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.     

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.     

An autoregulation loop in fust-1 for circular RNA regulation in Caenorhabditis elegans

Many circular RNAs (circRNAs) are differentially expressed in different tissues or cell types, suggestive of specific factors that regulate their biogenesis. Here, taking advantage of available mutation strains of RNA-binding proteins (RBPs) in Caenorhabditis elegans, I performed a screening of circRNA regulation in 13 conserved RBPs. Among them, loss of FUST-1, the homolog of Fused in Sarcoma (FUS), caused downregulation of multiple circRNAs. By rescue experiments, I confirmed FUST-1 as a circRNA regulator. Through RNA sequencing using circRNA-enriched samples, circRNAs targets regulated by FUST-1 were identified globally, with hundreds of them significantly altered. Furthermore, I showed that FUST-1 regulates circRNA formation with only small to little effect on the cognate linear mRNAs. When recognizing circRNA pre-mRNAs, FUST-1 can affect both exon-skipping and circRNA in the same genes. Moreover, I identified an autoregulation loop in fust-1, where FUST-1, isoform a (FUST-1A) promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b (FUST-1B) with different N-terminal sequences. FUST-1A is the functional isoform in circRNA regulation. Although FUST-1B has the same functional domains as FUST-1A, it cannot regulate either exon-skipping or circRNA formation. This study provided an in vivo investigation of circRNA regulation, which will be helpful to understand the mechanisms that govern circRNA formation.     

The human MAPT locus generates circular RNAs

The microtubule-associated protein Tau, generated by the MAPT gene is involved in dozens of neurodegenerative conditions (“tauopathies”), including Alzheimer's disease (AD) and frontotemporal lobar degeneration/frontotemporal dementia (FTLD/FTD). The pre-mRNA of MAPT is well studied and its aberrant pre-mRNA splicing is associated with frontotemporal dementia. Using a PCR screen of RNA from human brain tissues, we found that the MAPT locus generates circular RNAs through a backsplicing mechanism from exon 12 to either exon 10 or 7. MAPT circular RNAs are localized in the cytosol and contain open reading frames encoding Tau protein fragments. The MAPT exon 10 is alternatively spliced and proteins involved in its regulation, such as CLK2, SRSF7/9G8, PP1 (protein phosphatase 1) and NIPP1 (nuclear inhibitor of PP1) reduce the abundance of the circular MAPT exon 12?→?10 backsplice RNA after being transfected into cultured HEK293 cells. In summary, we report the identification of new bona fide human brain RNAs produced from the MAPT locus. These may be a component of normal human brain Tau regulation and, since the circular RNAs could generate high molecular weight proteins with multiple microtubule binding sites, they could contribute to taupathies.     

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.     

CircRNAs: a new target for the diagnosis and treatment of digestive system neoplasms

A circRNA is a type of endogenous noncoding RNA that consists of a closed circular RNA molecule formed by reverse splicing; these RNAs are widely distributed in a variety of biological cells. In contrast to linear RNAs, circRNAs have no 5′ cap or 3′ poly(A) tail. They have a stable structure, a high degree of conservation, and high stability, and they are richly and specifically expressed in certain tissues and developmental stages. CircRNAs play a very important role in the occurrence and progression of malignant tumors. According to their origins, circRNAs can be divided into four types: exon-derived circRNAs (ecRNAs), intron-derived circRNAs (ciRNAs), circRNAs containing both exons and introns (EIciRNAs) and intergenic circRNAs. A large number of studies have shown that circRNAs have a variety of biological functions, participate in the regulation of gene expression and play an important role in the occurrence and progression of tumors. In this paper, the structure and function of circRNAs are reviewed, along with their biological role in malignant tumors of the digestive tract, in order to provide a reference for the diagnosis and treatment of digestive system neoplasms.Subject terms: Tumour biomarkers, Non-coding RNAs     

DEBKS: A Tool to Detect Differentially Expressed Circular RNAs

Circular RNAs (circRNAs) are involved in various biological processes and disease pathogenesis. However, only a small number of functional circRNAs have been identified among hundreds of thousands of circRNA species, partly because most current methods are based on circular junction counts and overlook the fact that a circRNA is formed from the host gene by back-splicing (BS). To distinguish the expression difference originating from BS or the host gene, we present differentially expressed back-splicing (DEBKS), a software program to streamline the discovery of differential BS events between two rRNA-depleted RNA sequencing (RNA-seq) sample groups. By applying to real and simulated data and employing RT-qPCR for validation, we demonstrate that DEBKS is efficient and accurate in detecting circRNAs with differential BS events between paired and unpaired sample groups. DEBKS is available at https://github.com/yangence/DEBKS as open-source software.     

环状RNA翻译蛋白在癌症中的研究进展北大核心CSCD

环状RNA(circular RNA,circRNA)是一种单链环状闭合RNA分子,由线性RNA通过反向剪接形成,具有稳定、高度保守、组织特异性等特点。circRNA能够通过形成竞争性内源性RNA、结合蛋白等多种方式参与机体的生理、病理过程。最近发现,circRNA分子可以通过翻译形成多肽或蛋白参与癌症的发生和发展。circRNA是人类癌症中有前途的诊断和预后标志物,也是癌症治疗的潜在药物靶点。本文重点介绍了circRNAs编码的多肽和蛋白质在多种癌症中的相关研究进展。这些多肽和蛋白质分别依赖内部核糖体进入位点和m6A两种不同的机制进行翻译。我们还总结了circRNA编码的多肽和蛋白质在各种癌症的诊断、治疗、预后和机制研究中的潜在用途。     

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。     

Analysis of co-expression networks for circular RNAs and mRNAs reveals that circular RNAs hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 are candidate oncogenes in gastric cancer

Accumulating evidence has suggested that circular RNAs (circRNAs) play important roles in oncogenesis and tumor progression. However, our knowledge of circRNAs in gastric cancer (GC) remains limited. To investigate circRNAs involved in GC oncogenesis, we examined differentially-expressed circRNAs and mRNAs in GC tissues and paired noncancerous mucosa tissues using circRNA and mRNA microarrays. Next, we built gene co-expression networks according to the degree of correlation to predict the critical circRNAs in GC. Through bioinformatics analysis, we observed three newly identified circRNAs that are substantially upregulated in GC: hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15. Additionally, hsa_circ_0047905 and hsa_circ_0138960 positively correlated with their parental gene mRNA. Knockdown of hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 in GC cells, resulted in downregulation of parental gene expression. Functional assays suggested that inhibition of these three circular RNAs suppresses GC cell proliferation and invasion in vitro. Those findings suggest that hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 might act as tumor promoters in the pathogenesis of gastric cancer.     

RNA circularization strategies in vivo and in vitro

In the plenitude of naturally occurring RNAs, circular RNAs (circRNAs) and their biological role were underestimated for years. However, circRNAs are ubiquitous in all domains of life, including eukaryotes, archaea, bacteria and viruses, where they can fulfill diverse biological functions. Some of those functions, as for example playing a role in the life cycle of viral and viroid genomes or in the maturation of tRNA genes, have been elucidated; other putative functions still remain elusive. Due to the resistance to exonucleases, circRNAs are promising tools for in vivo application as aptamers, trans-cleaving ribozymes or siRNAs. How are circRNAs generated in vivo and what approaches do exist to produce ring-shaped RNAs in vitro? In this review we illustrate the occurrence and mechanisms of RNA circularization in vivo, survey methods for the generation of circRNA in vitro and provide appropriate protocols.     

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.