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Circular RNA is a group of covalently closed, single-stranded transcripts with unique biogenesis, stability, and conformation that play distinct roles in modulating cellular functions and also possess a great potential for developing circular RNA-based therapies. Importantly, due to its circular conformation, circular RNA generates distinct intramolecular base pairing that is different from the linear transcript. In this perspective, we review how circular RNA conformation can affect its turnover and modes of action, as well as what factors can modulate circular RNA conformation. We also discuss how understanding circular RNA conformation can facilitate learning about their functions as well as the remaining technological issues to further address their conformation. These efforts will ultimately inform the design of circular RNA-based platforms for biomedical applications., Competing Interests: Declaration of interests L.-L.C. is a member of the Molecular Cell advisory board and a scientific co-founder of RiboX therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Circular RNAs (circRNAs) have emerged as a promising alternative to linear mRNA, owing to their unique properties and potential therapeutic applications, driving the development of novel approaches for their production. This study introduces a cis-splicing system that efficiently produces circRNAs by incorporating a ribozyme core at one end of the precursor, thereby eliminating the need for additional spacer elements between the ribozyme and the gene of interest (GOI). In this cis-splicing system, sequences resembling homologous arms at both ends of the precursor are crucial for forming the P9.0 duplex, which in turn facilitates effective self-splicing and circularization. We demonstrate that the precise recognition of the second transesterification site depends more on the structural characteristics of P9.0 adjacent to the ωG position than on the nucleotide composition of the P9.0-ωG itself. Further optimization of structural elements, like P10 and P1-ex, significantly improves circularization efficiency. The circRNAs generated through the cis-splicing system exhibit prolonged protein expression and minimal activation of the innate immune response. This study provides a comprehensive exploration of circRNA generation via a novel strategy and offers valuable insights into the structural engineering of RNA, paving the way for future advancements in circRNA-based applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Synthetic messenger RNA (mRNA) has been focused on as an emerging application for mRNA-based therapies and vaccinations. Recently, synthetic circular RNAs (circRNAs) have shown promise as a new class of synthetic mRNA that enables superior stability and persistent gene expression in cells. However, translational control of circRNA remained challenging. Here, we develop 'circRNA switches' capable of controlling protein expression from circRNA by sensing intracellular RNA or proteins. We designed microRNA (miRNA) and protein-responsive circRNA switches by inserting miRNA-binding or protein-binding sequences into untranslated regions (UTRs), or Coxsackievirus B3 Internal Ribosome Entry Site (CVB3 IRES), respectively. Engineered circRNAs efficiently expressed reporter proteins without inducing severe cell cytotoxicity and immunogenicity, and responded to target miRNAs or proteins, controlling translation levels from circRNA in a cell type-specific manner. Moreover, we constructed circRNA-based gene circuits that selectively activated translation by detecting endogenous miRNA, by connecting miRNA and protein-responsive circRNAs. The designed circRNA circuits performed better than the linear mRNA-based circuits in terms of persistent expression levels. Synthetic circRNA devices provide new insights into RNA engineering and have a potential for RNA synthetic biology and therapies., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

3D structures of RNAs are the basis for understanding their biological functions. However, experimentally solved RNA 3D structures are very limited. Therefore, many computational methods have been proposed to solve this problem, including our 3dRNA. 3dRNA is an automated template-based method of building RNA 3D structures from sequences and secondary structures by using the smallest secondary elements (SSEs) (http://biophy.hust.edu.cn/new/3dRNA). The first version of 3dRNA simply predicts an assembled structure for a target RNA. Later, it is improved to generate a set of assembled models and a method to further optimize them using experimental or theoretical restraints. In particular, pseudoknot base pairings are treated as restraints to solve the problem of no 3D templates for pseudoknots. Here 3dRNA is further extended to predict the 3D structures of circular RNAs since thousands of circular RNAs have been found recently but no 3D structures of them have been determined up to now. We show that circular RNAs can be divided into four types and two types show similar 3D structures with their linear counterparts while two types very different. We also show that the predicted structures of circular RNAs can bind to their ligands more stable than those of their linear counterparts, consistent with experimental results., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hepatitis delta virus (HDV) is a significant human pathogen that causes acute and chronic liver disease; there is no licensed therapy. HDV is a circular negative-sense single-stranded RNA (ssRNA) virus that produces three RNAs in infected cells, genome, antigenome, and mRNA; the latter encodes hepatitis delta antigen (HDAg), the viral protein. These RNAs are synthesized by host DNA-dependent RNA polymerase acting as an RNA-dependent RNA polymerase. Although HDV genome RNA accumulates to high levels in infected cells, the mechanism by which this process occurs remains poorly understood. For example, the nature of the 5' end of the genome, including the synthesis start site and its chemical composition, is not known. Analysis of this process has been challenging because the initiation site is part of an unstable precursor in the rolling-circle mechanism by which HDV genome RNA is synthesized. In this study, circular HDV antigenome RNAs synthesized in vitro were used to directly initiate HDV genome RNA synthesis in transfected cells, thus enabling the detection of the 5' end of the genome RNA. The 5' end of this RNA is capped, as expected for a polymerase II product. Initiation begins at position 1646 on the genome, which is located near the loop end proximal to the start site for HDAg mRNA synthesis. Unexpectedly, synthesis begins with a guanosine that is not conventionally templated by the HDV RNA. IMPORTANCE Hepatitis delta virus (HDV) is a unique virus that causes severe liver disease. It uses host RNA polymerase II to copy its circular RNA genome in a unique and poorly understood process. Although the virus RNA accumulates to high levels within infected cells, it is not known how the synthesis of the viral RNA begins or even where on the genome synthesis starts. Here, we identify the start site for the initiation of HDV genome RNA synthesis as position 1646, which is at one end of the closed-hairpin-like structure of the viral RNA. The 5' end of the RNA is capped, as expected for polymerase II products. However, RNA synthesis begins with a guanosine that is not present in the genome. Thus, although HDV uses polymerase II to synthesize the viral genome, some details of the initiation process are different. These differences could be important for successfully targeting virus replication.

Circular RNAs (circRNAs) are non-coding RNAs with a special circular structure produced formed by the reverse splicing mechanism. Increasing evidence shows that circular RNAs can directly bind to RNA-binding proteins (RBP) and play an important role in a variety of biological activities. The interactions between circRNAs and RBPs are key to comprehending the mechanism of posttranscriptional regulation. Accurately identifying binding sites is very useful for analyzing interactions. In past research, some predictors on the basis of machine learning (ML) have been presented, but prediction accuracy still needs to be ameliorated. Therefore, we present a novel calculation model, CRBPDL, which uses an Adaboost integrated deep hierarchical network to identify the binding sites of circular RNA-RBP. CRBPDL combines five different feature encoding schemes to encode the original RNA sequence, uses deep multiscale residual networks (MSRN) and bidirectional gating recurrent units (BiGRUs) to effectively learn high-level feature representations, it is sufficient to extract local and global context information at the same time. Additionally, a self-attention mechanism is employed to train the robustness of the CRBPDL. Ultimately, the Adaboost algorithm is applied to integrate deep learning (DL) model to improve prediction performance and reliability of the model. To verify the usefulness of CRBPDL, we compared the efficiency with state-of-the-art methods on 37 circular RNA data sets and 31 linear RNA data sets. Moreover, results display that CRBPDL is capable of performing universal, reliable, and robust. The code and data sets are obtainable at https://github.com/nmt315320/CRBPDL.git., Competing Interests: The authors have declared no competing interests.

