Bmal1 integrates circadian function and temperature sensing in the suprachiasmatic nucleus.

Circadian regulation and temperature dependency are important orchestrators of molecular pathways. How the integration between these two drivers is achieved, is not understood. We monitored circadian- and temperature-dependent effects on transcription dynamics of cold-response protein RNA Binding Motif 3 (Rbm3). Temperature changes in the mammalian master circadian pacemaker, the suprachiasmatic nucleus (SCN), induced Rbm3 transcription and regulated its circadian periodicity, whereas the core clock gene Per2 was unaffected. Rbm3 induction depended on a full Brain And Muscle ARNT-Like Protein 1 (Bmal1) complement: reduced Bmal1 erased Rbm3 responses and weakened SCN circuit resilience to temperature changes. By focusing on circadian and temperature dependency, we highlight weakened transmission between core clock and downstream pathways as a potential route for reduced circadian resilience.

Timeless noncoding DNA contains cell-type preferential enhancers important for proper Drosophila circadian regulation.

To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E-box region and an upstream E-box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E-box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E-box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (assay for transposase-accessible chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E-box region.

Single cell and bulk RNA expression analyses identify enhanced hexosamine biosynthetic pathway and O-GlcNAcylation in acute myeloid leukemia blasts and stem cells.

Introduction: Acute myeloid leukemia (AML) is the most common acute leukemia in adults with an overall poor prognosis and high relapse rate. Multiple factors including genetic abnormalities, differentiation defects and altered cellular metabolism contribute to AML development and progression. Though the roles of oxidative phosphorylation and glycolysis are defined in AML, the role of the hexosamine biosynthetic pathway (HBP), which regulates the O-GlcNAcylation of cytoplasmic and nuclear proteins, remains poorly defined. Methods: We studied the expression of the key enzymes involved in the HBP in AML blasts and stem cells by RNA sequencing at the single-cell and bulk level. We performed flow cytometry to study OGT protein expression and global O-GlcNAcylation. We studied the functional effects of inhibiting O-GlcNAcylation on transcriptional activation in AML cells by Western blotting and real time PCR and on cell cycle by flow cytometry. Results: We found higher expression levels of the key enzymes in the HBP in AML as compared to healthy donors in whole blood. We observed elevated O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) expression in AML stem and bulk cells as compared to normal hematopoietic stem and progenitor cells (HSPCs). We also found that both AML bulk cells and stem cells show significantly enhanced OGT protein expression and global O-GlcNAcylation as compared to normal HSPCs, validating our in silico findings. Gene set analysis showed substantial enrichment of the NF-κB pathway in AML cells expressing high OGT levels. Inhibition of O-GlcNAcylation decreased NF-κB nuclear translocation and the expression of selected NF-κB-dependent genes controlling cell cycle. It also blocked cell cycle progression suggesting a link between enhanced O-GlcNAcylation and NF-κB activation in AML cell survival and proliferation. Discussion: Our study suggests the HBP may prove a potential target, alone or in combination with other therapeutic approaches, to impact both AML blasts and stem cells. Moreover, as insufficient targeting of AML stem cells by traditional chemotherapy is thought to lead to relapse, blocking HBP and O-GlcNAcylation in AML stem cells may represent a novel promising target to control relapse.

An RNA-hydrolyzing recombinant minibody prevents both influenza A virus and coronavirus in co-infection models.

