Increased ß2-adrenergic signaling promotes fracture healing through callus neovascularization in mice.

Traumatic brain injury (TBI) leads to skeletal changes, including bone loss in the unfractured skeleton, and paradoxically accelerates healing of bone fractures; however, the mechanisms remain unclear. TBI is associated with a hyperadrenergic state characterized by increased norepinephrine release. Here, we identified the ß2-adrenergic receptor (ADRB2) as a mediator of skeletal changes in response to increased norepinephrine. In a murine model of femoral osteotomy combined with cortical impact brain injury, TBI was associated with ADRB2-dependent enhanced fracture healing compared with osteotomy alone. In the unfractured 12-week-old mouse skeleton, ADRB2 was required for TBI-induced decrease in bone formation and increased bone resorption. Adult 30-week-old mice had higher bone concentrations of norepinephrine, and ADRB2 expression was associated with decreased bone volume in the unfractured skeleton and better fracture healing in the injured skeleton. Norepinephrine stimulated expression of vascular endothelial growth factor A and calcitonin gene-related peptide-α (αCGRP) in periosteal cells through ADRB2, promoting formation of osteogenic type-H vessels in the fracture callus. Both ADRB2 and αCGRP were required for the beneficial effect of TBI on bone repair. Adult mice deficient in ADRB2 without TBI developed fracture nonunion despite high bone formation in uninjured bone. Blocking ADRB2 with propranolol impaired fracture healing in mice, whereas the ADRB2 agonist formoterol promoted fracture healing by regulating callus neovascularization. A retrospective cohort analysis of 72 patients with long bone fractures indicated improved callus formation in 36 patients treated with intravenous norepinephrine. These findings suggest that ADRB2 is a potential therapeutic target for promoting bone healing.

Loss-of-function variants affecting the STAGA complex component SUPT7L cause a developmental disorder with generalized lipodystrophy.

Generalized lipodystrophy is a feature of various hereditary disorders, often leading to a progeroid appearance. In the present study we identified a missense and a frameshift variant in a compound heterozygous state in SUPT7L in a boy with intrauterine growth retardation, generalized lipodystrophy, and additional progeroid features. SUPT7L encodes a component of the transcriptional coactivator complex STAGA. By transcriptome sequencing, we showed the predicted missense variant to cause aberrant splicing, leading to exon truncation and thereby to a complete absence of SUPT7L in dermal fibroblasts. In addition, we found altered expression of genes encoding DNA repair pathway components. This pathway was further investigated and an increased rate of DNA damage was detected in proband-derived fibroblasts and genome-edited HeLa cells. Finally, we performed transient overexpression of wildtype SUPT7L in both cellular systems, which normalizes the number of DNA damage events. Our findings suggest SUPT7L as a novel disease gene and underline the link between genome instability and progeroid phenotypes.

Loss of microglial MCT4 leads to defective synaptic pruning and anxiety-like behavior in mice.

Microglia, the innate immune cells of the central nervous system, actively participate in brain development by supporting neuronal maturation and refining synaptic connections. These cells are emerging as highly metabolically flexible, able to oxidize different energetic substrates to meet their energy demand. Lactate is particularly abundant in the brain, but whether microglia use it as a metabolic fuel has been poorly explored. Here we show that microglia can import lactate, and this is coupled with increased lysosomal acidification. In vitro, loss of the monocarboxylate transporter MCT4 in microglia prevents lactate-induced lysosomal modulation and leads to defective cargo degradation. Microglial depletion of MCT4 in vivo leads to impaired synaptic pruning, associated with increased excitation in hippocampal neurons, enhanced AMPA/GABA ratio, vulnerability to seizures and anxiety-like phenotype. Overall, these findings show that selective disruption of the MCT4 transporter in microglia is sufficient to alter synapse refinement and to induce defects in mouse brain development and adult behavior.

Increased beta2-adrenergic signaling is a targetable stimulus essential for bone healing by promoting callus neovascularization.