We introduce ViroidDB, a value-added database that attempts to collect all known viroid and viroid-like circular RNA sequences into a single resource. Spanning about 10 000 unique sequences, ViroidDB includes viroids, retroviroid-like elements, small circular satellite RNAs, ribozyviruses, and retrozymes. Each sequence's secondary structure, ribozyme content, and cluster membership are predicted via a custom pipeline optimized for handling circular RNAs. The data can be explored via a purpose-built user interface that features visualizations, multiple sequence alignments, and a portal for downloading bulk data. Users can browse the data by sequence type, taxon, or typo-tolerant search of metadata fields. The database is freely accessible at https://viroids.org., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

Here, we performed N6-methyladenosine (m6A) RNA sequencing to determine the circRNA m6A methylation changes in the placentas during the pathogenesis of preeclampsia (PE). We verified the expression of the circRNA circPAPPA2 using quantitative reverse transcription-PCR. An invasion assay was carried out to identify the role of circPAPPA2 in the development of PE. Mechanistically, we investigated the cause of the altered m6A modification of circPAPPA2 through overexpression and knockdown cell experiments, RNA immunoprecipitation, fluorescence in situ hybridization and RNA stability experiments. We found that increases in m6A-modified circRNAs are prevalent in PE placentas and that the main changes in methylation occur in the 3'UTR and near the start codon, implicating the involvement of these changes in PE development. We also found that the levels of circPAPPA2 are decreased but that m6A modification is augmented. Furthermore, we discovered that methyltransferase‑like 14 (METTL14) increases the level of circPAPPA2 m6A methylation and that insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) maintains circPAPPA2 stability. Decreases in IGF2BP3 levels lead to declines in circPAPPA2 levels. In summary, we provide a new vision and strategy for the study of PE pathology and report that placental circRNA m6A modification appears to be an important regulatory mechanism., (© 2021. The Author(s).)

Circular RNA is progressively reported to occur in various species including mammals where it is thought to be involved in the post-transcriptional regulation of gene expression, partly via interactions with microRNA. Here, we asked whether the circular topology causes functional differences to linear forms when interacting with short RNA strands in vitro and in human cells. Kinetic studies with human bladder cancer-derived synthetic circular RNA versus linear transcripts, respectively, with short oligoribonucleotides showed similar association rates for both topologies. Conversely, a substantial topology-related difference was measured for the activation entropy and the activation enthalpy of RNA-RNA annealing. This finding strongly indicates a significant difference of the mechanism of RNA-RNA interactions. To investigate whether these characteristics of circular RNA are biologically meaningful we performed transient transfection experiments with a microRNA-regulated expression system for luciferase in bladder cancer-derived cells. We co-transfected linear or circular RNA containing one microRNA binding site for the target-suppressing microRNA mlet7a. Here, the circular isoform showed a strongly increased competition with microRNA function versus linear versions. In summary, this study suggests novel topology-related characteristics of RNA-RNA interactions involving circRNA in vitro and in living cells.

Circular RNA (circRNA) is a large class of covalently closed circRNA. As a member of competitive endogenous RNA, it participates in the regulation of circRNA-miRNA-mRNA network and plays an important role in the regulation of physiology and pathology. CircRNA is produced by the reverse splicing of exon, intron or both, forming exon or intron circRNA. Studies have shown that circRNA is a ubiquitous molecule, which exceeds the linear mRNA distributed in human cells. Because of its covalent closed-loop structure, circRNA is resistant to RNase R, which is more stable than linear mRNA; circRNA is highly conserved in different species. It was found that circRNA competitively adsorbs miRNA, as a miRNA sponge, to involve in the expression regulation of a variety of genes and plays an important role in tumor development, invasion, metastasis and other processes. These molecules offer new potential opportunities for therapeutic intervention and serve as biomarkers for diagnosis. In this paper, the origin, characteristics and functions of circRNA and its role in tumor development, invasion and metastasis, diagnosis and prognosis are reviewed., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Circular RNAs (circRNAs) are widely expressed in highly diverged eukaryotes. Although circRNAs have been known for many years, their function remains unclear. Interaction with RNA-binding protein (RBP) to influence post-transcriptional regulation is considered to be an important pathway for circRNA function, such as acting as an oncogenic RBP sponge to inhibit cancer. In this study, we design a deep learning framework, CRPBsites, to predict the binding sites of RBPs on circRNAs. In this model, the sequences of variable-length binding sites are transformed into embedding vectors by word2vec model. Bidirectional LSTM is used to encode the embedding vectors of binding sites, and then they are fed into another LSTM decoder for decoding and classification tasks. To train and test the model, we construct four datasets that contain sequences of variable-length binding sites on circRNAs, and each set corresponds to an RBP, which is overexpressed in bladder cancer tissues. Experimental results on four datasets and comparison with other existing models show that CRPBsites has superior performance. Afterwards, we found that there were highly similar binding motifs in the four binding site datasets. Finally, we applied well-trained CRPBsites to identify the binding sites of IGF2BP1 on circCDYL, and the results proved the effectiveness of this method. In conclusion, CRPBsites is an effective prediction model for circRNA-RBP interaction site identification. We hope that CRPBsites can provide valuable guidance for experimental studies on the influence of circRNA on post-transcriptional regulation., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Circular RNAs (circRNAs) act through multiple mechanisms via their sequence features to fine-tune gene expression networks. Due to overlapping sequences with linear cognates, identifying internal sequences of circRNAs remains a challenge, which hinders a comprehensive understanding of circRNA functions and mechanisms. Here, based on rolling circular reverse transcription and nanopore sequencing, we developed circFL-seq, a full-length circRNA sequencing method, to profile circRNA at the isoform level. With a customized computational pipeline to directly identify full-length sequences from rolling circular reads, we reconstructed 77,606 high-quality circRNAs from seven human cell lines and two human tissues. circFL-seq benefits from rolling circles and long-read sequencing, and the results showed more than tenfold enrichment of circRNA reads and advantages for both detection and quantification at the isoform level compared to those for short-read RNA sequencing. The concordance of the RT-qPCR and circFL-seq results for the identification of differential alternative splicing suggested wide application prospects for functional studies of internal variants in circRNAs. Moreover, the detection of fusion circRNAs at the omics scale may further expand the application of circFL-seq. Taken together, the accurate identification and quantification of full-length circRNAs make circFL-seq a potential tool for large-scale screening of functional circRNAs., Competing Interests: ZL, CT, SL, MD, YB, XH, YL, JC, EY No competing interests declared, (© 2021, Liu et al.)