With the lifting of COVID-19 non-pharmaceutical interventions, the resurgence of common viral respiratory infections was recorded in several countries worldwide. It facilitates viral co-infection, further burdens the already over-stretched healthcare systems. Racing to find co-infection-associated efficacy therapeutic agents need to be rapidly established. However, it has encountered numerous challenges that necessitate careful investigation. Here, we introduce a potential recombinant minibody-associated treatment, 3D8 single chain variable fragment (scFv), which has been developed as a broad-spectrum antiviral drug that acts via its nucleic acid catalytic and cell penetration abilities. In this research, we demonstrated that 3D8 scFv exerted antiviral activity simultaneously against both influenza A viruses (IAVs) and coronaviruses in three established co-infection models comprising two types of coronaviruses [beta coronavirus-human coronavirus OC43 (hCoV-OC43) and alpha coronavirus-porcine epidemic diarrhea virus (PEDV)] in Vero E6 cells, two IAVs [A/Puerto Rico/8/1934 H1N1 (H1N1/PR8) and A/X-31 (H3N2/X-31)] in MDCK cells, and a combination of coronavirus and IAV (hCoV-OC43 and adapted-H1N1) in Vero E6 cells by a statistically significant reduction in viral gene expression, proteins level, and approximately around 85%, 65%, and 80% of the progeny of 'hCoV-OC43-PEDV', 'H1N1/PR8-H3N2/X-31', and 'hCoV-OC43-adapted-H1N1', respectively, were decimated in the presence of 3D8 scFv. Taken together, we propose that 3D8 scFv is a promising broad-spectrum drug for treatment against RNA viruses in co-infection.

Circular RNAs: Biogenesis, Functions, and Role in Myocardial Hypertrophy.

Circular RNAs (circRNAs) are a large class of endogenous single-stranded covalently closed RNA molecules. High-throughput RNA sequencing and bioinformatic algorithms have identified thousands of eukaryotic circRNAs characterized by high stability and tissue-specific expression pattern. Recent studies have shown that circRNAs play an important role in the regulation of physiological processes in the norm and in various diseases, including cardiovascular disorders. The review presents current concepts of circRNA biogenesis, structural features, and biological functions, describes the methods of circRNA analysis, and summarizes the results of studies on the role of circRNAs in the pathogenesis of hypertrophic cardiomyopathy, the most common inherited heart disease.

Differential gene expression in decidualized human endometrial stromal cells induced by different stimuli.

Decidualization can be induced by culturing human endometrial stromal cells (ESCs) with several decidualization stimuli, such as cAMP, medroxyprogesterone acetate (MPA) or Estradiol (E2). However, it has been unclear how decidualized cells induced by different stimuli are different. We compared transcriptomes and cellular functions of decidualized ESCs induced by different stimuli (MPA, E2 + MPA, cAMP, and cAMP + MPA). We also investigated which decidualization stimulus induces a closer in vivo decidualization. Differentially expressed genes (DEGs) and altered cellular functions by each decidualization stimuli were identified by RNA-sequence and gene-ontology analysis. DEGs was about two times higher for stimuli that use cAMP (cAMP and cAMP + MPA) than for stimuli that did not use cAMP (MPA and E2 + MPA). cAMP-using stimuli altered the cellular functions including angiogenesis, inflammation, immune system, and embryo implantation whereas MPA-using stimuli (MPA, E2 + MPA, and cAMP + MPA) altered the cellular functions associated with insulin signaling. A public single-cell RNA-sequence data of the human endometrium was utilized to analyze in vivo decidualization. The altered cellular functions by in vivo decidualization were close to those observed by cAMP + MPA-induced decidualization. In conclusion, decidualized cells induced by different stimuli have different transcriptome and cellular functions. cAMP + MPA may induce a decidualization most closely to in vivo decidualization.

Loss-of-function mutation in PRMT9 causes abnormal synapse development by dysregulation of RNA alternative splicing.

Protein arginine methyltransferase 9 (PRMT9) is a recently identified member of the PRMT family, yet its biological function remains largely unknown. Here, by characterizing an intellectual disability associated PRMT9 mutation (G189R) and establishing a Prmt9 conditional knockout (cKO) mouse model, we uncover an important function of PRMT9 in neuronal development. The G189R mutation abolishes PRMT9 methyltransferase activity and reduces its protein stability. Knockout of Prmt9 in hippocampal neurons causes alternative splicing of ~1900 genes, which likely accounts for the aberrant synapse development and impaired learning and memory in the Prmt9 cKO mice. Mechanistically, we discover a methylation-sensitive protein-RNA interaction between the arginine 508 (R508) of the splicing factor 3B subunit 2 (SF3B2), the site that is exclusively methylated by PRMT9, and the pre-mRNA anchoring site, a cis-regulatory element that is critical for RNA splicing. Additionally, using human and mouse cell lines, as well as an SF3B2 arginine methylation-deficient mouse model, we provide strong evidence that SF3B2 is the primary methylation substrate of PRMT9, thus highlighting the conserved function of the PRMT9/SF3B2 axis in regulating pre-mRNA splicing.

