Use Of RNA Research Articles

Use of double-stranded RNA targeting β2 divergent nicotinic acetylcholine receptor subunit to control pea aphid Acyrthosiphon pisum at larval and adult stages.

In recent years, the use of the RNA interference technology (RNAi) has emerged as one of the new strategies for species-specific control of insect pests. Its specificity depends on the distinctiveness of the target gene sequence for a given species. In this work, we assessed in the pea aphid Acyrthosiphon pisum (A. pisum) the use of a double-stranded RNA (dsRNA) that targets the β2 divergent nicotinic acetylcholine receptor (nAChR) subunit (dsRNA-β2), which shares low sequence identity with other subunits, to control populations of this pest at different developmental stages. Because nAChRs are targeted by neonicotinoid insecticides such as imidacloprid, we also assessed the effect of dsRNA-β2 coupled to this insecticide on aphid survival. Finally, because the effect of a control agent on beneficial insect must be considered before any use of new pest management strategies, the acute toxicity of dsRNA-β2 combined with imidacloprid was evaluated on honeybee Apis mellifera. In this work, we demonstrated that dsRNA-β2 alone has an insecticidal effect on aphid larvae and adults. Moreover, dsRNA-β2 and imidacloprid effects on aphid larvae and adults were additive, meaning that dsRNA-β2 did not alter the efficacy of imidacloprid on these two developmental stages. Also, no obvious acute toxicity on Apis mellifera was reported. Using RNAi that targets β2 divergent nAChR subunit is effective alone or combined with imidacloprid to control A. pisum at larval and adult stages. Because no obvious Apis mellifera mortality has been reported, this RNAi-based pest management strategy should be considered to control insect pest. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

A simplified protocol for gene expression-based biological dosimetry using peripheral whole blood

Purpose Assessing alterations in the expression of radiation-responsive genes in peripheral blood cells is considered a promising approach for high-throughput radiation biodosimetry. However, optimization of conditions for storage and transport of blood samples would be critical for obtaining reliable results. Recent studies involved the incubation of isolated peripheral blood mononuclear cells (in cell culture medium) and/or use of RNA stabilizing agents for sample storage, immediately after the ex vivo irradiation of whole blood. We used a simpler protocol by incubating undiluted peripheral whole blood without any RNA stabilizing agent, and studied the impact of storage temperature and incubation time on the expression levels of 19 known radiation responsive genes. Materials & methods Peripheral whole blood was γ-irradiated ex vivo at room temperature at low (0.5 Gy), moderate (1 Gy, 2 Gy) and high (4 Gy) doses and immediately incubated at two different temperatures at 4 °C or 37 °C for 2h, 4h and 24 h. Using qRT-PCR, mRNA expression levels of CDKN1A, DDB2, GADD45A, FDXR, BAX, BBC3, MYC, PCNA, XPC, ZMAT3, AEN, TRIAP1, CCNG1, RPS27L, CD70, EI24, C12orf5, TNFRSF10B, ASCC3 were analyzed at respective time-points and compared with the sham-irradiated controls. Results Transcriptional responses of all 19 genes did not alter significantly upon incubation of whole blood samples at 4 °C, as compared to untreated controls. However, incubation at 37 °C for 24 h resulted in significant radiation-induced overexpression in 14 out of the 19 genes analyzed (except CDKN1A, BBC3, MYC, CD 70 and EI24). Detailed patterns during incubation at 37 °C revealed time-dependent up-regulation of these genes, with DDB2 and FDXR showing significant up-regulation both at 4 and 24 h with the highest fold-change observed. Conclusion Overall, the undiluted whole blood incubated at 37 °C for 24 h was found to elicit most optimal transcriptional response in the genes studied, with most profound overexpression of DDB2 and FDXR. We propose that sample storage/transport/post-transit incubation at the physiological temperature for up to 24 h may enhance the sensitivity of gene expression based biodosimetry and facilitate its usage for triage application.