Non-coding RNAs are emerging as critical molecules in the genesis, progression, and therapy resistance of cutaneous melanoma. This includes circular RNAs (circRNAs), a class of non-coding RNAs with distinct characteristics that forms through non-canonical back-splicing. In this review, we summarize the features and functions of circRNAs and introduce the current knowledge of the roles of circRNAs in melanoma. We also highlight the various mechanisms of action of the well-studied circRNA CDR1as and describe how it acts as a melanoma tumor suppressor. We further discuss the utility of circRNAs as biomarkers, therapeutic targets, and therapeutic agents in melanoma and outline challenges that must be overcome to comprehensively characterize circRNA functions., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Rheumatic arthritis (RA) is an autoimmune disease with bad effects. Recent researches have shown that circular RNAs (circRNAs) could affect the progress of RA, but the mechanism still indistinct. In this work, we explored the roles of circ_0025908 in RA., Methods: The levels of circ_0025908, microRNA-137 (miR-137), and mRNA of homeodomain-interacting protein kinase 2 (HIPK2) were detected by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) in RA tissues. Meanwhile, the level of HIPK2 was quantified by Western blot analysis. Besides, the cell functions were examined by CCK8 assay, EdU assay, flow cytometry assay, ELISA, and Western blot. Furthermore, the interplay between miR-137 and circ_0025908 or HIPK2 was detected by dual-luciferase reporter assay., Results: The levels of circ_0025908 and HIPK2 were upregulated, and the miR-137 level was decreased in RA tissues in contrast to that in normal tissues. For functional analysis, circ_0025908 deficiency inhibited cell vitality, cell mitotic cycle, cell proliferation, and immunoreaction in RA cells, whereas promoted cell apoptosis. Moreover, miR-137 was confirmed to repress the progression of RA cells by suppressing HIPK2. In mechanism, circ_0025908 acted as a miR-137 sponge to regulate the level of HIPK2., Conclusion: Circ_0025908 facilitates the development of RA through increasing HIPK2 expression by regulating miR-137, which also offered an underlying targeted therapy for RA treatment., (© 2021. The Author(s).)

Rational design of aptamers to incorporate unnatural nucleotides and special chemical moieties can expand their functional complexity and diversity. Spiegelmer (L-RNA aptamer) is a unique class of aptamer that is composed of unnatural L-RNA nucleotides, and so far there are limited L-RNA aptamer candidates and applications being reported. Moreover, the target binding properties of current L-RNA aptamers require significant improvement. Here, using L-Apt.4-1c as an example, we develop a simple and robust strategy to generate the first circular L-RNA aptamer, cycL-Apt.4-1c, quantitatively, demonstrate substantial enhancement in binding affinity and selectivity toward its target, and notably report novel applications of circular L-RNA aptamer in controlling RNA-protein interaction, and gene activity including telomerase activity and gene expression. Our approach and findings will be applicable to any L-RNA aptamers and open up a new avenue for diverse applications., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Circular RNAs (circRNAs) are highly expressed in the brain and their expression increases during neuronal differentiation. The factors regulating circRNAs in the developing mouse brain are unknown. NOVA1 and NOVA2 are neural-enriched RNA-binding proteins with well-characterized roles in alternative splicing. Profiling of circRNAs from RNA-seq data revealed that global circRNA levels were reduced in embryonic cortex of Nova2 but not Nova1 knockout mice. Analysis of isolated inhibitory and excitatory cortical neurons lacking NOVA2 revealed an even more dramatic reduction of circRNAs and establishes a widespread role for NOVA2 in enhancing circRNA biogenesis. To investigate the cis-elements controlling NOVA2-regulation of circRNA biogenesis, we generated a backsplicing reporter based on the Efnb2 gene. We found that NOVA2-mediated backsplicing of circEfnb2 was impaired when YCAY clusters located in flanking introns were mutagenized. CLIP (cross-linking and immunoprecipitation) and additional reporter analyses demonstrated the importance of NOVA2 binding sites located in both flanking introns of circRNA loci. NOVA2 is the first RNA-binding protein identified to globally promote circRNA biogenesis in the developing brain., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Prediction of protein-RNA interactions is important to understand post-transcriptional events taking place in the cell. Here we introduce catRAPID omics v2.0, an update of our web server dedicated to the computation of protein-RNA interaction propensities at the transcriptome- and RNA-binding proteome-level in 8 model organisms. The server accepts multiple input protein or RNA sequences and computes their catRAPID interaction scores on updated precompiled libraries. Additionally, it is now possible to predict the interactions between a custom protein set and a custom RNA set. Considerable effort has been put into the generation of a new database of RNA-binding motifs that are searched within the predicted RNA targets of proteins. In this update, the sequence fragmentation scheme of the catRAPID fragment module has been included, which allows the server to handle long linear RNAs and to analyse circular RNAs. For the top-scoring protein-RNA pairs, the web server shows the predicted binding sites in both protein and RNA sequences and reports whether the predicted interactions are conserved in orthologous protein-RNA pairs. The catRAPID omics v2.0 web server is a powerful tool for the characterization and classification of RNA-protein interactions and is freely available at http://service.tartaglialab.com/page/catrapid_omics2_group along with documentation and tutorial., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Self-cleaving ribozymes are catalytic RNAs that cut themselves at a specific inter-nucleotide linkage. They serve as a model of RNA catalysis, and as an important tool in biotechnology. For most of the nine known structural classes of self-cleaving ribozymes, at least hundreds of examples are known, and some are present in multiple domains of life. By contrast, only four unique examples of the hairpin ribozyme class are known, despite its discovery in 1986. We bioinformatically predicted 941 unique hairpin ribozymes of a different permuted form from the four previously known hairpin ribozymes, and experimentally confirmed several diverse predictions. These results profoundly expand the number of natural hairpin ribozymes, enabling biochemical analysis based on natural sequences, and suggest that a distinct permuted form is more biologically relevant. Moreover, all novel hairpins were discovered in metatranscriptomes. They apparently reside in RNA molecules that vary both in size-from 381 to 5170 nucleotides-and in protein content. The RNA molecules likely replicate as circular single-stranded RNAs, and potentially provide a dramatic increase in diversity of such RNAs. Moreover, these organisms have eluded previous attempts to isolate RNA viruses from metatranscriptomes-suggesting a significant untapped universe of viruses or other organisms hidden within metatranscriptome sequences., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The application of micro-Raman spectroscopy was used for characterization of structural features of the high-k stack (h-k) layer of "silicon-on-insulator" (SOI) nanowire (NW) chip (h-k-SOI-NW chip), including Al 2 O 3 and HfO 2 in various combinations after heat treatment from 425 to 1000 °C. After that, the NW structures h-k-SOI-NW chip was created using gas plasma etching optical lithography. The stability of the signals from the monocrine phase of HfO 2 was shown. Significant differences were found in the elastic stresses of the silicon layers for very thick (>200 nm) Al 2 O 3 layers. In the UV spectra of SOI layers of a silicon substrate with HfO 2 , shoulders in the Raman spectrum were observed at 480-490 cm -1 of single-phonon scattering. The h-k-SOI-NW chip created in this way has been used for the detection of DNA-oligonucleotide sequences (oDNA), that became a synthetic analog of circular RNA-circ-SHKBP1 associated with the development of glioma at a concentration of 1.1 × 10 -16 M. The possibility of using such h-k-SOI NW chips for the detection of circ-SHKBP1 in blood plasma of patients diagnosed with neoplasm of uncertain nature of the brain and central nervous system was shown.

