Identification of potential proteins translated from circular RNA splice variants

Circular RNAs (circRNAs) are covalently closed RNA molecules generated from precursor RNAs by the head-to-tail backsplicing of exons. Hundreds of studies demonstrated that circRNAs are ubiquitously expressed and regulate cellular events by modulating microRNA (miRNA) and RNA-binding protein (RBP) activities. A few circRNAs are also known to translate into functional polypeptides regulating cellular physiology. All these functions primarily depend on the full-length sequence of the circRNAs. CircRNA backsplice junction sequence is the key to identifying circRNAs and their full-length mature sequence. However, some multi-exonic circRNAs exist in different isoforms sharing identical backsplice junction sequences and are termed circRNA splice variants. Here, we analyzed the previously published HeLa cell RNA-seq datasets to identify circRNA splice variants using the de novo module of the CIRCexplorer2 circRNA annotation pipeline. A subset of circRNAs with splice variants was validated by the circRNA-rolling circle amplification (circRNA-RCA) method. Interestingly, several validated circRNAs were predicted to translate into proteins by the riboCIRC database. Furthermore, polyribosome fractionation followed by quantitative PCR confirmed the association of a subset of circRNAs with polyribosome supporting their protein-coding potential. Finally, bioinformatics analysis of proteins derived from splice variants of circCORO1C and circASPH suggested altered protein sequences and structures that could affect their physiological functions. Together, our study identified novel circRNA splice variants and their potential translation into protein isoforms which may regulate various physiological processes.     

CircRNA expression profile and functional analysis in retinal ischemia-reperfusion injury

Retinal ischemia-reperfusion (I/R) is involved in the pathogenesis of many vision-threatening diseases. circRNAs act as key players in gene regulation and human diseases. However, the global circRNA expression profile in retinal I/R injury has not been fully uncovered. Herein, we established a murine model of retinal I/R injury and performed circRNA microarrays to identify I/R-related circRNAs. 1265 differentially expressed circRNAs were identified between I/R retinas and normal retinas. Notably, the detection of cWDR37 level in aqueous humor could discriminate glaucoma patients from cataract patients (AUC = 0.9367). cWdr37 silencing protected against hypoxic stress- or oxidative stress-induced retinal ganglion cell (RGC) injury. cWdr37 silencing alleviated IR-induced retinal neurodegeneration as shown by increased NeuN staining, reduced retinal reactive gliosis, and decreased retinal apoptosis. Collectively, this study provides a novel insight into the pathogenesis of retinal I/R injury. cWdr37 is a promising target for the diagnosis or treatment of I/R-related ocular diseases.     

条纹斑竹鲨肝脏环形RNA的筛选、鉴定及功能分析北大核心CSCD

为筛选和验证条纹斑竹鲨肝脏中环形RNA(circRNA)及探究其在肝癌细胞HepG2中过表达对肝癌细胞增殖、迁移能力的影响,本研究主要进行了两项实验:对条纹斑竹鲨肝脏circRNA进行高通量测序和预测,随后设计正、反向引物验证其真实性;构建circRNA过表达载体,将其瞬时转染进肝癌细胞HepG2,进行CCK-8和划痕实验来评价其对肝癌细胞增殖和迁移能力的影响。结果显示:预测到有4558条circRNAs,并确认了14条circRNAs的真实性;qRT-PCR实验表明在肝癌细胞HepG2中能瞬时过表达circRNA 13-566、circRNA 4-475、circRNA 5-402、circRNA 294-177、circRNA 30-219;且CCK-8和划痕实验显示,这5条circRNAs过表达后,均能不同程度地抑制肝癌细胞的增殖和迁移能力,其中circRNA 4-475、circRNA 294-177作用尤为显著。上述结果为深入研究条纹斑竹鲨肝脏中circRNA及其在肝再生、肝癌治疗方面的功能提供了新思路和基础。     

Circular RNA expression profiles and features in human tissues: a study using RNA-seq data

Background

Circular RNA (circRNA) is one type of noncoding RNA that forms a covalently closed continuous loop. Similar to long noncoding RNA (lncRNA), circRNA can act as microRNA (miRNA) ‘sponges’ to regulate gene expression, and its abnormal expression is related to diseases such as atherosclerosis, nervous system disorders and cancer. So far, there have been no systematic studies on circRNA abundance and expression profiles in human adult and fetal tissues.

Results

We explored circRNA expression profiles using RNA-seq data for six adult and fetal normal tissues (colon, heart, kidney, liver, lung, and stomach) and four gland normal tissues (adrenal gland, mammary gland, pancreas, and thyroid gland). A total of 8120, 25,933 and 14,433 circRNAs were detected by at least two supporting junction reads in adult, fetal and gland tissues, respectively. Among them, 3092, 14,241 and 6879 circRNAs were novel when compared to the published results. In each adult tissue type, we found at least 1000 circRNAs, among which 36.97–50.04% were tissue-specific. We reported 33 circRNAs that were ubiquitously expressed in all the adult tissues we examined. To further explore the potential “housekeeping” function of these circRNAs, we constructed a circRNA-miRNA-mRNA regulatory network containing 17 circRNAs, 22 miRNAs and 90 mRNAs. Furthermore, we found that both the abundance and the relative expression level of circRNAs were higher in fetal tissue than adult tissue. The number of circRNAs in gland tissues, especially in mammary gland (9665 circRNA candidates), was higher than that of other adult tissues (1160–3777).

Conclusions

We systematically investigated circRNA expression in a variety of human adult and fetal tissues. Our observation of different expression level of circRNAs in adult and fetal tissues suggested that circRNAs might play their role in a tissue-specific and development-specific fashion. Analysis of circRNA-miRNA-mRNA network provided potential targets of circRNAs. High expression level of circRNAs in mammary gland might be attributed to the rich innervation.

