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Circular RNA (circRNA) is a noncoding RNA class with important implications for gene expression regulation, mostly by interaction with other RNA species or RNA-binding proteins. While the commonly applied short-read Illumina RNA-sequencing techniques can be used to detect circRNAs, their full sequence is not revealed. However, the complete sequence information is needed to analyze potential interactions and thus the mechanism of action of circRNAs. Here, we present an improved protocol to enrich and sequence full-length circRNAs by using the Oxford Nanopore long-read sequencing platform. The protocol involves an enrichment of lowly abundant circRNAs by exonuclease treatment and negative selection of linear RNAs. Then, a cDNA library is created and amplified by PCR. This protocol provides enough material for several sequencing runs. The library is used as input for ligation-based sequencing together with native barcoding. Stringent quality control of the libraries is ensured by a combination of Qubit, Fragment Analyzer and qRT-PCR. Multiplexing of up to 4 libraries yields in total more than 1–2 Million reads per library, of which 1–2% are circRNA-specific reads with >99% of them full-length. The protocol works well with human cancer cell lines. We further provide suggestions for the bioinformatic analysis of the created data, as well as the limitations of our approach together with recommendations for troubleshooting and interpretation. Taken together, this protocol enables reliable full-length analysis of circRNAs, a noncoding RNA type involved in a growing number of physiologic and pathologic conditions. Metadata Associated content. https://dx.doi.org/10.17504/protocols.io.rm7vzy8r4lx1/v2. [ABSTRACT FROM AUTHOR]

Glioblastoma multiforme (GBM), the most prevalent primary malignant brain tumor in adults, exhibits a dismal 6.9% five-year survival rate post-diagnosis. Thymoquinone (TQ), the most abundant bioactive compound in Nigella sativa, has been extensively researched for its anticancer properties across various human cancers. However, its specific anti-cancer mechanisms and pathways in glioblastoma remain to be completely elucidated. In this study, we assessed the impact of different TQ concentrations on the viability of A172 cells using WST-8 and Toluidine blue assays, followed by RNA sequencing (RNA-Seq) to identify differentially expressed genes (DEGs). We confirmed their expression levels through quantitative RT-PCR and performed Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for these DEGs. RNA-seq revealed no significant gene expression changes at 2.5 μM and 5 μM TQ concentrations. However, at 25 μM and 50 μM, TQ significantly reduced cell viability dose-dependently. We identified 1548 DEGs at 25 μM TQ (684 up-regulated, 864 down-regulated) and 2797 DEGs at 50 μM TQ (1528 up-regulated, 1269 downregulated), with 1202 DEGs common to both concentrations. TQ inhibited key pathways such as PI3K-Akt signaling, calcium signaling, focal adhesion, and ECM-receptor interaction in A172 cells. It downregulated several potential oncogenes (e.g., AEBP1, MIAT) and genes linked to GBM proliferation and migration (e.g., SOCS2, HCP5) while modulating Wnt signaling and up-regulating tumor suppressor genes (e.g., SPRY4, BEX2). TQ also affected p53 downstream targets, maintaining p53 levels. This study elucidates the anti-cancer mechanisms of TQ in A172 GBM cells, underscoring its effects on multiple signaling pathways and positioning TQ as a promising candidate for innovative glioblastoma treatment strategies. [ABSTRACT FROM AUTHOR]

The mechanisms governing gene regulation in domestic Yuzhong pigeon breast muscle development remain largely elusive. Here, we conducted a comparative analysis using Iso-seq and RNA-seq data from domestic Yuzhong pigeons and European meat pigeons to uncover signaling pathways and genes involved in breast muscle development. The Iso-seq data from domestic Yuzhong pigeons yielded 131,377,075 subreads, resulting in 16,587 non-redundant high-quality full-length transcripts post-correction. Furthermore, utilizing pfam, CPC, PLEK, and CPAT, we predicted 5575, 4973, 2333, and 4336 lncRNAs, respectively. Notably, several genes potentially implicated in breast muscle development were identified, including tropomyosin beta chain, myosin regulatory light chain 2, and myosin binding protein C. KEGG enrichment analysis revealed critical signaling pathways in breast muscle development, spanning carbon metabolism, biosynthesis of amino acids, glycolysis/gluconeogenesis, estrogen signaling, PI3K-AKT signaling, protein processing in the endoplasmic reticulum, oxidative phosphorylation, pentose phosphate pathway, fructose and mannose metabolism, and tight junctions. These findings offer insights into the biological processes driving breast muscle development in domestic Yuzhong pigeon, contributing to our understanding of this complex phenomenon. [ABSTRACT FROM AUTHOR]