The capsid protein of dengue virus strain 2 (DENV2C) promotes nucleic acid structural rearrangements using chaperone activity. However, the role of DENV2C during the interaction of RNA elements in the conserved 5' untranslated region (5'UTR) to the 3' untranslated region (3'UTR) is still unclear. Thus, we investigated the effect of DENV2C on the annealing mechanism of two RNA hairpin elements from the 5'UTR to their complementary sequences during (+)/(-) ds-RNAformation and (+) RNA circularization. DENV2C was found to switch the annealing pathway for RNA elements involved in (+)/(-) ds-RNA formation, but not for RNA elements related to (+) RNA circularization. In addition, we also determined that DENV2C modulates intrinsic dynamics and reduces kinetically trapped unfavourable conformations of the 5'UTR sequence. Thus, our results provide mechanistic insights by which DENV2C chaperones the interactions between RNA elements at the 5' and 3' ends during genome recombination, a prerequisite for DENV replication.

Just as eukaryotic circular RNA (circRNA) is a product of intracellular backsplicing, custom circRNA can be synthesized in vitro using a transcription template in which transposed halves of a split group I intron flank the sequence of the RNA to be circularized. Such permuted intron-exon (PIE) constructs have been used to produce circRNA versions of ribozymes, mimics of viral RNA motifs, a streptavidin aptamer, and protein expression vectors for genetic engineering and vaccine development. One limitation of this approach is the obligatory incorporation of small RNA segments (E1 and E2) into nascent circRNA at the site of end-joining. This restriction may preclude synthesis of small circRNA therapeutics and RNA nanoparticles that are sensitive to extraneous sequence, as well as larger circRNA mimics whose sequences must precisely match those of the native species on which they are modelled. In this work, we used serial mutagenesis and in vitro selection to determine how varying E1 and E2 sequences in a thymidylate synthase (td) group I intron PIE transcription template construct affects circRNA synthesis yield. Based on our collective findings, we present guidelines for the design of custom-tailored PIE transcription templates from which synthetic circRNAs of almost any sequence may be efficiently synthesized., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

Temporal lobe epilepsy (TLE) is the most prevalent form of acquired epilepsy. Circular RNAs (circRNAs) have recently been highlighted as important regulators in TLE. Nevertheless, the role and mechanism of circRNA Drosha ribonuclease III (circ_DROSHA) in TLE pathogenesis are still unknown. Magnesium-free extracellular solution was used to establish the TLE cell model. The levels of circ_DROSHA, myocyte-specific enhancer factor 2C (MEF2C) and miR-106b-5p were determined by qRT-PCR and western blot. Cell proliferation was detected by the Cell Counting-8 Kit (CCK-8) assay, and cell apoptosis was measured by flow cytometry. Targeted relationships among circ_DROSHA, miR-106b-5p and MEF2C were confirmed by a dual-luciferase reporter or RNA immunoprecipitation (RIP) assay. Our data showed that circ_DROSHA was down-regulated in the serum samples of TLE patients and the TLE cell model. Circ_DROSHA up-regulation alleviated the cytotoxicity of the TLE cell model by enhancing cell proliferation and repressing cell apoptosis. Circ_DROSHA directly bound to miR-106b-5p. Moreover, miR-106b-5p represented a downstream effector of circ_DROSHA function. MEF2C was a direct target of miR-106b-5p, and miR-106b-5p knockdown relieved magnesium-free treatment-induced cell injury by up-regulating MEF2C. Furthermore, circ_DROSHA regulated MEF2C expression via sponging miR-106b-5p. Our study suggested that the enforced expression of circ_DROSHA alleviated the cell damage of the TLE cell model at least in part through the regulation of the miR-106b-5p/MEF2C axis., (Copyright © 2020 Elsevier Inc. All rights reserved.)

In this study, the potential of specific Circular RNAs (circRNAs) as novel peripheral blood biomarkers for myasthenia gravis (MG) was explored. We analyzed circRNAs in the peripheral blood of three normal controls and three MG patients using RNA microarray. Candidate circRNAs were validated in three independent cohorts by Quantitative Real-time polymerase chain reaction (qPCR). Eleven differentially expressed circRNAs were initially identified and four were confirmed in the first independent cohort. Hsa&#95;circ&#95;0076490 and hsa-circ&#95;5333-4 had the largest areas under the curve (AUCs) of the receiver operating characteristics (ROC) and were validated in the second cohort. In the third cohort, hsa-circRNA5333-4 had a larger AUC: 0.864 (95% confidence interval [CI] = 0.801-0.928, P < 0.001), a stronger correlation with the Quantitative Myasthenia Gravis Score (qMG): r = 0.505 (P < 0.001) and was correlated with gender and acetylcholine receptor antibody levels (P < 0.05). So hsa-circRNA5333-4 represents a novel biomarker for the diagnosis and monitoring of MG., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Mammalian circRNAs can influence different cellular processes by interacting with proteins and other nucleic acids. Here, we used ribonucleoprotein immunoprecipitation (RIP) analysis to identify systematically the circRNAs associated with the cancer-related protein AUF1. Among the circRNAs interacting with AUF1 in HeLa (human cervical carcinoma) cells, we focused on hsa&#95;circ&#95;0032434 (circPCNX), an abundant target of AUF1. Overexpression of circPCNX specifically interfered with the binding of AUF1 to p21 (CDKN1A) mRNA, thereby promoting p21 mRNA stability and elevating the production of p21, a major inhibitor of cell proliferation. Conversely, silencing circPCNX increased AUF1 binding to p21 mRNA, reducing p21 production and promoting cell division. Importantly, eliminating the AUF1-binding region of circPCNX abrogated the rise in p21 levels and rescued proliferation. Therefore, we propose that the interaction of circPCNX with AUF1 selectively prevents AUF1 binding to p21 mRNA, leading to enhanced p21 mRNA stability and p21 protein production, thereby suppressing cell growth., (Published by Oxford University Press on behalf of Nucleic Acids Research 2021.)

