Cellular Messenger Research Articles

Biomarkers in Kidney Transplantation: A Rapidly Evolving Landscape.

The last decade has seen an explosion in clinical research focusing on the use of noninvasive biomarkers in kidney transplantation. Much of the published literature focuses on donor-derived cell-free DNA (dd-cfDNA). Although initially studied as a noninvasive means of identifying acute rejection, it is now clear that dd-cfDNA is more appropriately described as a marker of severe injury and irrespective of the etiology, elevated dd-cfDNA ≥0.5% portends worse graft outcomes. Blood gene expression profiling is also commercially available and has mostly been studied in the context of early identification of subclinical rejection, although additional data is needed to validate these findings. Torque teno virus, a ubiquitous DNA virus, has emerged as a biomarker of immunosuppression exposure as peripheral blood Torque teno virus copy numbers might mirror the intensity of host immunosuppression. Urinary chemokine tests including C-X-C motif chemokine ligand 9 and C-X-C motif chemokine ligand 10 have recently been assessed in large clinical trials and hold promising potential for early diagnosis of both subclinical and acute rejection, as well as, for long-term prognosis. Urinary cellular messenger RNA and exosome vesicular RNA based studies require additional validation. Although current data does not lend itself to conclusion, future studies on multimodality testing may reveal the utility of serial surveillance for individualization of immunosuppression and identify windows of opportunity to intervene early and before the irreversible allograft injury sets in.

Theoretical insights into the linker effects on the turn-on fluorescence behaviors in pyridazinone-containing NO probes

Fluorescent probes with preferred photophysical properties have attracted considerable attention for their advantages in real-time and accurate detection of signalling molecules in living organisms. Nitric oxide (NO) is a ubiquitous cellular messenger closely associated with many physiological and pathological processes. A NO fluorescent probe, PYSNO, based on the pyridazinone (PY) scaffold with o-phenylenediamine as the receptor and thiophene (S) as the linker has been synthesized. Inspired by the experimental guidance, three other dyes (PYSSNO, PYSONO and PYONO) were theoretically designed by replacing the S linker with thieno[3,2-b]thiophene (SS), thieno[3,2-b]thiophene 1,1-dioxide (SO) and thiophene 1,1-dioxide (O) groups. The photophysical properties were theoretically investigated in aqueous solution, by the combined time-dependent density functional theory, polarizable continuum model and thermal vibration correlation function approaches. Our results indicate that the emission wavelengths of all the designed dyes show red shifts due to either an increase in the conjugation length or electron-accepting ability of the linkers compared to PYSNO. The photoinduced electron transfer (PET) processes are all absent in these systems. PYSSNO and PYSONO are theoretically expected to be promising candidates for novel NO fluorescent probes, but the suitability of PYONO as a NO probe is compromised by the predicted non-luminescent emission before and after reaction with NO. Our study not only offers valuable insights into the detailed structure–property relationships, but also opens a new avenue for the rational design of efficient fluorescent sensors for NO detection.

A quantitative comparison of urine centrifugation and filtration for the isolation and analysis of urinary nucleic acid biomarkers

Urine is a rich source of nucleic acid biomarkers including cell-free DNA (cfDNA) and RNA for monitoring the health of kidney allografts. In this study, we aimed to evaluate whether urine filtration can serve as an alternative to the commonly used method of centrifugation to collect urinary fluid and cell pellets for isolating cfDNA and cellular messenger RNA (mRNA). We collected urine specimens from kidney allograft recipients and obtained the urine supernatant and cell pellet from each specimen using both filtration and centrifugation for paired analyses. We performed DNA sequencing to characterize the origin and properties of cfDNA, as well as quantitative PCR of mRNAs extracted from cell fractions. Our results showed that the biophysical properties of cfDNA, the microbial DNA content, and the tissues of origin of cfDNA were comparable between samples processed using filtration and centrifugation method. Similarly, mRNA quality and quantity obtained using both methods met our criteria for downstream application and the Ct values for each mRNA were comparable between the two techniques.The Ct values demonstrated a high degree of correlation. These findings suggest that urine filtration is a viable alternative to urine centrifugation for isolation of nucleic acid biomarkers from urine specimens.

Roles of RNA scaffolding in nanoscale Gag multimerization and selective protein sorting at HIV membranes.