Clinical significance of RNA methylation in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a primary liver malignancy with high mortality rates and poor prognosis. Recent advances in high-throughput sequencing and bioinformatic technologies have greatly enhanced the understanding of the genetic and epigenetic changes in liver cancer. Among these changes, RNA methylation, the most prevalent internal RNA modification, has emerged as a significant contributor of the development and progression of HCC. Growing evidence has reported significantly abnormal levels of RNA methylation and dysregulation of RNA-methylation-related enzymes in HCC tissues and cell lines. These alterations in RNA methylation play a crucial role in the regulation of various genes and signaling pathways involved in HCC, thereby promoting tumor progression. Understanding the pathogenesis of RNA methylation in HCC would help in developing prognostic biomarkers and targeted therapies for HCC. Targeting RNA-methylation-related molecules has shown promising potential in the management of HCC, in terms of developing novel prognostic biomarkers and therapies for HCC. Exploring the clinical application of targeted RNA methylation may provide new insights and approaches for the management of HCC. Further research in this field is warranted to fully understand the functional roles and underlying mechanisms of RNA methylation in HCC. In this review, we described the multifaceted functional roles and potential mechanisms of RNA methylation in HCC. Moreover, the prospects of clinical application of targeted RNA methylation for HCC management are discussed, which may provide the basis for subsequent in-depth research on RNA methylation in HCC.

Proinflammatory cytokines suppress nonsense-mediated RNA decay to impair regulated transcript isoform processing in pancreatic ß cells.

Introduction: Proinflammatory cytokines are implicated in pancreatic ß cell failure in type 1 and type 2 diabetes and are known to stimulate alternative RNA splicing and the expression of nonsense-mediated RNA decay (NMD) components. Here, we investigate whether cytokines regulate NMD activity and identify transcript isoforms targeted in ß cells. Methods: A luciferase-based NMD reporter transiently expressed in rat INS1(832/13), human-derived EndoC-ßH3, or dispersed human islet cells is used to examine the effect of proinflammatory cytokines (Cyt) on NMD activity. The gain- or loss-of-function of two key NMD components, UPF3B and UPF2, is used to reveal the effect of cytokines on cell viability and function. RNA-sequencing and siRNA-mediated silencing are deployed using standard techniques. Results: Cyt attenuate NMD activity in insulin-producing cell lines and primary human ß cells. These effects are found to involve ER stress and are associated with the downregulation of UPF3B. Increases or decreases in NMD activity achieved by UPF3B overexpression (OE) or UPF2 silencing raise or lower Cyt-induced cell death, respectively, in EndoC-ßH3 cells and are associated with decreased or increased insulin content, respectively. No effects of these manipulations are observed on glucose-stimulated insulin secretion. Transcriptomic analysis reveals that Cyt increases alternative splicing (AS)-induced exon skipping in the transcript isoforms, and this is potentiated by UPF2 silencing. Gene enrichment analysis identifies transcripts regulated by UPF2 silencing whose proteins are localized and/or functional in the extracellular matrix (ECM), including the serine protease inhibitor SERPINA1/α-1-antitrypsin, whose silencing sensitizes ß-cells to Cyt cytotoxicity. Cytokines suppress NMD activity via UPR signaling, potentially serving as a protective response against Cyt-induced NMD component expression. Conclusion: Our findings highlight the central importance of RNA turnover in ß cell responses to inflammatory stress.

Regulation by the RNA-binding protein Unkempt at its effector interface.