Circular RNAs are a class of noncoding RNA with a widespread occurrence in eukaryote tissues, and with some having been demonstrated to have clear biological function. In sheep, little is known about the role of circular RNAs in mammary gland tissue, and therefore an RNA sequencing approach was used to compare mammary gland tissue expression of circular RNAs in 9 Small Tail Han sheep at peak lactation, and subsequently when they were not lactating. These 9 sheep had their RNA pooled for analysis into 3 libraries from peak lactation and 3 from the nonlactating period. A total of 3,278 and 1,756 circular RNAs were identified in the peak lactation and nonlactating mammary gland tissues, respectively, and the expression and identity of 9 of them was confirmed using reverse transcriptase-polymerase chain reaction analysis and DNA sequencing. The type, chromosomal location and length of the circular RNAs identified were ascertained. Forty upregulated and one downregulated circular RNAs were characterized in the mammary gland tissue at peak lactation compared with the nonlactating mammary gland tissue. Gene ontology enrichment analysis revealed that the parental genes of these differentially expressed circular RNAs were related to molecular function, binding, protein binding, ATP binding, and ion binding. Five differentially expression circular RNAs were selected for further analysis to predict their target microRNAs, and some microRNAs reportedly associated with the development of the mammary gland were found in the constructed circular RNA-microRNA network. This study reveals the expression profiles and characterization of circular RNAs at 2 key stages of mammary gland activity, thereby providing an improved understanding of the roles of circular RNAs in the mammary gland of sheep., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).)

Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), whether circular RNA (circRNA) is involved in this process remains unknown. In this study, we performed circRNA microarray profile and found an HBV-related circRNA, circ-ARL3 (hsa&#95;circ&#95;0092493). Stable knockdown of circ-ARL3 inhibited the proliferation and invasion of HBV + HCC cells. High circ-ARL3 was positively correlated with malignant clinical features and poor prognosis. In terms of mechanism, HBx protein upregulated N 6 -methyladenosine (m 6 A) methyltransferases METTL3 expression, increasing the m 6 A modification of circ-ARL3; then, m 6 A reader YTHDC1 bound to m 6 A-modified of circ-ARL3 and favored its reverse splicing and biogenesis. Furthermore, circ-ARL3 was able to sponge miR-1305, antagonizing the inhibitory effects of miR-1305 on a cohort of target oncogenes, thereby promoting HBV + HCC progression. Importantly, depletion of circ-ARL3 significantly retarded HBV + HCC cell growth in vivo, whereas this effect was evidently blocked after silencing of miR-1305. Collectively, our data suggest that circ-ARL3 is a critical regulator in HBV-related HCC, targeting the axis of circ-ARL3/miR-1305 may be a promising treatment for HBV + HCC patients., (© 2020 International Union of Biochemistry and Molecular Biology.)

Circular RNAs (circRNAs) are covalently closed RNA molecules that have been linked to various diseases, including cancer. However, a precise function and working mechanism are lacking for the larger majority. Following many different experimental and computational approaches to identify circRNAs, multiple circRNA databases were developed as well. Unfortunately, there are several major issues with the current circRNA databases, which substantially hamper progression in the field. First, as the overlap in content is limited, a true reference set of circRNAs is lacking. This results from the low abundance and highly specific expression of circRNAs, and varying sequencing methods, data-analysis pipelines, and circRNA detection tools. A second major issue is the use of ambiguous nomenclature. Thus, redundant or even conflicting names for circRNAs across different databases contribute to the reproducibility crisis. Third, circRNA databases, in essence, rely on the position of the circRNA back-splice junction, whereas alternative splicing could result in circRNAs with different length and sequence. To uniquely identify a circRNA molecule, the full circular sequence is required. Fourth, circRNA databases annotate circRNAs' microRNA binding and protein-coding potential, but these annotations are generally based on presumed circRNA sequences. Finally, several databases are not regularly updated, contain incomplete data or suffer from connectivity issues. In this review, we present a comprehensive overview of the current circRNA databases and their content, features, and usability. In addition to discussing the current issues regarding circRNA databases, we come with important suggestions to streamline further research in this growing field., (© The Author(s) 2020. Published by Oxford University Press.)

TransCirc (https://www.biosino.org/transcirc/) is a specialized database that provide comprehensive evidences supporting the translation potential of circular RNAs (circRNAs). This database was generated by integrating various direct and indirect evidences to predict coding potential of each human circRNA and the putative translation products. Seven types of evidences for circRNA translation were included: (i) ribosome/polysome binding evidences supporting the occupancy of ribosomes onto circRNAs; (ii) experimentally mapped translation initiation sites on circRNAs; (iii) internal ribosome entry site on circRNAs; (iv) published N-6-methyladenosine modification data in circRNA that promote translation initiation; (v) lengths of the circRNA specific open reading frames; (vi) sequence composition scores from a machine learning prediction of all potential open reading frames; (vii) mass spectrometry data that directly support the circRNA encoded peptides across back-splice junctions. TransCirc provides a user-friendly searching/browsing interface and independent lines of evidences to predicte how likely a circRNA can be translated. In addition, several flexible tools have been developed to aid retrieval and analysis of the data. TransCirc can serve as an important resource for investigating the translation capacity of circRNAs and the potential circRNA-encoded peptides, and can be expanded to include new evidences or additional species in the future., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Analysis of circular RNA (circRNA) expression from RNA-Seq data can be performed with different algorithms and analysis pipelines, tools allowing the extraction of heterogeneous information on the expression of this novel class of RNAs. Computational pipelines were developed to facilitate the analysis of circRNA expression by leveraging different public tools in easy-to-use pipelines. This chapter describes the complete workflow for a computationally reproducible analysis of circRNA expression starting for a public RNA-Seq experiment. The main steps of circRNA prediction, annotation, classification, sequence reconstruction, quantification, and differential expression are illustrated.