HIV-1 Gag proteins can multimerize upon the viral genomic RNA or multiple random cellular messenger RNAs to form a virus particle or a virus-like particle, respectively. To date, whether the two types of particles form via the same Gag multimerization process has remained unclarified. Using photoactivated localization microscopy to illuminate Gag organizations and dynamics at the nanoscale, here, we showed that genomic RNA mediates Gag multimerization in a more cluster-centric, cooperative, and spatiotemporally coordinated fashion, with the ability to drive dense Gag clustering dependent on its ability to act as a long-stranded scaffold not easily attainable by cellular messenger RNAs. These differences in Gag multimerization were further shown to affect downstream selective protein sorting into HIV membranes, indicating that the choice of RNA for packaging can modulate viral membrane compositions. These findings should advance the understanding of HIV assembly and further benefit the development of virus-like particle-based therapeutics.

Exhaled Nitric Oxide as Biomarker of Type 2 Diseases.

Nitric oxide (NO) is a short-lived gas molecule which has been studied for its role as a signaling molecule in the vasculature and later, in a broader view, as a cellular messenger in many other biological processes such as immunity and inflammation, cell survival, apoptosis, and aging. Fractional exhaled nitric oxide (FeNO) is a convenient, easy-to-obtain, and non-invasive method for assessing active, mainly Th2-driven, airway inflammation, which is sensitive to treatment with standard anti-inflammatory therapy. Consequently, FeNO serves as a valued tool to aid the diagnosis and monitoring of several asthma phenotypes. More recently, FeNO has been evaluated in several other respiratory and/or immunological conditions, including allergic rhinitis, chronic rhinosinusitis with/without nasal polyps, atopic dermatitis, eosinophilic esophagitis, and food allergy. In this review, we aim to provide an extensive overview of the current state of knowledge about FeNO as a biomarker in type 2 inflammation, outlining past and recent data on the application of its measurement in patients affected by a broad variety of atopic/allergic disorders.

The Battle for Survival: The Role of RNA Non-Canonical Tails in the Virus-Host Interaction.

Terminal nucleotidyltransferases (TENTs) could generate a 'mixed tail' or 'U-rich tail' consisting of different nucleotides at the 3' end of RNA by non-templated nucleotide addition to protect or degrade cellular messenger RNA. Recently, there has been increasing evidence that the decoration of virus RNA terminus with a mixed tail or U-rich tail is a critical way to affect viral RNA stability in virus-infected cells. This paper first briefly introduces the cellular function of the TENT family and non-canonical tails, then comprehensively reviews their roles in virus invasion and antiviral immunity, as well as the significance of the TENT family in antiviral therapy. This review will contribute to understanding the role and mechanism of non-canonical RNA tailing in survival competition between the virus and host.

Subcellular second messenger networks drive distinct repellent-induced axon behaviors

Second messengers, including cAMP, cGMP and Ca2+ are often placed in an integrating position to combine the extracellular cues that orient growing axons in the developing brain. This view suggests that axon repellents share the same set of cellular messenger signals and that axon attractants evoke opposite cAMP, cGMP and Ca2+ changes. Investigating the confinement of these second messengers in cellular nanodomains, we instead demonstrate that two repellent cues, ephrin-A5 and Slit1, induce spatially segregated signals. These guidance molecules activate subcellular-specific second messenger crosstalk, each signaling network controlling distinct axonal morphology changes in vitro and pathfinding decisions in vivo.

A6A-seq: N6-allyladenosine-based cellular messenger RNA metabolic labelling and sequencing

The integration of RNA metabolic labelling by nucleoside analogues with high-throughput RNA sequencing has been harnessed to study RNA dynamics. The immunoprecipitation purification or chemical pulldown technique is generally required to enrich the analogue-labelled RNAs. Here we developed an a6A-seq method, which takes advantage of N6-allyladenosine (a6A) metabolic labelling on cellular mRNAs and profiles them in an immunoprecipitation-free and mutation-based manner. a6A plays a role as a chemical sequencing tag in that the iodination of a6A in mRNAs results in 1,N6-cyclized adenosine (cyc-A), which induces base misincorporation during RNA reverse transcription, thus making a6A-labelled mRNAs detectable by sequencing. A nucleic acid melting assay was utilized to investigate why cyc-A prefers to be paired with guanine. a6A-seq was utilized to study cellular gene expression changes under a methionine-free stress condition. Compared with regular RNA-seq, a6A-seq could more sensitively detect the change of mRNA production over a time scale. The experiment of a6A-containing mRNA immunoprecipitation followed by qPCR successfully validated the high-throughput a6A-seq data. Together, our results show a6A-seq is an effective tool to study RNA dynamics.