Intracellular RNA and DNA tracking by uridine-rich internal loop tagging with fluorogenic bPNA

The most widely used method for intracellular RNA fluorescence labeling is MS2 labeling, which generally relies on the use of multiple protein labels targeted to multiple RNA (MS2) hairpin structures installed on the RNA of interest (ROI). While effective and conveniently applied in cell biology labs, the protein labels add significant mass to the bound RNA, which potentially impacts steric accessibility and native RNA biology. We have previously demonstrated that internal, genetically encoded, uridine-rich internal loops (URILs) comprised of four contiguous UU pairs (8 nt) in RNA may be targeted with minimal structural perturbation by triplex hybridization with 1 kD bifacial peptide nucleic acids (bPNAs). A URIL-targeting strategy for RNA and DNA tracking would avoid the use of cumbersome protein fusion labels and minimize structural alterations to the RNA of interest. Here we show that URIL-targeting fluorogenic bPNA probes in cell media can penetrate cell membranes and effectively label RNAs and RNPs in fixed and live cells. This method, which we call fluorogenic U-rich internal loop (FLURIL) tagging, was internally validated through the use of RNAs bearing both URIL and MS2 labeling sites. Notably, a direct comparison of CRISPR-dCas labeled genomic loci in live U2OS cells revealed that FLURIL-tagged gRNA yielded loci with signal to background up to 7X greater than loci targeted by guide RNA modified with an array of eight MS2 hairpins. Together, these data show that FLURIL tagging provides a versatile scope of intracellular RNA and DNA tracking while maintaining a light molecular footprint and compatibility with existing methods.

The immunomodulatory effects of RNA-based biomaterials on bone regeneration.

The use of RNA as therapeutic agents is a visionary idea in contemporary medicine. Some forms of RNA can modulate the immune response of the host to enhance tissue regeneration events such as osteogenesis. Herein, RNA molecules commercially available for immunomodulatory applications (imRNA) were used to prepare biomaterials for bone regeneration. The polyanionic imRNA stabilized calcium phosphate ionic clusters to produce imRNA-ACP that had the capacity to mineralize the intrafibrillar compartments of collagen fibrils. For the first time, it was shown that incorporating imRNA-ACP into collagen scaffolds resulted in rapid new bone formation in cranial defects of mice. Both in vivo and in vitro results demonstrated that macrophage polarization was highly-sensitive to the imRNA-ACP containing collagen scaffolds. Macrophages were polarized into the anti-inflammatory M2 phenotype that produced anti-inflammation cytokines and growth factors. The favorable osteoimmunological microenvironment created by the scaffolds prevented their immunorejection and facilitated osteogenesis. The potential of RNA in creating immunomodulatory biomaterials has been underestimated in the past. The overall aim of this study was to explore the potential application of imRNA-based biomaterials in bone tissue engineering, with the competitive edge of facile synthesis and excellent biocompatibility. STATEMENT OF SIGNIFICANCE: : In this work, commercially available RNA extracted from bovine spleens for immunomodulatory applications (imRNA) were used to stabilize amorphous calcium phosphate (ACP) and induce mineralization within collagen fibrils. Incorporation of imRNA-ACP into collagen scaffolds regenerated new bone in-situ. Because of its immunomodulatory effects, the imRNA-ACP that was incorporated into collagen scaffolds modulated the local immune environment of murine cranial defects by altering the macrophage phenotype through JAK2/STAT3 signaling pathway. The novelty of this work exists in the discovery of RNA's potential in creating immunomodulatory biomaterials. With the competitive edge of facile synthesis and excellent biocompatibility, the imRNA-based biomaterials are potentially useful for future bone tissue engineering applications.

Characterization of expression and prognostic implications of GD2 and GD3 synthase in canine histiocytic sarcoma

GD2 and GD3 are disialoganglioside oncofetal antigens important in oncogenesis. GD2 synthase (GD2S) and GD3 synthase (GD3S) are needed for GD2 and GD3 production. The objectives of this study are to validate the use of RNA in situ hybridization (RNAscope®) in the detection of GD2S and GD3S in canine histiocytic sarcoma (HS) in vitro and optimize this technique in canine formalin-fixed paraffin-embedded (FFPE) tissues. A secondary objective is to evaluate the prognostic significance of GD2S and GD3S on survival. Quantitative RT-PCR compared GD2S and GD3S mRNA expression between three HS cell lines followed by RNAscope® in fixed cell pellets from the DH82 cell line and FFPE tissues. Variables prognostic for survival were determined with Cox proportional hazard model. RNAscope® was validated for detection of GD2S and GD3S and optimized in FFPE tissues. GD2S and GD3S mRNA expression was variable between cell lines. GD2S and GD3S mRNA expression was detected and measured in all tumor tissues; there was no association with prognosis. GD2S and GD3S are expressed in canine HS and successfully detected using the high throughput technique of RNAscope® in FFPE samples. This study provides the foundation for future prospective research of GD2S and GD3S utilizing RNAscope®.