RNA helicase A (RHA) is a DExH-box helicase that plays regulatory roles in a variety of cellular processes, including transcription, translation, RNA splicing, editing, transport, and processing, microRNA genesis and maintenance of genomic stability. It is involved in virus replication, oncogenesis, and innate immune response. RHA can unwind nucleic acid duplex by nucleoside triphosphate hydrolysis. The insight into the molecular mechanism of helicase activity is fundamental to understanding the role of RHA in the cell. Herein, we reviewed the current advances on the helicase activity of RHA and its relevance to gene expression, particularly, to the genesis of circular RNA., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Circular RNAs (circRNAs) are a class of noncoding RNAs, generated from pre-mRNAs by circular splicing of exons and functionally largely uncharacterized. Here we report on the design, expression, and characterization of artificial circRNAs that act as protein sponges, specifically binding and functionally inactivating hnRNP (heterogeneous nuclear ribonucleoprotein) L. HnRNP L regulates alternative splicing, depending on short CA-rich RNA elements. We demonstrate that designer hnRNP L-sponge circRNAs with CA-repeat or CA-rich sequence clusters can efficiently and specifically modulate splicing-regulatory networks in mammalian cells, including alternative splicing patterns and the cellular distribution of a splicing factor. This new strategy can in principle be applied to any RNA-binding protein, opening up new therapeutic strategies in molecular medicine., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background and Aims: Accumulating evidence suggests that the primary and acquired resistance of hepatocellular carcinoma (HCC) to sorafenib is mediated by multiple molecular, cellular, and microenvironmental mechanisms. Understanding these mechanisms will enhance the likelihood of effective sorafenib therapy., Methods: In vitro and in vivo experiments were performed and clinical samples and online databases were acquired for clinical investigation., Results: In this study, we found that a circular RNA, circRNA-SORE, which is up-regulated in sorafenib-resistant HCC cells, was necessary for the maintenance of sorafenib resistance, and that silencing circRNA-SORE substantially increased the efficacy of sorafenib-induced apoptosis. Mechanistic studies determined that circRNA-SORE sequestered miR-103a-2-5p and miR-660-3p by acting as a microRNA sponge, thereby competitively activating the Wnt/β-catenin pathway and inducing sorafenib resistance. The increased level of circRNA-SORE in sorafenib-resistant cells resulted from increased RNA stability. This was caused by an increased level of N 6 -methyladenosine (m 6 A) at a specific adenosine in circRNA-SORE. In vivo delivery of circRNA-SORE interfering RNA by local short hairpin RNA lentivirus injection substantially enhanced sorafenib efficacy in animal models., Conclusions: This work indicates a novel mechanism for maintaining sorafenib resistance and is a proof-of-concept study for targeting circRNA-SORE in sorafenib-treated HCC patients as a novel pharmaceutical intervention for advanced HCC.

Human dental pulp stem cells (DPSCs) hold great promise in bone regeneration. However, the exact mechanism of osteogenic differentiation of DPSCs remains unknown, especially the role of exosomes played in. The DPSCs were cultured and received osteogenic induction; then, exosomes from osteogenic-induced DPSCs (OI-DPSC-Ex) at different time intervals were isolated and sequenced for circular RNA (circRNA) expression profiles. Gradually, increased circular lysophosphatidic acid receptor 1 (circLPAR1) expression was found in the OI-DPSC-Ex coincidentally with the degree of osteogenic differentiation. Meanwhile, results from osteogenic differentiation examinations showed that the OI-DPSC-Ex had osteogenic effect on the recipient homotypic DPSCs. To investigate the mechanism of exosomal circLPAR1 on osteogenic differentiation, we verified that circLPAR1 could competently bind to hsa-miR-31, by eliminating the inhibitory effect of hsa-miR-31 on osteogenesis, therefore promoting osteogenic differentiation of the recipient homotypic DPSCs. Our study showed that exosomal circRNA played an important role in osteogenic differentiation of DPSCs and provided a novel way of utilization of exosomes for the treatment of bone deficiencies., Competing Interests: There are no conflicts of interest from any author., (Copyright © 2020 Liangkun Xie et al.)

Lung cancer is one of the main causes of cancer-related death in the world, especially due to its frequency and ineffective therapeutically approaches in the late stages of the disease. Despite the recent advent of promising new targeted therapies, lung cancer diagnostic strategies still have difficulty in identifying the disease at an early stage. Therefore, the characterizations of more sensible and specific cancer biomarkers have become an important goal for clinicians. Circular RNAs (circRNAs), a type of RNA with covalently closed continuous loop structures that display high structural resistance and tissue specificity pointed toward a potential biomarker role. Current investigations have identified that circRNAs have a prominent function in the regulation of oncogenic pathways, by regulating gene expression both at transcriptional and post-transcriptional level. The aim of this review is to provide novel information regarding the implications of circRNAs in lung cancer, with an emphasis on the role in disease development and progression. Initially, we explored the potential utility of circRNAs as biomarkers, focusing on function, mechanisms, and correlation with disease progression in lung cancer. Further, we will describe the interaction between circRNAs and other non-coding species of RNA (particularly microRNA) and their biological significance in lung cancer. Describing the nature of these interactions and their therapeutic potential will provide additional insight regarding the altered molecular landscape of lung cancer and consolidate the potential clinical value of these circular transcripts. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA in Disease and Development > RNA in Disease RNA in Disease and Development > RNA in Development., (© 2020 Wiley Periodicals, Inc.)

Background: Deregulated circular RNAs (circRNAs) are associated with the development of cancer and therapy resistance. However, functional research of circRNAs mostly focus on potential miRNA or protein binding and more potential regulation of circRNA on host gene DNA in cancers are yet to be inspected., Method: We performed total RNA sequencing on clinical breast cancer samples and identified the expression patterns of circRNAs and corresponding host genes in patient blood, tumor and adjacent normal tissues. qPCR, northern blot and in situ hybridization were used to validate the dysregulation of circRNA circSMARCA5. A series of procedures including R-loop dot-blotting, DNA-RNA immunoprecipitation and mass spectrum, etc. were conducted to explore the regulation of circSMARCA5 on the transcription of exon 15 of SMARCA5. Moreover, immunofluorescence and in vivo experiments were executed to investigate the overexpression of circSMARCA5 with drug sensitivities., Results: We found that circRNAs has average higher expression over its host linear genes in peripheral blood. Compared to adjacent normal tissues, circSMARCA5 is decreased in breast cancer tissues, contrary to host gene SMARCA5. The enforced expression of circSMARCA5 induced drug sensitivity of breast cancer cell lines in vitro and in vivo. Furthermore, we demonstrated that circSMARCA5 can bind to its parent gene locus, forming an R-loop, which results in transcriptional pausing at exon 15 of SMARCA5. CircSMARCA5 expression resulted in the downregulation of SMARCA5 and the production of a truncated nonfunctional protein, and the overexpression of circSMARCA5 was sufficient to improve sensitivity to cytotoxic drugs., Conclusion: Our results revealed a new regulatory mechanism for circRNA on its host gene and provided evidence that circSMARCA5 may serve as a therapeutic target for drug-resistant breast cancer patients.

Circular RNAs (circRNAs) are a distinct family of RNAs derived from alternative splicing which play a crucial role in regulating gene expression by acting as microRNA (miRNA) and RNA binding protein (RBP) sponges. However, recent studies have also reported the multifunctional potential of these particles. Under different conditions, circRNAs not only regulate protein synthesis, destination, and degradation but can serve as protein scaffolds or recruiters and are also able to produce short peptides with active biological functions. circRNAs are under ongoing investigation because of their close association with the development of diseases. Some circRNAs are reportedly expressed in a tissue- and development stage-specific manner. Furthermore, due to other features of circRNAs, including their stability, conservation, and high abundance in bodily fluids, they are believed to be potential biomarkers for various diseases, including cancers. In this review, we focus on providing a summary of the current knowledge on circRNA-protein interactions. We present the properties and functions of circRNAs, the possible mechanisms of their translation abilities, and the emerging functions of circRNA-derived peptides in human pathologies.