Tangeretin (Tan), a citrus flavonoid, possesses a strong anti-tumor efficacy in various human cancers. However, the precise role of Tan in the development of esophageal squamous cell carcinoma (ESCC) remains unclear. RNA sequencing (RNA-seq) analysis was performed to observe the Tan-related genes in Tan-treated TE-1 cells. The direct relationship between GLI family zinc finger 2 (GLI2) and the promoter of glycoprotein non-metastatic melanoma protein B (GPNMB) was predicted by bioinformatics analysis and validated by luciferase reporter and chromatin immunoprecipitation (ChIP) assays. Cell survival after Tan treatment was assessed by CCK8 assay. Gene expression levels were evaluated by a qRT-PCR, western blot, or immunofluorescence method. Cell migration and invasion were detected by wound-healing and transwell assays. The function of Tan in vivo was examined using xenograft studies. Our data indicated anti-migration and anti-invasion functions of Tan in ESCC cells in vitro. Tan also diminished tumor growth in vivo. Mechanistically, Tan diminished the expression and transcriptional activity of GLI2 in ESCC cells. Silencing of GLI2 resulted in decreased expression of GPNMB by inhibiting GPNMB transcription via the binding site at the GPNMB promoter at position +(1539–1550). Moreover, Tan down-regulated GPNMB expression in ESCC cells, and re-expression of GPNMB reversed anti-migration and anti-invasion functions of Tan in ESCC cells. Our findings uncover anti-migration and anti-invasion effects of Tan in ESCC cells by down-regulating GPNMB by suppressing GLI2-mediated GPNMB transcription, providing new evidence that Tan can function as a therapeutic agent against ESCC. [ABSTRACT FROM AUTHOR]

The MADS-box gene family controls plant flowering and floral organ development; therefore, it is particularly important in ornamental plants. To investigate the genes associated with the MADS-box family in Clematis courtoisii, we performed full-length transcriptome sequencing on C. courtoisii using the PacBio Sequel third-generation sequencing platform, as no reference genome data was available. A total of 12.38 Gb of data, containing 9,476,585 subreads and 50,439 Unigenes were obtained. According to functional annotation, a total of 37,923 Unigenes (75.18% of the total) were assigned with functional annotations, and 50 Unigenes were identified as MADS-box related genes. Subsequently, we employed hmmerscan to perform protein sequence similarity search for the translated Unigene sequences and successfully identified 19 Unigenes associated with the MADS-box gene family, including MIKC*(1) and MIKCC (18) genes. Furthermore, within the MIKCC group, six subclasses can be further distinguished. [ABSTRACT FROM AUTHOR]

Translation initiation is a complex and highly regulated process that represents an important mechanism, controlling gene expression. eIF2A was proposed as an alternative initiation factor, however, its role and biological targets remain to be discovered. To further gain insight into the function of eIF2A in Saccharomyces cerevisiae, we identified mRNAs associated with the eIF2A complex and showed that 24% of the most enriched mRNAs encode proteins related to cell wall biogenesis and maintenance. In agreement with this result, we showed that an eIF2A deletion sensitized cells to cell wall damage induced by calcofluor white. eIF2A overexpression led to a growth defect, correlated with decreased synthesis of several cell wall proteins. In contrast, no changes were observed in the transcriptome, suggesting that eIF2A controls the expression of cell wall-related proteins at a translational level. The biochemical characterization of the eIF2A complex revealed that it strongly interacts with the RNA binding protein, Ssd1, which is a negative translational regulator, controlling the expression of cell wall-related genes. Interestingly, eIF2A and Ssd1 bind several common mRNA targets and we found that the binding of eIF2A to some targets was mediated by Ssd1. Surprisingly, we further showed that eIF2A is physically and functionally associated with the exonuclease Xrn1 and other mRNA degradation factors, suggesting an additional level of regulation. Altogether, our results highlight new aspects of this complex and redundant fine-tuned regulation of proteins expression related to the cell wall, a structure required to maintain cell shape and rigidity, providing protection against harmful environmental stress. [ABSTRACT FROM AUTHOR]