Optimization of RNA Extraction from Dry Blood Spots for Nanostring Analysis.

Dry blood spot (DBS) technology has been widely used since the 1960's for the detection of protein biomarkers associated with various disease states. In this manuscript we report a revised approach using DBS samples to extract total RNA for use in downstream multiplex RNA detection methodology (Nanostring). To accomplish this objective, we have used commercially available supplies, kits, and equipment to ensure that the procedure described in this report can be adopted by any laboratory. The methods described in this report allow for the extraction of high-quality, total RNA from a minimal volume (∼200 µl) of DBS spots. The isolated RNA can be analyzed using a multiplex, Nanostring system to yield results for up to 800 RNA targets. Additional bioinformatics and pathway annotation can be conducted to determine changes in biological signaling pathways. © 2023 Wiley Periodicals LLC. Basic Protocol: RNA extraction from DBS for multiplex RNA nanostring analysis Support Protocol 1: RNA extraction from PAXgene blood Support Protocol 2: Concentration of DBS RNA.

Wastewater monitoring of SARS-CoV-2 RNA at K-12 schools: comparison to pooled clinical testing data.

Wastewater measurements of SARS-CoV-2 RNA have been extensively used to supplement clinical data on COVID-19. Most examples in the literature that describe wastewater monitoring for SARS-CoV-2 RNA use samples from wastewater treatment plants and individual buildings that serve as the primary residence of community members. However, wastewater surveillance can be an attractive supplement to clinical testing in K-12 schools where individuals only spend a portion of their time but interact with others in close proximity, increasing risk of potential transmission of disease. Wastewater samples were collected from two K-12 schools in California and divided into solid and liquid fractions to be processed for detection of SARS-CoV-2. The resulting detection rate in each wastewater fraction was compared to each other and the detection rate in pooled clinical specimens. Most wastewater samples were positive for SARS-CoV-2 RNA when clinical testing was positive (75% for solid samples and 100% for liquid samples). Wastewater samples continued to test positive for SARS-CoV-2 RNA when clinical testing was negative or in absence of clinical testing (83% for both solid and liquid samples), indicating presence of infected individuals in the schools. Wastewater solids had a higher concentration of SARS-CoV-2 than wastewater liquids on an equivalent mass basis by three orders of magnitude.

Review of the current and potential use of biological and molecular methods for the estimation of the postmortem interval in animals and humans.

We provide here an overview of the state of applied techniques in the estimation of the early period of the postmortem interval (PMI). The biological methods included consist of body cooling, CSF potassium, body cooling combined with CSF potassium, and tissue autolysis. For each method, we present its application in human and veterinary medicine and provide current methodology, strengths, and weaknesses, as well as target areas for improvement. We examine current and future molecular methods as they pertain to DNA and primarily to messenger RNA degradation for the estimation of the PMI, as well as the use of RNA in aging wounds, aging blood stains, and the identification of body fluids. Various types of RNA have different lengths, structures, and functions in cells. These differences in RNAs determine various intrinsic properties, such as their half-lives in cells, and, hence, their decay rate as well as their unique use for specific forensic tests. Future applications and refinements of RNA-based techniques provide opportunities for the use of molecular methods in the estimation of PMI and other general forensic applications.

EXPLORING THE POTENTIAL OF NEXT GENERATION SEQUENCING IN PLANT BREEDING AND GENETICS

Next generation sequencing (NGS) technologies have had a significant impact on plant breeding and genetics, enabling researchers to rapidly and accurately analyze large amounts of genetic data and identify and characterize important plant traits. NGS technologies, such as the study of gene expression and regulation in plants has made extensive use of RNA and genome sequencing, revealing important details about the mechanisms governing these activities. Plant-pathogen interactions have been studied using NGS, which has also been utilized to find pertinent genes. By identifying the genes and genetic variants that are associated with plant resistance to diseases, researchers can cross different plant varieties that are more disease- and pest-resistant. This can help to reduce crop losses and improve crop yields. NGS has been used to study the genetics of plant populations, enabling researchers to identify genetic variations that are associated with important plant traits and to develop new plant varieties with improved performance or desirable characteristics. Despite some challenges, such as complexity of NGS technologies and the limited availability of reference genomes for many plant species, NGS has had a major impact on plant breeding and genetics and is likely to continue to play a significant part in the future evolution of novel plant varieties.

RNA Nanostructures: From Structure to Function.