The elaborate control of biogenesis and turnover is essential for circular RNAs (circRNAs) to exert their functions properly in eukaryotic cells, whereas how circRNAs are degraded remains unclear. A recent study by Fischer et al. reveals a novel structure-mediated circRNA decay that selectively degrades highly structured RNAs by UPF1 and G3BP1., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Circular RNAs (circRNAs) sequester microRNAs (miRNAs) and repress their endogenous activity. We hypothesized that artificial circRNA sponges (circmiRs) can be constructed to target miRNAs therapeutically, with a low dosage requirement and extended half-lives compared to current alternatives. This could present a new treatment approach for critical global pathologies, including cardiovascular disease. Here, we constructed a circmiR sponge to target known cardiac pro-hypertrophic miR-132 and -212. Expressed circmiRs competitively inhibited miR-132 and -212 activity in luciferase rescue assays and showed greater stability than linear sponges. A design containing 12 bulged binding sites with 12 nucleotides spacing was determined to be optimal. Adeno-associated viruses (AAVs) were used to deliver circmiRs to cardiomyocytes in vivo in a transverse aortic constriction (TAC) mouse model of cardiac disease. Hypertrophic disease characteristics were attenuated, and cardiac function was preserved in treated mice, demonstrating the potential of circmiRs as novel therapeutic tools. Subsequently, group I permutated intron-exon sequences were used to directly synthesize exogenous circmiRs, which showed greater in vitro efficacy than the current gold standard antagomiRs in inhibiting miRNA function. Engineered circRNAs thus offer exciting potential as future therapeutics., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Circular nucleic acids (CNAs) are nucleic acid molecules with a closed-loop structure. This feature comes with a number of advantages including complete resistance to exonuclease degradation, much better thermodynamic stability, and the capability of being replicated by a DNA polymerase in a rolling circle manner. Circular functional nucleic acids, CNAs containing at least a ribozyme/DNAzyme or a DNA/RNA aptamer, not only inherit the advantages of CNAs but also offer some unique application opportunities, such as the design of topology-controlled or enabled molecular devices. This article will begin by summarizing the discovery, biogenesis, and applications of naturally occurring CNAs, followed by discussing the methods for constructing artificial CNAs. The exploitation of circular functional nucleic acids for applications in nanodevice engineering, biosensing, and drug delivery will be reviewed next. Finally, the efforts to couple functional nucleic acids with rolling circle amplification for ultra-sensitive biosensing and for synthesizing multivalent molecular scaffolds for unique applications in biosensing and drug delivery will be recapitulated., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rings of single-stranded RNA are promising for many practical applications, but the methods to prepare them in preparative scale have never been established. Previously, RNA circularization was achieved by T4 RNA ligase 2 (Rnl2, a dsRNA ligase) using splints, but the yield was low due to concurrent intermolecular polymerization. Here, various functional RNAs (siRNA, miRNA, ribozyme, etc.) are dominantly converted by Rnl2 to the rings without significant limitations in sizes and sequences. The key is to design a precursor RNA, which is highly activated for the efficient circularization without any splint. First, secondary structure of target RNA ring is simulated by Mfold, and then hypothetically cut at one site so that a few intramolecular base pairs are formed at the terminal. Simply by treating this RNA with Rnl2, the target ring was selectively and efficiently produced. Unexpectedly, circular RNA can be obtained in high yield (>90%), even when only 2 bp form in the 3'-OH side and no full match base pair forms in the 5'-phosphate side. Formation of polymeric by-products was further suppressed by diluting conventional Rnl2 buffer to abnormally low concentrations. Even at high-RNA concentrations (e.g. 50 μM), enormously high selectivity (>95%) was accomplished., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Duchenne muscular dystrophy (DMD) is an X-linked genetic neuromuscular disease that is characterized by progressive muscle wasting and by defects in the regenerative capacity and inflammatory infiltration of muscle. Many noncoding RNAs (ncRNAs) participate in the pathophysiological mechanisms of this disease. To explore the role of circular RNAs (circRNAs), a type of ncRNAs, in DMD, microarray analysis was performed to explore the expression patterns of circRNAs in the gastrocnemius muscles in mdx mice, a DMD animal model, and C57 mice. The microarray data were validated by qRT-PCR. Further, gene ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to predict the function of the differentially expressed circRNAs (DEcRNAs). A circRNA/microRNA (miRNA) interaction network was predicted by bioinformatics. We also predicted the protein-coding ability of the circRNAs based on their N 6 -methyladenosine motifs and open-reading frames. We identified 197 differentially expressed circRNAs between mdx mice and C57 mice. Of the 197 DEcRNAs, 6 circRNAs were randomly selected to validate the microarray data, and twenty-two circRNAs were randomly selected to construct a circRNA/miRNA interaction network. Bioinformatics analysis showed that the linear counterparts of the DEcRNAs were mainly associated with muscle structure, nervous system development, and the cAMP signaling pathway. A total of 189 circRNAs were predicted to have protein-coding potential, and there were 98 circRNAs that could potentially be translated into polypeptides with 150 or more amino acids. This work described the expression pattern of circRNAs in mdx mice and indicated that circRNAs may play pivotal roles in the pathophysiological mechanisms of DMD.

The most frequent kind of malignancy in the universe is skin cancer, which has been categorized into non-melanoma and melanoma skin cancer. There are no complete information of the skin carcinogenesis process. A variety of external and internal agents contribute to the non-melanoma and melanoma skin cancer pathogenesis. These factors are epigenetic changes, X-rays, genetic, arsenic compounds, UV rays, and additional chemical products. It was found that there could be a relationship between the appearing novel and more suitable therapies for participants in this class of diseases and detection of basic molecular paths. A covalently closed loop structure bond connecting the 5' and 3' ends characterizes a new group of extensively expressed endogenous regulatory RNAs, which are called circular RNAs (circRNAs). Mammals commonly express circRNAs. They are of high importance in tumorigenesis. Multiple lines evidence indicated that a variety of circular RNAs are associated with initiation and development of skin-related diseases such as skin cancers. Given that different circular RNAs (hsa&#95;circ&#95;0025039, hsa&#95;circRNA006612, circRNA005537, and circANRIL) via targeting various cellular and molecular targets (e.g., CDK4, DAB2IP, ZEB1, miR-889, and let-7c-3p) exert their effects on skin cancers progression. Herein, for first time, we summarized different circular RNAs in skin cancers and noncancerous diseases. Moreover, we highlighted crosstalk between circular RNAs and ceRNAs in cancerous conditions.