How RNA-binding proteins (RBPs) convey regulatory instructions to the core effectors of RNA processing is unclear. Here, we document the existence and functions of a multivalent RBP-effector interface. We show that the effector interface of a conserved RBP with an essential role in metazoan development, Unkempt, is mediated by a novel type of 'dual-purpose' peptide motifs that can contact two different surfaces of interacting proteins. Unexpectedly, we find that the multivalent contacts do not merely serve effector recruitment but are required for the accuracy of RNA recognition by Unkempt. Systems analyses reveal that multivalent RBP-effector contacts can repurpose the principal activity of an effector for a different function, as we demonstrate for the reuse of the central eukaryotic mRNA decay factor CCR4-NOT in translational control. Our study establishes the molecular assembly and functional principles of an RBP-effector interface.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

N6-methyladenosine-modified circSLCO1B3 promotes intrahepatic cholangiocarcinoma progression via regulating HOXC8 and PD-L1.

BACKGROUND: Refractoriness to surgical resection and chemotherapy makes intrahepatic cholangiocarcinoma (ICC) a fatal cancer of the digestive system with high mortality and poor prognosis. Important function invests circRNAs with tremendous potential in biomarkers and therapeutic targets. Nevertheless, it is still unknown how circRNAs contribute to the evolution of ICC. METHODS: CircRNAs in paired ICC and adjacent tissues were screened by circRNAs sequencing. To explore the impact of circRNAs on ICC development, experiments involving gain and loss of function were conducted. Various experimental techniques, including quantitative real-time PCR (qPCR), western blotting, RNA immunoprecipitation (RIP), luciferase reporter assays, RNA pull-down, chromatin immunoprecipitation (ChIP), ubiquitination assays and so on were employed to identify the molecular regulatory role of circRNAs. RESULTS: Herein, we reported a new circRNA, which originates from exon 9 to exon 15 of the SLCO1B3 gene (named circSLCO1B3), orchestrated ICC progression by promoting tumor proliferation, metastasis and immune evasion. We found that the circSLCO1B3 gene was highly overexpressed in ICC tissues and related to lymphatic metastasis, tumor sizes, and tumor differentiation. Mechanically, circSLCO1B3 not only promoted ICC proliferation and metastasis via miR-502-5p/HOXC8/SMAD3 axis, but also eradicated anti-tumor immunity via suppressing ubiquitin-proteasome-dependent degradation of PD-L1 by E3 ubiquitin ligase SPOP. We further found that methyltransferase like 3 (METTL3) mediated the m6A methylation of circSLCO1B3 and stabilizes its expression. Our findings indicate that circSLCO1B3 is a potential prognostic marker and therapeutic target in ICC patients. CONCLUSIONS: Taken together, m6A-modified circSLCO1B3 was correlated with poor prognosis in ICC and promoted ICC progression not only by enhancing proliferation and metastasis via potentiating HOXC8 expression, but also by inducing immune evasion via antagonizing PD-L1 degradation. These results suggest that circSLCO1B3 is a potential prognostic marker and therapeutic target for ICC.

RT-qPCR based quantitative analysis of ARO and ADH genes in Saccharomyces cerevisiae and Metschnikowia pulcherrima strains growth white grape juice.

BACKGROUND: Yeast biosynthesizes fusel alcohols in fermentation through amino acid catabolism via the Ehrlich pathway. ARO8 and ARO9 genes are involved in the first step of the Ehrlich pathway, while ADH2 and ADH5 genes are involved in the last step. In this study, we describe RT-qPCR methods to determine the gene expression level of genes (ARO8, ARO9, ADH2, ADH5) found in Saccharomyces cerevisiae (Sc) and Metschnikowia pulcherrima (Mp) strains growth pasteurized white grape juice. METHODS AND RESULTS: We used RNA extraction and cDNA synthesis protocols. The RT-qPCR efficiency of primer pairs was evaluated by generating a standard curve through serial dilution of yeast-derived cDNA. Method performance criteria were determined for each RT-qPCR assay. Then, we evaluated the gene expression levels of the four genes in all samples. RNA extraction and cDNA synthesis from yeast samples demonstrated the method's capability to generate high-yield, high-purity nucleic acids, supporting further RT-qPCR analysis. The highest normalized gene expression levels of ARO8 and ARO9 were observed in SC1, SC4, and SC5 samples. No significant difference in ADH2 gene expression among Mp strains was observed during the examination of ADH2 and ADH5 genes (p < 0.05). We observed no expression of the ADH5 gene in Mp strains except MP6 strain. The expression of ADH2 and ADH5 genes was higher in Sc strains compared to Mp strains. CONCLUSIONS: The results suggest that the proposed RT-qPCR methods can measure gene expression of ARO8, ARO9, ADH2, and ADH5 in Sc and Mp strains growing in pasteurized white grape juice.