African swine fever virus I73R is a critical virulence-related gene: A potential target for attenuation

African swine fever virus (ASFV) is a large, double-stranded DNA virus that causes a fatal disease in pigs, posing a threat to the global pig industry. Whereas some ASFV proteins have been found to play important roles in ASFV-host interaction, the functional roles of many proteins are still largely unknown. In this study, we identified I73R, an early viral gene in the replication cycle of ASFV, as a key virulence factor. Our findings demonstrate that pI73R suppresses the host innate immune response by broadly inhibiting the synthesis of host proteins, including antiviral proteins. Crystallization and structural characterization results suggest that pI73R is a nucleic-acid-binding protein containing a Zα domain. It localizes in the nucleus and inhibits host protein synthesis by suppressing the nuclear export of cellular messenger RNA (mRNAs). While pI73R promotes viral replication, the deletion of the gene showed that it is a nonessential gene for virus replication. Invivo safety and immunogenicity evaluation results demonstrate that the deletion mutant ASFV-GZΔI73R is completely nonpathogenic and provides effective protection to pigs against wild-type ASFV. These results reveal I73R as a virulence-related gene critical for ASFV pathogenesis and suggest that it is a potential target for virus attenuation. Accordingly, the deletion mutant ASFV-GZΔI73R can be a potent live-attenuated vaccine candidate.

Loss-of-function of MEDIATOR 12 or 13 subunits causes the swelling of root hairs in response to sucrose and abscisic acid in Arabidopsis

ABSTRACT Root hairs are epidermal cell extensions that increase the root surface for water and nutrient acquisition. Thus, both the initiation and elongation of root hairs are critical for soil exploration and plant adaptation to ever changing growth conditions. Here, we describe the critical roles of two subunits of the Mediator complex, MED12 and MED13, in root hair growth in response to sucrose and abscisic acid, which are tightly linked to abiotic stress resistance. When compared to the WT, med12 and med13 mutants showed increased sensitivity to sucrose and ABA treatments on root meristem and elongation zones that were accompanied with alterations in root hair length and morphology, leading to the isodiametric growth of these structures. The swollen root hair phenotype appeared to be specific, since med8 or med16 mutants did not develop rounded hairs when supplied with 4.8% sucrose. Under standard growth medium, MED12 and MED13 were mainly expressed in root vascular tissues and cotyledons, and their expression was repressed by sucrose or ABA. Interestingly, med12 and med13 mutants manifested exacerbated levels of nitric oxide under normal growth conditions, and upon sucrose supplementation in trichoblast cells, which coincided with root hair deformation. Our results indicate that MED12 and MED13 play non-redundant functions for maintenance of root hair integrity in response to sucrose and ABA and involve nitric oxide as a cellular messenger in Arabidopsis thaliana.

Innate and Adaptive Immunity during SARS-CoV-2 Infection: Biomolecular Cellular Markers and Mechanisms.

The coronavirus 2019 (COVID-19) pandemic was caused by a positive sense single-stranded RNA (ssRNA) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, other human coronaviruses (hCoVs) exist. Historical pandemics include smallpox and influenza, with efficacious therapeutics utilized to reduce overall disease burden through effectively targeting a competent host immune system response. The immune system is composed of primary/secondary lymphoid structures with initially eight types of immune cell types, and many other subtypes, traversing cell membranes utilizing cell signaling cascades that contribute towards clearance of pathogenic proteins. Other proteins discussed include cluster of differentiation (CD) markers, major histocompatibility complexes (MHC), pleiotropic interleukins (IL), and chemokines (CXC). The historical concepts of host immunity are the innate and adaptive immune systems. The adaptive immune system is represented by T cells, B cells, and antibodies. The innate immune system is represented by macrophages, neutrophils, dendritic cells, and the complement system. Other viruses can affect and regulate cell cycle progression for example, in cancers that include human papillomavirus (HPV: cervical carcinoma), Epstein-Barr virus (EBV: lymphoma), Hepatitis B and C (HB/HC: hepatocellular carcinoma) and human T cell Leukemia Virus-1 (T cell leukemia). Bacterial infections also increase the risk of developing cancer (e.g., Helicobacter pylori). Viral and bacterial factors can cause both morbidity and mortality alongside being transmitted within clinical and community settings through affecting a host immune response. Therefore, it is appropriate to contextualize advances in single cell sequencing in conjunction with other laboratory techniques allowing insights into immune cell characterization. These developments offer improved clarity and understanding that overlap with autoimmune conditions that could be affected by innate B cells (B1+ or marginal zone cells) or adaptive T cell responses to SARS-CoV-2 infection and other pathologies. Thus, this review starts with an introduction into host respiratory infection before examining invaluable cellular messenger proteins and then individual immune cell markers.