Modulation of odorant receptors mRNA induced by prolonged odor exposure is highly correlated with ligand-receptor interactions in Drosophila as well as mammals of the Muridae family. If this response feature is conserved in other organisms, this presents an intriguing initial screening tool when searching for novel receptor-ligand interactions in species with predominantly orphan olfactory receptors. We demonstrate that mRNA modulation in response to 1-octen-3-ol odor exposure occurs in a time- and concentration-dependent manner in Aedes aegypti mosquitoes. To investigate gene expression patterns at a global level, we generated an odor-evoked transcriptome associated with 1-octen-3-ol odor exposure. Transcriptomic data revealed that ORs and OBPs were transcriptionally responsive whereas other chemosensory gene families showed little to no differential expression. Alongside chemosensory gene expression changes, transcriptomic analysis found that prolonged exposure to 1-octen-3-ol modulated xenobiotic response genes, primarily members of the cytochrome P450, insect cuticle proteins, and glucuronosyltransferases families. Together, these findings suggest that mRNA transcriptional modulation of olfactory receptors caused by prolonged odor exposure is pervasive across taxa and can be accompanied by the activation of xenobiotic responses. [ABSTRACT FROM AUTHOR]

Myelofibrosis is a myeloproliferative neoplasm (MPN) which typically results in reduced length and quality of life due to systemic symptoms and blood count changes arising from fibrotic changes in the bone marrow. While the JAK2 inhibitor ruxolitinib provides some clinical benefit, there remains a substantial unmet need for novel targeted therapies to better modify the disease process or eradicate the cells at the heart of myelofibrosis pathology. Repurposing drugs bypasses many of the hurdles present in drug development, such as toxicity and pharmacodynamic profiling. To this end we undertook a re-analysis of our pre-existing proteomic data sets to identify perturbed biochemical pathways and their associated drugs/inhibitors to potentially target the cells driving myelofibrosis. This approach identified CBL0137 as a candidate for targeting Jak2 mutation-driven malignancies. CBL0137 is a drug derived from curaxin targeting the Facilitates Chromatin Transcription (FACT) complex. It is reported to trap the FACT complex on chromatin thereby activating p53 and inhibiting NF-kB activity. We therefore assessed the activity of CBL0137 in primary patient samples and murine models of Jak2-mutated MPN and found it preferentially targets CD34+ stem and progenitor cells from myelofibrosis patients by comparison with healthy control cells. Further we investigate its mechanism of action in primary haemopoietic progenitor cells and demonstrate its ability to reduce splenomegaly and reticulocyte number in a transgenic murine model of myeloproliferative neoplasms. [ABSTRACT FROM AUTHOR]

Zinc is an essential metal for cells, but excess amounts are toxic. Other than by regulating the intracellular zinc concentration by zinc uptake or efflux, the mechanisms underlying bacterial resistance to excess zinc are unknown. In the present study, we searched for zinc-resistant mutant strains from the Keio collection, a gene knockout library of Escherichia coli, a model gram-negative bacteria. We found that knockout mutant of RpmJ (L36), a 50S ribosomal protein, exhibited zinc resistance. The rpmJ mutant was sensitive to protein synthesis inhibitors and had altered translation fidelity, indicating ribosomal dysfunction. In the rpmJ mutant, the intracellular zinc concentration was decreased under excess zinc conditions. Knockout of ZntA, a zinc efflux pump, abolished the zinc-resistant phenotype of the rpmJ mutant. RNA sequence analysis revealed that the rpmJ mutant exhibited altered gene expression of diverse functional categories, including translation, energy metabolism, and stress response. These findings suggest that knocking out RpmJ alters gene expression patterns and causes zinc resistance by lowering the intracellular zinc concentration. Knockouts of other ribosomal proteins, including RplA, RpmE, RpmI, and RpsT, also led to a zinc-resistant phenotype, suggesting that deletion of ribosomal proteins is closely related to zinc resistance. [ABSTRACT FROM AUTHOR]

A symbiotic relationship is observed between Armillaria and the Chinese herbal medicine Gastrodia elata (G. elata). Armillaria is a nutrient source for the growth of G. elata, and its nutrient metabolism efficiency affects the growth and development of G. elata. Auxin has been reported to stimulate Armillaria species, but the molecular mechanism remains unknown. We found that naphthalene acetic acid (NAA) can also promote the growth of A. gallica. Moreover, we identified a total of 2071 differentially expressed genes (DEGs) by analyzing the transcriptome sequencing data of A. gallica at 5 and 10 hour of NAA treatment. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses showed that these unigenes were significantly enriched in the metabolism pathways of arginine, proline, propanoate, phenylalanine and tryptophan. The expression levels of the general amino acid permease (Gap), ammonium transporter (AMT), glutamate dehydrogenase (GDH), glutamine synthetase (GS), Zn(II) 2Cys6 and C2H2 transcription factor genes were upregulated. Our transcriptome analysis showed that the amino acid and nitrogen metabolism pathways in Armillaria were rapidly induced within hours after NAA treatment. These results provide valuable insights into the molecular mechanisms by which NAA promotes the growth of Armillaria species. [ABSTRACT FROM AUTHOR]