N6-adenosine methylation and the regulatory mechanism on LINE-1.

Long interspersed elements-1(LINE-1) is the only autonomous transposon in human genome，and its retrotransposition results in change of cellular genome structure and function, leading occurrence of various severe diseases. As a central key intermediated component during life cycle of LINE-1 retrotransposition, the host modification of LINE-1 mRNA affects the LINE-1 transposition directly. N6-adenosine methylation(m6A), the most abundant epigenetic modification on eukaryotic RNA, is dynamically reversible. m6A modification is also found on LINE-1 mRNA, and it participants regulation of the whole LINE-1 replication cycle, with affecting LINE-1 retrotransposition as well as its adjacent genes expression, followed by influencing genomic stability, cellular self-renewal, and differentiation potential, which plays important roles in human development and diseases. In this review, we summarize the research progress in LINE-1 m6A modification, including its modification positions, patterns and related mechanisms, hoping to provide a new sight on the mechanism research and treatment of related diseases.

Effects of silencing hsa_circ_0015326 on proliferation, migration, invasion, and apoptosis of epithelial ovarian cancer cells.

Although the treatment of ovarian cancer has made great progress, there are still many patients who are not timely detected and given targeted therapy due to unknown pathogenesis. Recent studies have found that hsa_circ_0015326 is upregulated in ovarian cancer and is involved in the proliferation, invasion, and migration of ovarian cancer cells. However, whether hsa_circ_0015326 can be used as a new target of ovarian cancer needs further investigation. Therefore, the effect of hsa_circ_0015326 on epithelial ovarian cancer was investigated in this study. At first, si-hsa_circ_0015326 lentivirus was transfected into epithelial ovarian cancer cells. Then real-time fluorescence quantitative PCR (qRT-PCR) was used to detect hsa_circ_0015326 level. The proliferation of ovarian cancer cells was detected by CCK-8 assay. The horizontal and vertical migration abilities of the cells were detected by wound-healing assay and Transwell assay, respectively. Transwell assay was also used to determine the invasion rate. As for the apoptosis rate, it was assessed by flow cytometry. As a result, the expression level of hsa_circ_0015326 in A2780 and SKOV3 was found to be higher than that in IOSE-80. However, after transfecting si-hsa_circ_0015326 and si-NC into the cells, the proliferation, migration, and invasion abilities of A2780 and SKOV3 cells in the si-hsa_circ_0015326 group were significantly reduced in comparison to those in the si-NC and mock groups, while their apoptosis rates were elevated. Collectively, silencing hsa_circ_0015326 bears the capability of inhibiting the proliferation, migration, and invasion of ovarian cancer cells while increasing apoptosis rate. It can be concluded that hsa_circ_0015326 promotes the malignant biological activities of epithelial ovarian cancer cells.

Revealing myopathy spectrum: integrating transcriptional and clinical features of human skeletal muscles with varying health conditions.

