAseq) of etoposide and cisplatin resistant NB and their parental cells. Our analysis showed a clear separation of resistant and parental cells for both conditions by identifying 8 distinct tumor clusters in etoposide-resistant/parental and 7 in cisplatin-resistant/parental cells. We discovered that drug resistance can affect NB cell identities; highlighting the bi-directional ability of adrenergic-to-mesenchymal transition of NB cells. The biological processes driving the identified resistant cell subpopulations revealed genes such as (BARD1, BRCA1, PARP1, HISTH1 axis, members of RPL family), suggesting a potential drug resistance due to the acquisition of DNA repair mechanisms and to the modification of the drug targets. Deconvolution analysis of bulk RNAseq data from 498 tumors with cell subpopulation signatures showed that the transcriptional heterogeneity of our cellular models reflected the ITH of NB tumors and allowed the identification of clusters associated with worse/better survival. Our study demonstrates the distinct cell populations characterized by genes involved in different biological processes can have a role in NB drug treatment failure. These findings evidence the importance of ITH in NB drug resistance studies and the chance that scRNA-seq analysis offers in the identification of genes and pathways liable for drug resistance.

• Publication type
• Journal Article MeSH

• MeSH
• Cell Cycle physiology   MeSH
• Cell Cycle Proteins physiology   MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Buněčná biologie. Cytologie
• NML Fields
• biochemie
• biologie

• MeSH
• DNA genetics   MeSH
• Genetic Phenomena MeSH
• Genome MeSH
• Genes physiology   MeSH
• Proteins genetics   MeSH
• RNA genetics   MeSH
• Publication type
• Monograph MeSH

• Conspectus
• Obecná genetika. Obecná cytogenetika. Evoluce
• NML Fields
• genetika, lékařská genetika