Gastric epithelial response to milk fat using the semi-dynamic INFOGEST digestion model coupled with NCI-N87 cells

The stomach is a relevant spot of lipolysis for milk fat, but research on the effect of digested milk fat in the gastric epithelium is scarce and difficult to evaluate. In the present study, we implemented the semi-dynamic in vitro digestion model of INFOGEST, combined with gastric NCI-N87 cells, to study the effect of fat-free, whole conventional, and whole pasture-based milk on gastric epithelium. Cellular messenger ribonucleic acid (mRNA) expression of membrane fatty acids receptors (GPR41, GPR84), antioxidant enzymes (CAT, SOD, GPX), and inflammatory molecules (NF-κB p65, IL-1β, IL-6, IL-8 and TNF-α) was assessed. No significant differences were observed in mRNA expression of GPR41, GPR84, SOD, GPX, IL-6, IL-8, and TNF-α, after exposure of the NCI-N87 cells to milk digesta samples (p > 0.05). An increase of CAT mRNA expression was observed (p < 0.05), at a similar level, for all milk types. Whole milk digested samples induced higher mRNA expression of NF-κB p65 and IL-1β than fat-free milk (p < 0.05); while no differences were observed between whole conventional and whole pasture-based milk (p > 0.05). Moreover, the effect of milk digesta on gastric mRNA expression was studied in a scenario of subsequent stimulation of NCI-N87 monolayer with the pro-inflammatory cytokine IFN-γ. In these conditions, milk digesta samples increased CAT mRNA expression (p < 0.05), but had no effect in the expression of NF-κB p65 and IL-1β (p > 0.05). The increase of CAT mRNA expression suggests that milk fatty acids are used for energy production by gastric epithelial cells. Cellular antioxidant response to higher milk fatty acids availability could be associated to gastric epithelial inflammation, but did not contribute to increased inflammation in case of an external contact with IFN-γ. Besides, a conventional or a pasture-based origin did not affect the impact of whole milk in the NCI-N87 monolayer. The combined model responded to differences in milk fat content, which indicates its usefulness to study effects of foods at the gastric level.

Human cytomegalovirus infection perturbs neural progenitor cell fate via the expression of viral microRNAs.

Human cytomegalovirus (HCMV) preferentially targets neural progenitor cells (NPCs) in congenitally infected fetal brains, inducing neurodevelopmental disorders. While HCMV expresses several microRNAs (miRNAs) during infection, their roles in NPC infection are unclear. Here, we characterized expression of cellular and viral miRNAs in HCMV-infected NPCs during early infection by microarray and identified seven differentially expressed cellular miRNAs and six significantly upregulated HCMV miRNAs. Deep learning approaches were used to identify potential targets of significantly upregulated HCMV miRNAs against differentially expressed cellular mRNAs, and the associations with miRNA-mRNA expression changes were observed. Gene ontology enrichment analysis indicated cellular gene targets were significantly enriched in pathways involved in neurodevelopment and cell-cycle processes. Viral modulation of selected miRNAs and cellular gene targets involved in neurodevelopmental processes were further validated by qRT-PCR. Finally, a predicted 3' UTR target site of hcmv-miR-US25-1 in Jag1, a factor important for neurogenesis, was confirmed by mutagenesis. Reduction of Jag1 RNA and protein levels in NPCs was observed in response to transient expression of hcmv-miR-US25-1. A hcmv-miR-US25-1 mutant virus (ΔmiR-US25) displayed limited ability to downregulate Jag1 mRNA levels and protein levels during the early infection stage compared with the wild type virus. Our collective experimental and computational investigation of miRNAs and cellular mRNAs expression in HCMV-infected NPCs yields new insights into the roles of viral miRNAs in regulating NPC fate and their contributions to HCMV neuropathogenesis. This article is protected by copyright. All rights reserved.

Druggable targets for the immunopathy of Alzheimer's disease.