Myopathy refers to a large group of heterogeneous, rare muscle diseases. Bulk RNA-sequencing has been utilized for the diagnosis and research of these diseases for many years. However, the existing valuable sequencing data often lack integration and clinical interpretation. In this study, we integrated bulk RNA-sequencing data from 1221 human skeletal muscles (292 with myopathies, 929 controls) from both databases and our local samples. By applying a method similar to single-cell analysis, we revealed a general spectrum of muscle diseases, ranging from healthy to mild disease, moderate muscle wasting, and severe muscle disease. This spectrum was further partly validated in three specific myopathies (97 muscles) through clinical features including trinucleotide repeat expansion, magnetic resonance imaging fat fraction, pathology, and clinical severity scores. This spectrum helped us identify 234 genuinely healthy muscles as unprecedented controls, providing a new perspective for deciphering the hallmark genes and pathways among different myopathies. The newly identified featured genes of general myopathy, inclusion body myositis, and titinopathy were highly expressed in our local muscles, as validated by quantitative polymerase chain reaction.

Glutaminolysis regulates endometrial fibrosis in intrauterine adhesion via modulating mitochondrial function.

BACKGROUND: Endometrial fibrosis, a significant characteristic of intrauterine adhesion (IUA), is caused by the excessive differentiation and activation of endometrial stromal cells (ESCs). Glutaminolysis is the metabolic process of glutamine (Gln), which has been implicated in multiple types of organ fibrosis. So far, little is known about whether glutaminolysis plays a role in endometrial fibrosis. METHODS: The activation model of ESCs was constructed by TGF-ß1, followed by RNA-sequencing analysis. Changes in glutaminase1 (GLS1) expression at RNA and protein levels in activated ESCs were verified experimentally. Human IUA samples were collected to verify GLS1 expression in endometrial fibrosis. GLS1 inhibitor and glutamine deprivation were applied to ESCs models to investigate the biological functions and mechanisms of glutaminolysis in ESCs activation. The IUA mice model was established to explore the effect of glutaminolysis inhibition on endometrial fibrosis. RESULTS: We found that GLS1 expression was significantly increased in activated ESCs models and fibrotic endometrium. Glutaminolysis inhibition by GLS1 inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl) ethyl sulfide (BPTES or glutamine deprivation treatment suppressed the expression of two fibrotic markers, α-SMA and collagen I, as well as the mitochondrial function and mTORC1 signaling in ESCs. Furthermore, inhibition of the mTORC1 signaling pathway by rapamycin suppressed ESCs activation. In IUA mice models, BPTES treatment significantly ameliorated endometrial fibrosis and improved pregnancy outcomes. CONCLUSION: Glutaminolysis and glutaminolysis-associated mTOR signaling play a role in the activation of ESCs and the pathogenesis of endometrial fibrosis through regulating mitochondrial function. Glutaminolysis inhibition suppresses the activation of ESCs, which might be a novel therapeutic strategy for IUA.

Impact of microglia isolation and culture methodology on transcriptional profile and function.

BACKGROUND: Microglial isolation and culturing methods continue to be explored to maximize cellular yield, purity, responsiveness to stimulation and similarity to in vivo microglia. This study aims to evaluate five different microglia isolation methods-three variants of microglia isolation from neonatal mice and two variants of microglia isolation from adult mice-on transcriptional profile and response to HMGB1. METHODS: Microglia from neonatal mice, age 0-3 days (P0-P3) were isolated from mixed glial cultures (MGC). We included three variations of this protocol that differed by use of GM-CSF in culture (No GM-CSF or 500 pg/mL GM-CSF), and days of culture in MGC before microglial separation (10 or 21). Protocols for studying microglia from adult mice age 6-8 weeks included isolation by adherence properties followed by 7 days of culture with 100 ng/mL GM-CSF and 100 ng/mL M-CSF (Vijaya et al. in Front Cell Neurosci 17:1082180, 2023), or acute isolation using CD11b beads (Bordt et al. in STAR Protoc 1:100035, 2020. https://doi.org/10.1016/j.xpro.2020.100035 ). Purity, yield, and RNA quality of the isolated microglia were assessed by flow cytometry, hemocytometer counting, and Bioanalyzer, respectively. Microglial responsiveness to an inflammatory stimulus, HMGB1, was evaluated by measuring TNFα, IL1ß, and IFNß concentration in supernatant by ELISA and assessing gene expression patterns using bulk mRNA sequencing. RESULTS: All five methods demonstrated greater than 90% purity. Microglia from all cultures increased transcription and secretion of TNFα, IL1ß, and IFNß in response to HMGB1. RNA sequencing showed a larger number of differentially expressed genes in response to HMGB1 treatment in microglia cultured from neonates than from adult mice, with sparse changes among the three MGC culturing conditions. Additionally, cultured microglia derived from adult and microglia derived from MGCs from neonates display transcriptional signatures corresponding to an earlier developmental stage. CONCLUSION: These findings suggest that while all methods provided high purity, the choice of protocol may significantly influence yield, RNA quality, baseline transcriptional profile and response to stimulation. This comparative study provides valuable insights to inform the choice of microglial isolation and culture method.