Traumatic brain injury (TBI) is associated with a hyperadrenergic state and paradoxically causes systemic bone loss while accelerating fracture healing. Here, we identify the beta2-adrenergic receptor (Adrb2) as a central mediator of these skeletal manifestations. While the negative effects of TBI on the unfractured skeleton can be explained by the established impact of Adrb2 signaling on bone formation, Adrb2 promotes neovascularization of the fracture callus under conditions of high sympathetic tone, including TBI and advanced age. Mechanistically, norepinephrine stimulates the expression of Vegfa and Cgrp primarily in periosteal cells via Adrb2, both of which synergistically promote the formation of osteogenic type-H vessels in the fracture callus. Accordingly, the beneficial effect of TBI on bone repair is abolished in mice lacking Adrb2 or Cgrp, and aged Adrb2-deficient mice without TBI develop fracture nonunions despite high bone formation in uninjured bone. Pharmacologically, the Adrb2 antagonist propranolol impairs, and the agonist formoterol promotes fracture healing in aged mice by regulating callus neovascularization. Clinically, intravenous beta-adrenergic sympathomimetics are associated with improved callus formation in trauma patients with long bone fractures. Thus, Adrb2 is a novel target for promoting bone healing, and widely used beta-blockers may cause fracture nonunion under conditions of increased sympathetic tone.

A proteomics analysis of 5xFAD mouse brain regions reveals the lysosome-associated protein Arl8b as a candidate biomarker for Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is characterized by the intra- and extracellular accumulation of amyloid-ß (Aß) peptides. How Aß aggregates perturb the proteome in brains of patients and AD transgenic mouse models, remains largely unclear. State-of-the-art mass spectrometry (MS) methods can comprehensively detect proteomic alterations, providing relevant insights unobtainable with transcriptomics investigations. Analyses of the relationship between progressive Aß aggregation and protein abundance changes in brains of 5xFAD transgenic mice have not been reported previously. METHODS: We quantified progressive Aß aggregation in hippocampus and cortex of 5xFAD mice and controls with immunohistochemistry and membrane filter assays. Protein changes in different mouse tissues were analyzed by MS-based proteomics using label-free quantification; resulting MS data were processed using an established pipeline. Results were contrasted with existing proteomic data sets from postmortem AD patient brains. Finally, abundance changes in the candidate marker Arl8b were validated in cerebrospinal fluid (CSF) from AD patients and controls using ELISAs. RESULTS: Experiments revealed faster accumulation of Aß42 peptides in hippocampus than in cortex of 5xFAD mice, with more protein abundance changes in hippocampus, indicating that Aß42 aggregate deposition is associated with brain region-specific proteome perturbations. Generating time-resolved data sets, we defined Aß aggregate-correlated and anticorrelated proteome changes, a fraction of which was conserved in postmortem AD patient brain tissue, suggesting that proteome changes in 5xFAD mice mimic disease-relevant changes in human AD. We detected a positive correlation between Aß42 aggregate deposition in the hippocampus of 5xFAD mice and the abundance of the lysosome-associated small GTPase Arl8b, which accumulated together with axonal lysosomal membranes in close proximity of extracellular Aß plaques in 5xFAD brains. Abnormal aggregation of Arl8b was observed in human AD brain tissue. Arl8b protein levels were significantly increased in CSF of AD patients. CONCLUSIONS: We report a comprehensive biochemical and proteomic investigation of hippocampal and cortical brain tissue derived from 5xFAD transgenic mice, providing a valuable resource to the neuroscientific community. We identified Arl8b, with significant abundance changes in 5xFAD and AD patient brains. Arl8b might enable the measurement of progressive lysosome accumulation in AD patients and have clinical utility as a candidate biomarker.

Diverse but unique astrocytic phenotypes during embryonic stem cell differentiation, culturing and development.