Nucleic-acid nanostructures, which have been designed and constructed with atomic precision, have been used as scaffolds for different molecules and proteins, as nanomachines, as computational components, and more. In particular, RNA has garnered tremendous interest as a building block for the self-assembly of sophisticated and functional nanostructures by virtue of its ease of synthesis by in vivo or in vitro transcription, its superior mechanical and thermodynamic properties, and its functional roles in nature. In this Topical Review, we describe recent developments in the use of RNA for the design and construction of nanostructures. We discuss the differences between RNA and DNA that make RNA attractive as a building block for the construction of nucleic-acid nanostructures, and we present the uses of different nanostructures─RNA alone, RNA-DNA, and functional RNA nanostructures.

SMaRT modulation of tau isoforms rescues cognitive and motor impairments in a preclinical model of tauopathy.

Tau is a microtubule-associated protein predominantly expressed in neurons, which participates in microtubule polymerization and axonal transport. Abnormal tau metabolism leads to neurodegenerative diseases named tauopathies, such as Alzheimer’s disease and frontotemporal dementia. The alternative splicing of exon 10 (E10) in the primary transcript produces tau protein isoforms with three (3R) or four (4R) microtubule binding repeats, which are found in equal amounts in the normal adult human brain. Several tauopathies are associated with abnormal E10 alternative splicing, leading to an imbalance between 3R and 4R isoforms, which underlies disease. Correction of such imbalance represents a potential disease-modifying therapy for those tauopathies. We have previously optimized a trans-splicing RNA reprogramming strategy to modulate the 3R:4R tau content in a mouse model of tauopathy related to tau mis-splicing (htau mice), and showed that local modulation of E10 inclusion in the prefrontal cortex prevents cognitive decline, neuronal firing impairments and hyperphosphorylated tau accumulation. Furthermore, local shifting of 3R–4R tau into the striatum of htau mice prevented motor coordination deficits. However, a major bottleneck of our previous work is that local splicing regulation was performed in young mice, before the onset of pathological phenotypes. Here we tested whether regulation of tau E10 splicing could rescue tau pathology phenotypes in htau mice, after the onset of cognitive and motor impairments, comparable to early stages of human tauopathies. To determine phenotypic time course and affected brain nuclei, we assessed htau mice using behavioural tests and microPET FDG imaging over time, similarly to diagnosis methods used in patients. Based on these analyses, we performed local delivery of pre-trans splicing molecules to regulate E10 inclusion either into the medial prefrontal cortex (mPFC) or the striatum at 6-month-old once behavioral phenotypes and metabolic changes were detected. Tau isoforms modulation into the mPFC restored cognitive performance in mice that previously showed mild to severe memory impairment while motor coordination deficit was rescued after striatal injection of trans-splicing molecules. Our data suggest that tau regulation could recover pathological phenotypes early after phenotypic onset, raising promising perspectives for the use of RNA based therapies in tauopathies related to MAPT abnormal splicing.

Development and Characterization of Synthetic Norovirus RNA for Use in Molecular Detection Methods.

Reference materials are essential for the quality assurance of molecular detection methods. We developed and characterized synthetic norovirus GI and GII RNA reference materials. Norovirus GI and GII RNA sequences including the ORF1-ORF2 junction region were designed based on 1,495 reported norovirus sequences and synthesized via plasmid preparation and in vitro transcription. The synthetic norovirus GI and GII RNAs were evaluated using six commercial norovirus detection kits used in Korea and subjected to homogeneity and stability analyses. A multicenter study involving five laboratories and using four commercial real-time PCR norovirus detection assays was conducted for synthetic norovirus RNA characterization and uncertainty measurements. The synthetic norovirus GI and GII RNAs were positively detected using the six commercial norovirus detection kits and were homogeneous and stable for one year when stored at -20°C or -70°C. All data from the five laboratories were within a range of 1.0 log copies/μL difference for each RNA, and the overall mean concentrations for norovirus GI and GII RNAs were 7.90 log copies/μL and 6.96 log copies/μL, respectively. The synthetic norovirus GI and GII RNAs are adequate for quality control based on commercial molecular detection reagents for noroviruses with high sequence variability. The synthetic RNAs can be used as reference materials in norovirus molecular detection methods.