Utility of oligonucleotide in upregulating circular RNA production in a cellular model.

Circular RNAs (circRNAs), are a covalently closed, single-stranded RNA without 5'- and 3'-termini, commonly stem from the exons of precursor mRNAs (pre-mRNAs). They have recently garnered interest, with studies uncovering their pivotal roles in regulating various aspects of cell functions and disease progressions. A notable feature of circRNA lies in the mechanism of its biogenesis involving a specialized form of splicing: back-splicing. A splicing process that relies on interactions between introns flanking the circularizing exon to bring the up and downstream splice sites in proximity through the formation of a prerequisite hairpin structure, allowing the spliceosomes to join the two splice sites together to produce a circular RNA molecule. Based on this mechanism, we explored the feasibility of facilitating the formation of such a prerequisite hairpin structure by utilizing a newly designed oligonucleotide, CircuLarIzation Promoting OligoNucleotide (CLIP-ON), to promote the production of circRNA in cells. CLIP-ON was designed to hybridize with and physically bridge two distal sequences in the flanking introns of the circularizing exons. The feasibility of CLIP-ON was confirmed in HeLa cells using a model pre-mRNA, demonstrating the applicability of CLIP-ON as a trans-acting modulator to upregulate the production of circRNAs in a cellular environment.

Deciphering the possible role of RNA-helicase genes mechanism in response to abiotic stresses in rapeseed (Brassica napus L.).

BACKGROUND: Plants mediate several defense mechanisms to withstand abiotic stresses. Several gene families respond to stress as well as multiple transcription factors to minimize abiotic stresses without minimizing their effects on performance potential. RNA helicase (RH) is one of the foremost critical gene families that can play an influential role in tolerating abiotic stresses in plants. However, little knowledge is present about this protein family in rapeseed (canola). Here, we performed a comprehensive survey analysis of the RH protein family in rapeseed (Brassica napus L.). RESULTS: A total of 133 BnRHs genes have been discovered in this study. By phylogenetic analysis, RHs genes were divided into one main group and a subgroup. Examination of the chromosomal position of the identified genes showed that most of the genes (27%) were located on chromosome 3. All 133 identified sequences contained the main DEXDC domain, the HELICC domain, and a number of sub-domains. The results of biological process studies showed that about 17% of the proteins acted as RHs, 22% as ATP binding, and 14% as mRNA binding. Each part of the conserved motifs, communication network, and three-dimensional structure of the proteins were examined separately. The results showed that the RWC in leaf tissue decreased with higher levels of drought stress and in both root and leaf tissues sodium concentration was increased upon increased levels of salt stress treatments. The proline content were found to be increased in leaf and root with the increased level of stress treatment. Finally, the expression patterns of eight selected RHs genes that have been exposed to drought, salinity, cold, heat and cadmium stresses were investigated by qPCR. The results showed the effect of genes under stress. Examination of gene expression in the Hayola #4815 cultivar showed that all primers except primer #79 had less expression in both leaves and roots than the control level. CONCLUSIONS: New finding from the study have been presented new insights for better understanding the function and possible mechanism of RH in response to abiotic stress in rapeseed.