     

CircR2Disease v2.0: An Updated Web Server for Experimentally Validated circ RNA–disease Associations and Its Application

With accumulating dysregulated circular RNAs(circRNAs) in pathological processes,the regulatory functions of circRNAs, especially circRNAs as microRNA(miRNA) sponges and their interactions with RNA-binding proteins(RBPs), have been widely validated. However, the collected information on experimentally validated circRNA–disease associations is only preliminary.Therefore, an updated CircR2Disease database providing a comprehensive resource and web tool to clarify the relationships between circRNAs...     

NOVA2 regulates neural circRNA biogenesis

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.     

Genome-wide identification of circular RNAs in adult Schistosoma japonicum

Circular RNAs (circRNAs) are a class of novel, widespread, covalently closed RNAs that have played an essential role in animal gene regulation. To systematically explore circRNAs in the blood fluke Schistosoma japonicum, we performed RNA sequencing and bioinformatics analysis, and found that hundreds of circRNAs showed gender-associated expression. Among these identified circRNAs, more than 77.54% and 74.73% were putatively derived from the exon region of the genome and some circRNAs showed gender-associated expressions. The functional prediction of circRNAs (circ_003826 and circ_004690) showed potential binding sites and possibly acted as the sponge to regulate microRNAs (miRNAs) sja-miR-1, sja-miR-133 and sja-miR-3504. Altogether, these findings demonstrated that S. japonicum also contains circRNAs, which may have potential regulatory roles during schistosome development.     

circRNAs在植物中的研究进展

circular RNA(circRNA)是一类具有闭合环状结构的内源性非编码RNA,广泛存在于多种真核生物中,具有结构稳定、序列保守、表达特异性等特征。研究表明circRNAs可作为海绵(sponge)吸附microRNA(miRNA)并参与其表达调控过程,也可通过与蛋白互作调控基因表达等生物过程;发现circRNAs不仅参与植物激素信号转导等生理过程,而且还能在植物响应逆境胁迫中起到重要作用。该文主要对近年来国内外有关circRNAs的类型、形成机制、功能及其在植物生长发育过程中的研究进展进行了综述,并讨论了circRNAs的研究意义及存在的问题,为进一步研究circRNAs在植物中的作用机制及其基因调控网络提供参考。     

CIRCpedia v2: An Updated Database for Comprehensive Circular RNA Annotation and Expression Comparison

Circular RNAs (circRNAs) from back-splicing of exon(s) have been recently identified to be broadly expressed in eukaryotes, in tissue- and species-specific manners. Although functions of most circRNAs remain elusive, some circRNAs are shown to be functional in gene expression regulation and potentially relate to diseases. Due to their stability, circRNAs can also be used as biomarkers for diagnosis. Profiling circRNAs by integrating their expression among different samples thus provides molecular basis for further functional study of circRNAs and their potential application in clinic. Here, we report CIRCpedia v2, an updated database for comprehensive circRNA annotation from over 180 RNA-seq datasets across six different species. This atlas allows users to search, browse, and download circRNAs with expression features in various cell types/tissues, including disease samples. In addition, the updated database incorporates conservation analysis of circRNAs between humans and mice. Finally, the web interface also contains computational tools to compare circRNA expression among samples. CIRCpedia v2 is accessible at http://www.picb.ac.cn/rnomics/circpedia.     

CircPlant:An Integrated Tool for circRNA Detection and Functional Prediction in Plants

The recent discovery of circular RNAs (circRNAs) and characterization of their functional roles have opened a new avenue for understanding the biology of genomes. circRNAs have been implicated to play important roles in a variety of biological processes, but their precise functions remain largely elusive. Currently, a few approaches are available for novel circRNA prediction, but almost all these methods are intended for animal genomes. Considering that the major differences between the organization of plant and mammal genomes cannot be neglected, a plant-specific method is needed to enhance the validity of plant circRNA identification. In this study, we present CircPlant, an integrated tool for the exploration of plant circRNAs, potentially acting as competing endogenous RNAs (ceRNAs), and their potential functions. With the incorporation of several unique plant-specific criteria, CircPlant can accurately detect plant circRNAs from high-throughput RNA-seq data. Based on comparison tests on simulated and real RNA-seq datasets from Arabidopsis thaliana and Oryza sativa, we show that CircPlant outperforms all evaluated competing tools in both accuracy and efficiency. CircPlant is freely available at http://bis.zju.edu.cn/circplant.     

Circular RNA circDtx1 regulates IRF3-mediated antiviral immune responses through suppression of miR-15a-5p-dependent TRIF downregulation in teleost fish

Circular RNAs (circRNAs) represent a class of widespread and diverse covalently closed circular endogenous RNAs that exert crucial functions in regulating gene expression in mammals. However, the function and regulation mechanism of circRNAs in lower vertebrates are still unknown. Here, we discovered a novel circRNA derived from Deltex E3 ubiquitin ligase 1 (Dtx1) gene, namely, circDtx1, which was related to the antiviral responses in teleost fish. Results indicated that circDtx1 played essential roles in host antiviral immunity and inhibition of SCRV replication. Our study also found a microRNA miR-15a-5p, which could inhibit antiviral immune response and promote viral replication by targeting TRIF. Moreover, we also found that the antiviral effect inhibited by miR-15a-5p could be reversed with the circDtx1. In mechanism, our data revealed that circDtx1 was a competing endogenous RNA (ceRNA) of TRIF by sponging miR-15a-5p, leading to activation of the NF-κB/IRF3 pathway, and then enhancing the innate antiviral responses. Our results indicated that circRNAs played a regulatory role in immune responses in teleost fish.