Potential in anti-cancer therapy using RNA - overview of therapies

The main targets for the production of mRNA-based tumor vaccines are tumor-associated antigens and tumor-specific neoepitopes exposed on the surface of tumor cells. Their mission is to stimulate the cellular response in the body of a sick patient. In producing of mRNA vaccines it is important to use an appropriate vector and method of delivery of the vaccine to the body. Recent years a lot of interest has been seen in using dendritic cells as a vector. Ugur Sahin and his associates were the first to create procedures for creating personalized mRNA mutanomic vaccines. Apart from mutanomes, miRNA – the type of interference RNAs regulating gene expression - played a role in the development of mRNA vaccines. It has been shown that miRNA dysregulation is observed in some cancers and plays a role in invasion, angiogenesis, apoptosis, avoidance of immune destruction or sensitivity to growth factors. Many of the functions of LncRNA affect gene expression by regulating chromatin function, affecting the stability and translation of cytoplasmic mRNA, and signaling pathways, may ultimately lead to cancer formation – and also can be used in anti-cancer therapy and as sensitive biomarkers. These discoveries led to the rapid development of RNA-based anti-cancer therapies and the emergence of the first therapeutics. In this review, we present the use of RNA in anti-cancer therapies and the review of thrapies.

Evaluation of Whatman FTA cards for the preservation of yellow fever virus RNA for use in molecular diagnostics.

Yellow fever virus (YFV) is a flavivirus that frequently causes outbreaks of hemorrhagic fever in Africa and South America and is considered a reemerging public health threat. Accurate diagnosis of yellow fever (YF) disease is critical as one confirmed case constitutes an outbreak and may trigger a mass vaccination campaign. Highly sensitive and specific molecular diagnostics have been developed; however, these assays require maintenance of cold-chain during transport of specimens to prevent the degradation of viral RNA prior to testing. Such cold-chain requirements are difficult to meet in some regions. In this study, we investigated Whatman FTA cards as an alternative stabilization method of YFV RNA for use in molecular diagnosis. Using contrived specimens, linear regression analysis showed that RNA detection from a single 6mm FTA card punch was significantly less sensitive than traditional RNA extraction; however, pooling RNA extracted from two FTA punches significantly lowered the limit of detection to be equal to that of the traditional RNA extraction gold standard. In experiments addressing the ability of FTA card methodology to stabilize YFV RNA at variable temperature, RNA could be detected for more than two weeks following storage at 25°C. Even more promising, YFV RNA was detectable on cards held at 37°C from two days to over two weeks depending on viral input. FTA cards were also shown to stabilize YFV RNA at high humidity if cards were desiccated prior to inoculation. These results support that FTA cards could be cost effective and easy to use in molecular diagnosis of YF, preserving viral RNA to allow for positive diagnoses in situations where maintaining cold-chain is not feasible.

Nanomedicine for the Delivery of RNA in Cancer.

Simple SummaryCancer is a multifaceted, life-threatening, and genomically complex disease. The worldwide prevalence of cancer is so high that one in three people will develop cancer during their lifetime. Although the use of RNA therapy is promising to fight cancer, its efficient and safe delivery is still one of the significant challenges hampering its therapeutic application. Thus, the aim of the present review was to highlight the most recent developments in the field of nanomedicine RNA-associated therapies to fight cancer.The complexity, and the diversity of the different types of cancers allied to the tendency to form metastasis make treatment efficiency so tricky and often impossible due to the advanced stage of the disease in the diagnosis. In recent years, due to tremendous scientific breakthroughs, we have witnessed exponential growth in the elucidation of mechanisms that underlie carcinogenesis and metastasis. The development of more selective therapies made it possible to improve cancer treatment. Although interdisciplinary research leads to encouraging results, scientists still have a long exploration journey. RNA technology represents a promise as a therapeutic intervention for targeted gene silencing in cancer, and there are already some RNA-based formulations in clinical trials. However, the use of RNA as a therapeutic tool presents severe limitations, mainly related to its low stability and poor cellular uptake. Thus, the use of nanomedicine employing nanoparticles to encapsulate RNA may represent a suitable platform to address the major challenges hampering its therapeutic application. In this review, we have revisited the potential of RNA and RNA-associated therapies to fight cancer, also providing, as support, a general overview of nanoplatforms for RNA delivery.

The Progress and Promise of RNA Medicine─An Arsenal of Targeted Treatments.