Alzheimer's disease (AD) is one of the leading threats to the health and socioeconomic well-being of humankind. Though research to develop disease modifying therapies for AD has traditionally focussed on the misfolding and aggregation of proteins, this approach has failed to yield a definitively curative agent. Accordingly, the search for additional or alternative approaches is a medicinal chemistry priority. Dysfunction of the brain's neuroimmune-neuroinflammation axis has emerged as a leading contender. Neuroimmunity however is mechanistically complex, rendering the recognition of candidate receptors a challenging task. Herein, a review of the role of neuroimmunity in the biomolecular pathogenesis of AD is presented with the identification of a 'druggable dozen' targets; in turn, each identified target represents one or more discrete receptors centred on a common biochemical mechanism. The druggable dozen is composed of both cellular and molecular messenger targets, with a 'targetable ten' microglial targets as well as two cytokine-based targets. For each target, the underlying molecular basis, with a consideration of strengths and weaknesses, is considered.

Correlation of NF-kB Serum Level with the Expression Pattern of Microrna-221 in a Sample of Hcv-Exposed Iraqi Patients

Background: Hepatitis C virus (HCV) has the ability to change cellular messenger RNA transcription and translation by stimulating the synthesis of cellular microRNAs (miRNAs) that impair immune response and facilitate viral reproduction. One of the most important members of the immune response against HCV is nuclear factor-kappa B (NF-κB), which is regulated by cellular miRNAs. Aims: we aimed to investigate the correlation of NF-κB serum level with circulatory miRNA-221 (miR-221) fold change in HCV-exposed individuals. Materials and Methods: Serum level of NF-κB in 88 samples (22 patients with persistent HCV infection, 22 individuals with spontaneous HCV virus clearance, 22 individuals treated with direct-acting antivirals (DAAs) drugs, and 22 uninfected apparently healthy blood donors as control) was measured by enzyme-linked immunosorbent assay. Reverse transcriptase–polymerase chain reaction was used to quantify the expression fold of circulatory miR-221. Results: The results showed that there was a significant decrease in the mean level of NF-κB at P &lt; 0.000 among HCV-exposed patients (2.0058 ng/ml) as compared to the control group (2.9841 ng/ml). The mean fold change of miR-221 was significantly upregulated about six more times among HCV-exposed patients (mean = 6.3545) compared to the control group (mean = 1.1864). Furthermore, the mean ± standard deviation of miR-221 fold change in patients with persistent HCV infection was significantly higher compared to patients cured after DAA therapy (P = 0.048), there was a weak negative correlation (r = −0.246, P = 0.021) between NF-κB serum level and miR-221 folding level. Conclusion: HCV infection disrupts NF-κB activation, resulting in dysregulation of miR-221 that persists long after the virus has been cleared. Thus, quantification of serum NF-κB and miR-221in HCV-exposed patients could be used as noninvasive prognostic marker during long-term follow-up. Furthermore, a miRNAs profile analysis can help distinguish HCV-exposed from healthy individuals.

Role of calcium dysregulation in Alzheimer's disease and its therapeutic implications.

The increasing incidence of Alzheimer's disease (AD) coupled with the lack of therapeutics to address the underlying pathology of the disease has necessitated the need for exploring newer targets. Calcium dysregulation represents a relatively newer target associated with AD. Ca+2 serves as an important cellular messenger in neurons. The concentration of the Ca+2 ion needs to be regulated at optimal concentrations intracellularly for normal functioning of the neurons. This is achieved with the help of mitochondria, endoplasmic reticulum, and neuronal plasma membrane channel proteins. Disruption in normal calcium homeostasis can induce formation of amyloid beta plaques, accumulation of neurofibrillary tangles, and dysfunction of synaptic plasticity, which in turn can affect calcium homeostasis further, thus forming a vicious cycle. Hence, understanding calcium dysregulation can prove to be a key to develop newer therapeutics. This review provides detailed account of physiology of calcium homeostasis and its dysregulation associated with AD. Further, with an understanding of various receptors and organelles involved in these pathways, the review also discusses various calcium channel blockers explored in AD hand in hand with some multitarget molecules addressing calcium as one of the targets.

Roles and mechanisms of CircRNAs in ovarian cancer.

Ovarian cancer (OC) is one of the female malignancies with nearly 45% 5-year survival rate. Circular RNAs (circRNAs), a kind of single-stranded non-coding RNAs, are generated from the back-splicing of cellular housekeeping noncoding RNAs and precursor messenger RNAs. Recent studies revealed that circRNAs have different biological function, including sponging miRNAs, encoding micropeptides, regulating stability of cytoplasmic mRNAs, affecting transcription and splicing, via interacting with DNA, RNA and proteins. Due to their stability, circRNAs have the potential of acting as biomarkers and treatment targets. In this review, we briefly illustrate the biogenesis mechanism and biological function of circRNAs in OC, and make a perspective of circRNAs drug targeting immune responses and signaling pathways in OC. This article can provide a systematic view into the current situation and future of circRNAs in OC.