Astrocytes are resident glial cells of the central nervous system (CNS) that play complex and heterogeneous roles in brain development, homeostasis and disease. Since their vast involvement in health and disease is becoming increasingly recognized, suitable and reliable tools for studying these cells in vivo and in vitro are of utmost importance. One of the key challenges hereby is to adequately mimic their context-dependent in vivo phenotypes and functions in vitro. To better understand the spectrum of astrocytic variations in defined settings we performed a side-by-side-comparison of murine embryonic stem cell (ESC)-derived astrocytes as well as primary neonatal and adult astrocytes, revealing major differences on a functional and transcriptomic level, specifically on proliferation, migration, calcium signaling and cilium activity. Our results highlight the need to carefully consider the choice of astrocyte origin and phenotype with respect to age, isolation and culture protocols based on the respective biological question.

Non-redundant functions of group 2 innate lymphoid cells.

Protective immunity relies on the interplay of innate and adaptive immune cells with complementary and redundant functions. Innate lymphoid cells (ILCs) have recently emerged as tissue-resident, innate mirror images of the T cell system, with which they share lineage-specifying transcription factors and effector machinery1. Located at barrier surfaces, ILCs are among the first responders against invading pathogens and thus could potentially determine the outcome of the immune response2. However, so far it has not been possible to dissect the unique contributions of ILCs to protective immunity owing to limitations in specific targeting of ILC subsets. Thus, all of the available data have been generated either in mice lacking the adaptive immune system or with tools that also affect other immune cell subsets. In addition, it has been proposed that ILCs might be dispensable for a proper immune response because other immune cells could compensate for their absence3-7. Here we report the generation of a mouse model based on the neuromedin U receptor 1 (Nmur1) promoter as a driver for simultaneous expression of Cre recombinase and green fluorescent protein, which enables gene targeting in group 2 ILCs (ILC2s) without affecting other innate and adaptive immune cells. Using Cre-mediated gene deletion of Id2 and Gata3 in Nmur1-expressing cells, we generated mice with a selective and specific deficiency in ILC2s. ILC2-deficient mice have decreased eosinophil counts at steady state and are unable to recruit eosinophils to the airways in models of allergic asthma. Further, ILC2-deficient mice do not mount an appropriate immune and epithelial type 2 response, resulting in a profound defect in worm expulsion and a non-protective type 3 immune response. In total, our data establish non-redundant functions for ILC2s in the presence of adaptive immune cells at steady state and during disease and argue for a multilayered organization of the immune system on the basis of a spatiotemporal division of labour.

Analyses of circRNA Expression throughout the Light-Dark Cycle Reveal a Strong Regulation of Cdr1as, Associated with Light Entrainment in the SCN.

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that Cdr1as, a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, Cdr1as has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that Cdr1as is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of Cdr1as in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation.

Adolescence is a sensitive period for prefrontal microglia to act on cognitive development.

The prefrontal cortex (PFC) is a cortical brain region that regulates various cognitive functions. One distinctive feature of the PFC is its protracted adolescent maturation, which is necessary for acquiring mature cognitive abilities in adulthood. Here, we show that microglia, the brain's resident immune cells, contribute to this maturational process. We find that transient and cell-specific deficiency of prefrontal microglia in adolescence is sufficient to induce an adult emergence of PFC-associated impairments in cognitive functions, dendritic complexity, and synaptic structures. While prefrontal microglia deficiency in adolescence also altered the excitatory-inhibitory balance in adult prefrontal circuits, there were no cognitive sequelae when prefrontal microglia were depleted in adulthood. Thus, our findings identify adolescence as a sensitive period for prefrontal microglia to act on cognitive development.