In the past decade, there has been a shift in research, clinical development, and commercial activity to exploit the many physiological roles of RNA for use in medicine. With the rapid success in the development of lipid–RNA nanoparticles for mRNA vaccines against COVID-19 and with several approved RNA-based drugs, RNA has catapulted to the forefront of drug research. With diverse functions beyond the role of mRNA in producing antigens or therapeutic proteins, many classes of RNA serve regulatory roles in cells and tissues. These RNAs have potential as new therapeutics, with RNA itself serving as either a drug or a target. Here, based on the CAS Content Collection, we provide a landscape view of the current state and outline trends in RNA research in medicine across time, geography, therapeutic pipelines, chemical modifications, and delivery mechanisms.

RNA in situ hybridisation as a molecular diagnostic technique targeting IBA‐1 and CD204 in canine histiocytic sarcoma

BackgroundCanine histiocytic sarcoma (HS) is an aggressive cancer with morphologically variable features; therefore, obtaining a definitive diagnosis can be challenging. Two proteins, IBA‐1, ionised calcium‐binding adapter molecule 1, and CD204, a macrophage scavenger receptor, have been shown to be specific immunohistochemical markers helpful in distinguishing HS from other tumour types with similar morphological features.ObjectivesThis study was performed to demonstrate the use of RNA in situ hybridisation (ISH) technology allowing single‐molecule RNA visualisation in formalin‐fixed paraffin‐embedded (FFPE) tissues as a molecular tool for the diagnosis of canine HS.MethodsReverse transcription polymerase chain reaction (RT‐PCR) and western blot analysis for IBA‐1 and CD204 were performed to correlate gene expression and protein expression of these two markers in the histiocytic sarcoma DH82 cell line. RNA‐ISH for IBA‐1 and CD204 was performed on the DH82 cell line to validate the RNA‐ISH probes. RNA‐ISH and immunohistochemistry (IHC) were performed in clinical HS FFPE samples to demonstrate mRNA and protein expression of IBA‐1 and CD204. FFPE archived samples of canine round cell tumours, melanoma and anaplastic sarcoma were used as negative controls.ResultsRNA‐ISH and IHC showed moderate to strong expression for IBA‐1 and CD204 in the neoplastic cells in both the canine DH82 cell line and the archived canine HS samples. RNA‐ISH and IHC showed scattered positive staining in the control tumours samples, consistent with macrophagic infiltration.ConclusionRNA‐ISH for CD204 and IBA‐1 appeared to have a high specificity and sensitivity in our samples and may be an additional valuable diagnostic technique in identifying HS.

RNA-Based Strategies for Cell Reprogramming toward Pluripotency.

Cell therapy approaches to treat a wide range of pathologies have greatly benefited from cell reprogramming techniques that allow the conversion of a somatic cell into a pluripotent cell. Many technological developments have been made since the initial major discovery of this biological process. Recently reprogramming methods based on the use of RNA have emerged and seem very promising. Thus, in this review we will focus on presenting the interest of such methods for cell reprogramming but also how these RNA-based strategies can be extended to eventually lead to medical applications to improve healthspan and longevity.

Phase separation of RNA-binding protein promotes polymerase binding and transcription.

An RNA-involved phase-separation model has been proposed for transcription control. However, the molecular links that connect RNA to the transcription machinery remain missing. Here we find that RNA-binding proteins (RBPs) constitute half of the chromatin proteome in embryonic stem cells (ESCs), some being colocalized with RNA polymerase (Pol) II at promoters and enhancers. Biochemical analyses of representative RBPs show that the paraspeckle protein PSPC1 inhibits the RNA-induced premature release of Pol II, and makes use of RNA as multivalent molecules to enhance the formation of transcription condensates and subsequent phosphorylation and release of Pol II. This synergistic interplay enhances polymerase engagement and activity via the RNA-binding and phase-separation activities of PSPC1. In ESCs, auxin-induced acute degradation of PSPC1 leads to genome-wide defects in Pol II binding and nascent transcription. We propose that promoter-associated RNAs and their binding proteins synergize the phase separation of polymerase condensates to promote active transcription.

Nucleotide Metabolism Behind Epigenetics.

The mechanisms of epigenetic gene regulation—histone modifications, chromatin remodeling, DNA methylation, and noncoding RNA—use metabolites as enzymatic cofactors and substrates in reactions that allow chromatin formation, nucleotide biogenesis, transcription, RNA processing, and translation. Gene expression responds to demands from cellular processes that use specific metabolites and alters or maintains cellular metabolic status. However, the roles of metabolites—particularly nucleotides—as regulatory molecules in epigenetic regulation and biological processes remain largely unknown. Here we review the crosstalk between gene expression, nucleotide metabolism, and cellular processes, and explore the role of metabolism in epigenetics as a critical regulator of biological events.