Exosomal microRNAs and Progression of Nonalcoholic Steatohepatitis (NASH).

Nonalcoholic fatty liver disease (NAFLD)/metabolic associated fatty liver disease (MAFLD) is becoming a public health problem worldwide. Steatosis as the simple form and nonalcoholic steatohepatitis (NASH) as its progression form are commonly seen in liver biopsy specimens from patients with obesity, diabetes, hyperlipidemia, hypertension, and the use of certain drugs. Patients with NASH and advanced fibrosis were associated with increased risks of liver-related complications, including hepatocellular carcinoma (HCC). However, the mechanisms regarding the progression from simple steatosis to NASH fibrosis remain incompletely understood. Because NASH-caused liver injury is a complex process and multiple cell types are involved, intercellular communication is likely mediated by extracellular vesicles. Exosomes are a type of small extracellular vesicles and contain various cellular molecules, including proteins, messenger RNAs (mRNAs), and microRNAs (miRNAs). MiRNAs are short, non-coding RNA species that are important post-transcriptional regulators of gene expression and may play an important role in the pathogenesis of NALFD/NASH. In this article, we review the articles about NASH and exosomal miRNAs published in the most recent English literature through PubMed search and discuss the most recent criteria for histological diagnosis, pathogenesis from steatosis to NASH, roles of exosomal miRNAs in NASH pathogenesis and progression, as well as their potential in future clinical diagnosis and treatment for patients with NASH.

Bio-orthogonal Toolbox for Monitoring Nitric Oxide in Targeted Organelles of Live Cells and Zebrafishes.

Nitric oxide (NO), playing crucial roles as a cellular messenger and as a toxic ROS, is highly related to the physiological and pathological states of living systems. The very wide but very uneven distribution of this radical gas in the inhomogeneous biological microenvironment imposes big challenges for specifically detecting its local level in certain subcellular areas, which calls for a long list of NO probes for each target. In order to simplify the syntheses and designs of these probes, herein it is proposed to construct a versatile NO-sensing toolbox based on a bio-orthogonal concept, i.e., inverse electron demand Diels-Alder click reaction between tetrazine and strained alkyne BCN. On the one hand, rhodamine-o-phenylenediamine as the NO-responsive scaffold is coupled with a tetrazine unit to generate a general probe TMR-Tz-NO, which, to our knowledge, is the first case of the tetrazine-coupled analyte-responsive probe. On the other hand, the BCN moiety is connected to different targeting groups, such as TPP, morpholine, and Ac4ManN, targeting to mitochondria, lysosomes, and membranes, respectively. It works well to use TMR-Tz-NO to match with any targetable BCN counterpart in this toolbox to achieve the imaging of NO in the corresponding subcellular area. For example, through metabolism, Ac4ManN-BCN is effectively taken and grows on the cell membranes. The bio-orthogonal reaction between TMR-Tz-NO and Ac4ManN-BCN makes the NO probe anchored to the membrane surface permanently. The zebrafish experiment revealed that this bio-orthogonal pair can track and image the NO produced during inflammation in vivo.

Biogenesis and Regulatory Roles of Circular RNAs.

Covalently closed, single-stranded circular RNAs can be produced from viral RNA genomes as well as from the processing of cellular housekeeping noncoding RNAs and precursor messenger RNAs. Recent transcriptomic studies have surprisingly uncovered that many protein-coding genes can be subjected to backsplicing, leading to widespread expression of a specific type of circular RNAs (circRNAs) in eukaryotic cells. Here, we discuss experimental strategies used to discover and characterize diverse circRNAs at both the genome and individual gene scales. We further highlight the current understanding of how circRNAs are generated and how the mature transcripts function. Some circRNAs act as noncoding RNAs to impact gene regulation by serving as decoys or competitors for microRNAs and proteins. Others form extensive networks of ribonucleoprotein complexes or encode functional peptides that are translated in response to certain cellular stresses. Overall, circRNAs have emerged as an important class of RNAmolecules in gene expression regulation that impact many physiological processes, including early development, immune responses, neurogenesis, and tumorigenesis.