Dataset for: Modeling chemotherapy induced neurotoxicity with human induced pluripotent stem cell (iPSC)-derived sensory neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent and potentially irreversible adverse event of cytotoxic chemotherapy. We evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for chemotherapy induced neurotoxicity. Sensory neurons differentiated from two established induced pluripotent stem cell lines were used (s.c. BIHi005-A https://hpscreg.eu/cell-line/BIHi005-A and BIHi004-B https://hpscreg.eu/cell-line/BIHi004-B, Berlin Institute of Health Stem Cell Core Facility). Cell viability and cytotoxicity assays were performed, comparing susceptibility to four neurotoxic and two non-neurotoxic drugs. RNA sequencing analyses in paclitaxel vs. vehicle (DMSO)-treated sensory neurons were performed. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to a dose dependent decline of cell viability in clinically relevant IC50 ranges, which was not the case for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. RNA sequencing analyses at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways in response to 1 µM paclitaxel. Neuroprotective effects of lithium chloride co-incubation, which were previously shown in rodent dorsal root ganglia, could be replicated in human iPSC-DSN. Cell lines from the two different donors BIHi005-A and BIHi004-B showed different responses to the neurotoxic treatment in cell viability and cytotoxicity assays.

Modeling chemotherapy induced neurotoxicity with human induced pluripotent stem cell (iPSC) -derived sensory neurons.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent, potentially irreversible adverse effect of cytotoxic chemotherapy often leading to a reduction or discontinuation of treatment which negatively impacts patients' prognosis. To date, however, neither predictive biomarkers nor preventive treatments for CIPN are available, which is partially due to a lack of suitable experimental models. We therefore aimed to evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for CIPN. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to axonal blebbing and a dose dependent decline of cell viability in clinically relevant IC50 ranges, which was not observed for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. Paclitaxel treatment effects were less pronounced after 24 h but prominent when treatment was applied for 72 h. Global transcriptome analyses performed at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways. We further evaluated if known neuroprotective strategies can be reproduced in iPSC-DSN and observed protective effects of lithium replicating findings from rodent dorsal root ganglia cells. Comparing sensory neurons derived from two different healthy donors, we found preliminary evidence that these cell lines react differentially to neurotoxic drugs as expected from the variable presentation of CIPN in patients. In conclusion, iPSC-DSN are a promising platform to study the pathogenesis of CIPN and to evaluate neuroprotective treatment strategies. In the future, the application of patient-specific iPSC-DSN could open new avenues for personalized medicine with individual risk prediction, choice of chemotherapeutic compounds and preventive treatments.

Transcriptomic profiling of SARS-CoV-2 infected human cell lines identifies HSP90 as target for COVID-19 therapy.

Detailed knowledge of the molecular biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is crucial for understanding of viral replication, host responses, and disease progression. Here, we report gene expression profiles of three SARS-CoV- and SARS-CoV-2-infected human cell lines. SARS-CoV-2 elicited an approximately two-fold higher stimulation of the innate immune response compared to SARS-CoV in the human epithelial cell line Calu-3, including induction of miRNA-155. Single-cell RNA sequencing of infected cells showed that genes induced by virus infections were broadly upregulated, whereas interferon beta/lambda genes, a pro-inflammatory cytokines such as IL-6, were expressed only in small subsets of infected cells. Temporal analysis suggested that transcriptional activities of interferon regulatory factors precede those of nuclear factor κB. Lastly, we identified heat shock protein 90 (HSP90) as a protein relevant for the infection. Inhibition of the HSP90 activity resulted in a reduction of viral replication and pro-inflammatory cytokine expression in primary human airway epithelial cells.

Enzymatic Dissociation Induces Transcriptional and Proteotype Bias in Brain Cell Populations.

Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. Here, we provide a systematic investigation of the influence of different cell isolation protocols on transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis of microglia. We show that standard enzymatic digestion of brain tissue at 37 °C induces profound and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared to an optimized mechanical dissociation protocol at 4 °C. These findings emphasize the risk of introducing technical biases and biological artifacts when implementing enzymatic digestion-based isolation methods for brain cell analyses.

An exploratory investigation of brain collateral circulation plasticity after cerebral ischemia in two experimental C57BL/6 mouse models.