CircRNA is a specific type of non-coding RNA that has been shown to have an important role in mammary gland (MG) activity, but no study of MG circRNA activity in sheep so far. In this study, the expression profile of circRNAs was investigated using RNA-Seq in MG parenchyma at peak lactation from Small-Tailed Han sheep and Gansu Alpine Merino sheep with phenotypic differences in milk yield and components. A total of 4, 906 circRNAs were found and 33 of these were differentially expressed between breeds. GO and KEGG results showed that the parental genes of differentially expressed circRNAs were mainly enriched in heterocyclic compound binding, kinase activity, adherens junction, the TGF-β signaling pathway, and the MAPK signaling pathway. This study provides an overview of circRNA expression in the ovine MG and the interaction between some key circRNAs and their target miRNAs. It improves our knowledge of the role of circRNA in sheep milk synthesis., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Existing circular RNA (circRNA) databases have become essential for transcriptomics. However, most are unsuitable for mining in-depth information for candidate circRNA prioritization. To address this, we integrate circular transcript collections to develop the circAtlas database based on 1070 RNA-seq samples collected from 19 normal tissues across six vertebrate species. This database contains 1,007,087 highly reliable circRNAs, of which over 81.3% have been assembled into full-length sequences. We profile their expression pattern, conservation, and functional annotation. We describe a novel multiple conservation score, co-expression, and regulatory networks for circRNA annotation and prioritization. CircAtlas can be accessed at http://circatlas.biols.ac.cn/.

By interacting with proteins and nucleic acids, the vast family of mammalian circRNAs is proposed to influence many biological processes. Here, RNA sequencing analysis of circRNAs differentially expressed during myogenesis revealed that circSamd4 expression increased robustly in mouse C2C12 myoblasts differentiating into myotubes. Moreover, silencing circSamd4, which is conserved between human and mouse, delayed myogenesis and lowered the expression of myogenic markers in cultured myoblasts from both species. Affinity pulldown followed by mass spectrometry revealed that circSamd4 associated with PURA and PURB, two repressors of myogenesis that inhibit transcription of the myosin heavy chain (MHC) protein family. Supporting the hypothesis that circSamd4 might complex with PUR proteins and thereby prevent their interaction with DNA, silencing circSamd4 enhanced the association of PUR proteins with the Mhc promoter, while overexpressing circSamd4 interfered with the binding of PUR proteins to the Mhc promoter. These effects were abrogated when using a mutant circSamd4 lacking the PUR binding site. Our results indicate that the association of PUR proteins with circSamd4 enhances myogenesis by contributing to the derepression of MHC transcription., (© Published by Oxford University Press on behalf of Nucleic Acids Research 2020.)

Post-transcriptional mechanisms regulate the stability and, hence, expression of coding and noncoding RNAs. Sequence-specific features within the 3' untranslated region (3' UTR) often direct mRNAs for decay. Here, we characterize a genome-wide RNA decay pathway that reduces the half-lives of mRNAs based on overall 3' UTR structure formed by base pairing. The decay pathway is independent of specific single-stranded sequences, as regulation is maintained in both the original and reverse complement orientation. Regulation can be compromised by reducing the overall structure by fusing the 3' UTR with an unstructured sequence. Mutating base-paired RNA regions can also compromise this structure-mediated regulation, which can be restored by re-introducing base-paired structures of different sequences. The decay pathway requires the RNA-binding protein UPF1 and its associated protein G3BP1. Depletion of either protein increased steady-state levels of mRNAs with highly structured 3' UTRs as well as highly structured circular RNAs. This structure-dependent mechanism therefore enables cells to selectively regulate coding and noncoding RNAs., Competing Interests: Declaration of Interests Authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Circular RNA (circRNA) is a class of single-stranded molecules with tissue/development-specific expression patterns. Unlike linear RNA, circRNA forms a covalently closed loop produced from 'back-splicing' of primary transcripts, conferring on them inherent resistance to exonucleolytic RNA decay. Increasing evidence demonstrates that many circRNAs exert important biological functions by acting as miRNA inhibitors ('sponges'), protein 'decoys', or by encoding small peptides. Importantly, circRNAs are aberrantly expressed in cancer and play indispensable oncogenic or tumor suppressive roles during tumor development and progression. In this review, we summarize the biogenesis, turnover, and involvements of circRNAs in cancer and also discuss their potential as diagnostic biomarkers or therapeutic targets., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Marek's disease (MD), induced by Marek's disease virus (MDV), is a lymphotropic neoplastic disease and causes huge economic losses to the poultry industry. Non-coding RNAs (ncRNAs) play important regulatory roles in disease pathogenesis. To investigate host circular RNA (circRNA) and microRNA (miRNA) expression profile, RNA sequencing was performed in tumourous spleens (TS), spleens from the survivors (SS) without any lesion after MDV infection, and non-infected chicken spleens (NS). A total of 2,169 circRNAs were identified and more than 80% of circRNAs were derived from exon. The flanking introns of 1,744 exonic circRNAs possessed 579 reverse complementary matches (RCMs), which mainly overlapped with chicken repeat 1 family (CR1F). It suggested that CR1F mediated the cyclization of exons by intron pairing. Out of 2,169 circRNAs, 113 were differentially expressed circRNAs (DECs). The Q-PCR and Rnase R digestion experiments showed circRNA possessed high stability compared with their linear RNAs. Integrated with previous transcriptome data, we profiled regulatory networks of circRNA/long non-coding RNA (lncRNA)-miRNA-mRNA. Extensive competing endogenous RNA (ceRNA) networks were predicted to be involved in MD tumourigenesis. Interestingly, circZMYM3, an intronic circRNA, interacted with seven miRNAs which targeted some immune genes, such as SWAP70 and CCL4. Gga-miR-155 not only interacted with circGTDC1 and circMYO1B, but also targeted immune-related genes, such as GATA4, which indicated the roles of non-coding RNAs played to mediate immune responsive genes. Collectively, this is the first study that integrated RNA expression profiles in MD model. Our results provided comprehensive interactions of ncRNAs and mRNA in MD tumourigenesis.

Circular RNAs (circRNAs) have recently attracted significant interest in the realm of science and the evolution of species. Given the lack of information available on circRNAs due to various barriers related to sequencing techniques and bioinformatics tools, little regarding their function is known. It has been predicted that circRNAs contribute to gene expression regulation, but aside from a few specific cases, this contention has yet to be proven. Although the role of circRNAs in evolution remains elusive, from the few studies that have shown circRNA conservation in mammalian species, tissue specificity in brain regions, and the abundance of circRNAs in the brains of various species, the concept is becoming more likely with much gravitas. The proposed functional role of circRNAs being gene regulators is of great interest and would provide a basis to further understand not only the functional capabilities of organisms, but also the evolution of mammalian species., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The circular RNA renaissance is upon us. Recent reports demonstrate applications of synthetic circular RNA molecules as gene therapies and in the production of biologics from cell-based expression systems. Circular RNAs are covalently closed loop RNA species that are formed naturally through noncolinear splicing of pre-mRNA. Although once thought to be noncoding artefacts from splicing errors, it is now accepted that circular RNAs are abundant and have diverse functions in gene regulation and protein coding in eukaryotes. Numerous reports have investigated circular RNAs in various diseases, but the promise of synthetic circular RNAs in the production of recombinant proteins and as RNA-based therapies is only now coming into focus. This review highlights reported uses of synthetic circular RNAs and describes methods for generating these molecules., (Copyright © 2019 Elsevier Ltd. All rights reserved.)