Objective: To determine the gene expression profile in individuals with new latent tuberculosis infection (LTBI), and to compare them with people with active tuberculosis (TB) and those exposed to TB but not infected. Design: A prospective cohort study. Recruitment and follow-up were conducted between September 2016 to December 2018. Gene expression and data processing and analysis from April 2019 to April 2021. Setting: Two male Colombian prisons. Participants: 15 new tuberculin skin test (TST) converters (negative TST at baseline that became positive during follow-up), 11 people that continued with a negative TST after two years of follow-up, and 10 people with pulmonary ATB. Main outcome measures: Gene expression profile using RNA sequencing from PBMC samples. The differential expression was assessed using the DESeq2 package in Bioconductor. Genes with |logFC| >1.0 and an adjusted p-value < 0.1 were differentially expressed. We analyzed the differences in the enrichment of KEGG pathways in each group using InterMiner. Results: The gene expression was affected by the time of incarceration. We identified group-specific differentially expressed genes between the groups: 289 genes in people with a new LTBI and short incarceration (less than three months of incarceration), 117 in those with LTBI and long incarceration (one or more years of incarceration), 26 in ATB, and 276 in the exposed but non-infected individuals. Four pathways encompassed the largest number of down and up-regulated genes among individuals with LTBI and short incarceration: cytokine signaling, signal transduction, neutrophil degranulation, and innate immune system. In individuals with LTBI and long incarceration, the only enriched pathway within up-regulated genes was Emi1 phosphorylation. Conclusions: Recent infection with MTB is associated with an identifiable RNA pattern related to innate immune system pathways that can be used to prioritize LTBI treatment for those at greatest risk for developing active TB. [ABSTRACT FROM AUTHOR]

Natural silks crafted by spiders comprise some of the most versatile materials known. Artificial silks–based on the sequences of their natural brethren–replicate some desirable biophysical properties and are increasingly utilized in commercial and medical applications today. To characterize the repertoire of protein sequences giving silks their biophysical properties and to determine the set of expressed genes across each unique silk gland contributing to the formation of natural silks, we report here draft genomic and transcriptomic assemblies of Darwin's bark spider, Caerostris darwini, an orb-weaving spider whose dragline is one of the toughest known biomaterials on Earth. We identify at least 31 putative spidroin genes, with expansion of multiple spidroin gene classes relative to the golden orb-weaver, Trichonephila clavipes. We observed substantial sharing of spidroin repetitive sequence motifs between species as well as new motifs unique to C. darwini. Comparative gene expression analyses across six silk gland isolates in females plus a composite isolate of all silk glands in males demonstrated gland and sex-specific expression of spidroins, facilitating putative assignment of novel spidroin genes to classes. Broad expression of spidroins across silk gland types suggests that silks emanating from a given gland represent composite materials to a greater extent than previously appreciated. We hypothesize that the extraordinary toughness of C. darwini major ampullate dragline silk may relate to the unique protein composition of major ampullate spidroins, combined with the relatively high expression of stretchy flagelliform spidroins whose union into a single fiber may be aided by novel motifs and cassettes that act as molecule-binding helices. Our assemblies extend the catalog of sequences and sets of expressed genes that confer the unique biophysical properties observed in natural silks. [ABSTRACT FROM AUTHOR]

Understanding the cell molecular changes occurring as a results of climatic circumstances is crucial in the current days in which climate change and global warming are one of the most serious challenges that living organisms have to face. Sperm are one of the mammals' cells most sensitive to heat, therefore evaluating the impact of seasonal changes in terms of its transcriptional activity can contribute to elucidate how these cells cope with heat stress events. We sequenced the total sperm RNA from 64 ejaculates, 28 collected in summer and 36 collected in autumn, from 40 Manchega rams. A highly rich transcriptome (11,896 different transcripts) with 90 protein coding genes that exceed an average number of 5000 counts were found. Comparing transcriptome in the summer and autumn ejaculates, 236 significant differential abundance genes were assessed, most of them (228) downregulated. The main functions that these genes are related to sexual reproduction and negative regulation of protein metabolic processes and kinase activity. Sperm response to heat stress supposes a drastic decrease of the transcriptional activity, and the upregulation of only a few genes related with the basic functions to maintain the organisms' homeostasis and surviving. Rams' spermatozoids carry remnant mRNAs which are retrospectively indicators of events occurring along the spermatogenesis process, including abiotic factors such as environmental temperature. [ABSTRACT FROM AUTHOR]