Brain collateral circulation is an essential compensatory mechanism in response to acute brain ischemia. To study the temporal evolution of brain macro and microcollateral recruitment and their reciprocal interactions in response to different ischemic conditions, we applied a combination of complementary techniques (T2-weighted magnetic resonance imaging [MRI], time of flight [TOF] angiography [MRA], cerebral blood flow [CBF] imaging and histology) in two different mouse models. Hypoperfusion was either induced by permanent bilateral common carotid artery stenosis (BCCAS) or 60-min transient unilateral middle cerebral artery occlusion (MCAO). In both models, collateralization is a very dynamic phenomenon with a global effect affecting both hemispheres. Patency of ipsilateral posterior communicating artery (PcomA) represents the main variable survival mechanism and the main determinant of stroke lesion volume and recovery in MCAO, whereas the promptness of external carotid artery retrograde flow recruitment together with PcomA patency, critically influence survival, brain ischemic lesion volume and retinopathy in BCCAS mice. Finally, different ischemic gradients shape microcollateral density and size.

let-7 MicroRNAs Regulate Microglial Function and Suppress Glioma Growth through Toll-Like Receptor 7.

Microglia express Toll-like receptors (TLRs) that sense pathogen- and host-derived factors, including single-stranded RNA. In the brain, let-7 microRNA (miRNA) family members are abundantly expressed, and some have recently been shown to serve as TLR7 ligands. We investigated whether let-7 miRNA family members differentially control microglia biology in health and disease. We found that a subset of let-7 miRNA family members function as signaling molecules to induce microglial release of inflammatory cytokines, modulate antigen presentation, and attenuate cell migration in a TLR7-dependent manner. The capability of the let-7 miRNAs to control microglial function is sequence specific, mapping to a let-7 UUGU motif. In human and murine glioblastoma/glioma, let-7 miRNAs are differentially expressed and reduce murine GL261 glioma growth in the same sequence-specific fashion through microglial TLR7. Taken together, these data establish let-7 miRNAs as key TLR7 signaling activators that serve to regulate the diverse functions of microglia in health and glioma.

Transcriptional and Translational Differences of Microglia from Male and Female Brains.

Sex differences in brain structure and function are of substantial scientific interest because of sex-related susceptibility to psychiatric and neurological disorders. Neuroinflammation is a common denominator of many of these diseases, and thus microglia, as the brain's immunocompetent cells, have come into focus in sex-specific studies. Here, we show differences in the structure, function, and transcriptomic and proteomic profiles in microglia freshly isolated from male and female mouse brains. We show that male microglia are more frequent in specific brain areas, have a higher antigen-presenting capacity, and appear to have a higher potential to respond to stimuli such as ATP, reflected in higher baseline outward and inward currents and higher protein expression of purinergic receptors. Altogether, we provide a comprehensive resource to generate and validate hypotheses regarding brain sex differences.

Tumour ischaemia by interferon-γ resembles physiological blood vessel regression.

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ-GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.

Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers.

Insm1 cooperates with Neurod1 and Foxa2 to maintain mature pancreatic ß-cell function.

Key transcription factors control the gene expression program in mature pancreatic ß-cells, but their integration into regulatory networks is little understood. Here, we show that Insm1, Neurod1 and Foxa2 directly interact and together bind regulatory sequences in the genome of mature pancreatic ß-cells. We used Insm1 ablation in mature ß-cells in mice and found pronounced deficits in insulin secretion and gene expression. Insm1-dependent genes identified previously in developing ß-cells markedly differ from the ones identified in the adult. In particular, adult mutant ß-cells resemble immature ß-cells of newborn mice in gene expression and functional properties. We defined Insm1, Neurod1 and Foxa2 binding sites associated with genes deregulated in Insm1 mutant ß-cells. Remarkably, combinatorial binding of Insm1, Neurod1 and Foxa2 but not binding of Insm1 alone explained a significant fraction of gene expression changes. Human genomic sequences corresponding to the murine sites occupied by Insm1/Neurod1/Foxa2 were enriched in single nucleotide polymorphisms associated with glycolytic traits. Thus, our data explain part of the mechanisms by which ß-cells maintain maturity: Combinatorial Insm1/Neurod1/Foxa2 binding identifies regulatory sequences that maintain the mature gene expression program in ß-cells, and disruption of this network results in functional failure.

Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals.

Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions.