Plant circRNAs Research Articles

PlantCircRNA: a comprehensive database for plant circular RNAs.

Circular RNAs (circRNAs) represent recently discovered novel regulatory non-coding RNAs. While they are present in many eukaryotes, there has been limited research on plant circRNAs. We developed PlantCircRNA (https://plant.deepbiology.cn/PlantCircRNA/) to fill this gap. The two most important features of PlantCircRNA are (i) it incorporates circRNAs from 94 plant species based on 39 245 RNA-sequencing samples and (ii) it imports the original AtCircDB and CropCircDB databases. We manually curated all circRNAs from published articles, and imported them into the database. Furthermore, we added detailed information of tissue as well as abiotic stresses to the database. To help users understand these circRNAs, the database includes a detection score to measure their consistency and a naming system following the guidelines recently proposed for eukaryotes. Finally, we developed a comprehensive platform for users to visualize, analyze, and download data regarding specific circRNAs. This resource will serve as a home for plant circRNAs and provide the community with unprecedented insights into these mysterious molecule.

Vv-circSIZ1 mediated by pre-mRNA processing machinery contributes to salt tolerance.

CircRNAs exist widely in plants, but the regulatory mechanisms for the biogenesis and function of plant circRNAs remain largely unknown. Using extensive mutagenesis of expression plasmids and genetic transformation methods, we analyzed the biogenesis and anti-salt functions of a new grape circRNA Vv-circSIZ1. We identified Vv-circSIZ1 that is mainly expressed in the cytoplasm of xylem. CircSIZ1 is species-specific, and genomic circSIZ1-forming region of seven tested species could be backspliced in Nicotiana benthamiana, but not in Arabidopsis. The retention length of Vv-circSIZ1 flanking introns was significantly positively correlated with its generation efficiency. The precise splicing of Vv-circSIZ1 does not depend on its mature exon sequence or internal intron sequences, but on the AG/GT splicing signal sites and branch site of the flanking introns. The spliceosome activity was inversely proportional to the expression level of Vv-circSIZ1. Furthermore, RNA-binding proteins can regulate the expression of Vv-circSIZ1. The overexpression of Vv-circSIZ1 improved salt tolerance of grape and N. benthamiana. Additionally, Vv-circSIZ1 could relieve the repressive effect of VvmiR3631 on its target VvVHAc1. Vv-circSIZ1 also promoted transcription of its parental gene. Overall, these results broaden our understanding of circRNAs in plants.

CircPCBL: Identification of Plant CircRNAs with a CNN-BiGRU-GLT Model.

Circular RNAs (circRNAs), which are produced post-splicing of pre-mRNAs, are strongly linked to the emergence of several tumor types. The initial stage in conducting follow-up studies involves identifying circRNAs. Currently, animals are the primary target of most established circRNA recognition technologies. However, the sequence features of plant circRNAs differ from those of animal circRNAs, making it impossible to detect plant circRNAs. For example, there are non-GT/AG splicing signals at circRNA junction sites and few reverse complementary sequences and repetitive elements in the flanking intron sequences of plant circRNAs. In addition, there have been few studies on circRNAs in plants, and thus it is urgent to create a plant-specific method for identifying circRNAs. In this study, we propose CircPCBL, a deep-learning approach that only uses raw sequences to distinguish between circRNAs found in plants and other lncRNAs. CircPCBL comprises two separate detectors: a CNN-BiGRU detector and a GLT detector. The CNN-BiGRU detector takes in the one-hot encoding of the RNA sequence as the input, while the GLT detector uses k-mer (k = 1 - 4) features. The output matrices of the two submodels are then concatenated and ultimately pass through a fully connected layer to produce the final output. To verify the generalization performance of the model, we evaluated CircPCBL using several datasets, and the results revealed that it had an F1 of 85.40% on the validation dataset composed of six different plants species and 85.88%, 75.87%, and 86.83% on the three cross-species independent test sets composed of Cucumis sativus, Populus trichocarpa, and Gossypium raimondii, respectively. With an accuracy of 90.9% and 90%, respectively, CircPCBL successfully predicted ten of the eleven circRNAs of experimentally reported Poncirus trifoliata and nine of the ten lncRNAs of rice on the real set. CircPCBL could potentially contribute to the identification of circRNAs in plants. In addition, it is remarkable that CircPCBL also achieved an average accuracy of 94.08% on the human datasets, which is also an excellent result, implying its potential application in animal datasets. Ultimately, CircPCBL is available as a web server, from which the data and source code can also be downloaded free of charge.

Advances in Research Progress on the Biogenesis and Mechanism of Circular RNA in Plants

Circular RNA (circRNA) is a class of endogenous non-coding RNA which is widely existed in organisms. It is a closed RNA molecule formed by back splicing. CircRNAs have highly structural stability, sequence conservation and tissue specificity. A large number of circRNAs have been identified for the rapid development of high-throughput sequencing and other technologies in plants, which becomes a new field of non-coding RNA research. CircRNA functions acting as microRNA sponge, in regulating gene expression and splicing of transcripts. More and more research results reveal that circRNAs play an important role in organism development and resistance to the external environment. However, at present, we know very little about the biogenesis, function and molecular mechanism of circRNAs in plants, especially the regulation of back splicing of circRNAs. In this review, we summarize the advances in research progress of plant circRNAs, mainly focusing on the alternative splicing of circRNAs and their functions in flowering transition and stress responses. This review will provide an extended understanding of the regulation of biogenesis and molecular mechanism of circRNAs in plants.

Evolutionary Landscape of Tea Circular RNAs and Its Contribution to Chilling Tolerance of Tea Plant.

Chilling stress threatens the yield and distribution pattern of global crops, including the tea plant (Camellia sinensis), one of the most important cash crops around the world. Circular RNA (circRNA) plays roles in regulating plant growth and biotic/abiotic stress responses. Understanding the evolutionary characteristics of circRNA and its feedbacks to chilling stress in the tea plant will help to elucidate the vital roles of circRNAs. In the current report, we systematically identified 2702 high-confidence circRNAs under chilling stress in the tea plant, and interestingly found that the generation of tea plant circRNAs was associated with the length of their flanking introns. Repetitive sequences annotation and DNA methylation analysis revealed that the longer flanking introns of circRNAs present more repetitive sequences and higher methylation levels, which suggested that repeat-elements-mediated DNA methylation might promote the circRNAs biogenesis in the tea plant. We further detected 250 differentially expressed circRNAs under chilling stress, which were functionally enriched in GO terms related to cold/stress responses. Constructing a circRNA-miRNA-mRNA interaction network discovered 139 differentially expressed circRNAs harboring potential miRNA binding sites, which further identified 14 circRNAs that might contribute to tea plant chilling responses. We further characterized a key circRNA, CSS-circFAB1, which was significantly induced under chilling stress. FISH and silencing experiments revealed that CSS-circFAB1 was potentially involved in chilling tolerance of the tea plant. Our study emphasizes the importance of circRNA and its preliminary role against low-temperature stress, providing new insights for tea plant cold tolerance breeding.

Approaches for identification and analysis of plant circular RNAs and their role in stress responses

circRNAs comprise a large class of non-coding RNAs that lack free 3' and 5' ends, are covalently closed and produced by a non-canonical splicing event called backsplicing. Earlier circRNAs were regarded as worthless and called junk RNAs produced due to splicing errors but recent progress in circRNA research indicated their considerable roles in various biological processes. However, plant circRNA research is still in initial stages as compared to animal circRNAs. In this review, we have summarized the process of circRNA biogenesis, how plant circRNAs are different from animals, various modules that can be used for the identification of circRNAs and different circRNA databases. We have also provided an overview of recent progress on our understanding about the involvement of circRNA in various abiotic and biotic stress responses in plants. A proper understanding of the role of circRNAs in different aspects of plant development and stress response will provide novel ways to manipulate plants for their enhanced survivability and productivity in adverse climatic conditions. • circRNAs are long non-coding RNA produced by backsplicing process • circRNAs originate from exons, introns, exon-intron sequences • circRNAs play a key role in plant development and stress responses • PCirc and CircPlant are widely used for circRNA identification, specifically in plants

PlantcircBase 7.0: Full-length transcripts and conservation of plant circRNAs

Circular RNA (circRNA) is a special type of non-coding RNA that participates in diverse biological processes in both animals and plants. Five years ago, we developed a comprehensive plant circRNA database (PlantcircBase), which has attracted much attention from the plant circRNA community. Here, we report an updated PlantcircBase (v.7.0), which contains 171,118 circRNAs from 21 plant species. Over 31,000 of the circRNAs have full-length sequences constructed based on analysis of 749 bulk RNA sequencing (RNA-seq) datasets downloaded from the public domain and Nanopore long-read sequencing results of rice RNAs newly generated in this study. A plant multiple conservation score (PMCS), based on the conservation of both sequence and expression profiles, was calculated for each circRNA to quantify and compare the conservation of all circRNAs. A new parameter, plant circRNA confidence level (PCCL), is introduced to measure the identity reliability of each circRNA based on experimental validation results and the number of references that support the circRNA. All this information and other details of circRNAs can be browsed, searched, and downloaded from PlantcircBase 7.0, which also provides online bioinformatics tools for visualization and sequence alignment. PlantcircBase 7.0 is publicly and freely accessible at http://ibi.zju.edu.cn/plantcircbase/.

Progress in circular RNAs of plants

With the development of high-throughput sequencing technology, circular RNAs (circRNAs) have gradually become a hotspot in the research on non-coding RNA. CircRNAs are produced by the covalent circularization of a downstream 3' splice donor and an upstream 5' splice acceptor through backsplicing, and they are pervasive in eukaryotic cells. CircRNAs used to be considered byproducts of false splicing, whereas an explosion of related studies in recent years has disproved this misconception. Compared with the rich studies of circRNAs in animals, the study of circRNAs in plants is still in its infancy. In this review, we introduced the discovery of plant circRNAs, the discovery of plant circRNAs, the circularization feature, expression specificity, conservation, and stability of plant circRNAs and expounded the identification tools, main types, and biogenesis mechanisms of circRNAs. Furthermore, we summarized the potential roles of plant circRNAs as microRNA (miRNA) sponges and translation templates and in response to biotic/abiotic stress, and briefed the degradation and localization of plant circRNAs. Finally, we discussed the challenges and proposed the future directions in the research on plant circRNAs.

Exonic Circular RNAs Are Involved in Arabidopsis Immune Response Against Bacterial and Fungal Pathogens and Function Synergistically with Corresponding Linear RNAs.

Circular RNAs (circRNAs) are a group of covalently closed RNAs, and their biological function is largely unknown. In this study, we focused on circRNAs that are generated from exon back-splicing (exonic circRNAs). The linear RNA counterparts encode functional proteins so that we can compare and investigate the relationship between circular and linear RNAs. We compared circRNA expression profiles between untreated and Pseudomonas syringae-infected Arabidopsis and identified and experimentally validated differentially expressed exonic circRNAs by multiple approaches. We found that exonic circRNAs are preferentially enriched in biological processes that associate with biotic and abiotic stress responses. We discovered that circR194 and circR4022 are involved in plant response against P. syringae infection, whereas circR11208 is involved in response against Botrytis cinerea infection. Intriguingly, our results indicate that these exonic circRNAs function synergistically with their corresponding linear RNAs. Furthermore, circR4022 and circR11208 also play substantial roles in Arabidopsis tolerance to salt stress. This study extends our understanding of the molecular functions of plant circRNAs.

Recent origination of circular RNAs in plants

Circular RNA (circRNA) is a kind of new regulatory RNA with diverse biological functions. Numerous circRNAs have been identified in many plant species; however, evolution of plant circRNAs remains largely unknown. In this study, we assembled full-length sequences of 6519 rice (Oryza sativa) circRNAs and analyzed their conservation in another 46 plant species based on comparison of sequences and expression patterns. We found that, at the genomic level, 8.7% of the 6519 circRNAs were conserved in dicotyledonous plants and 49.1% in Oryza genus. Meanwhile, 57.8% of parental protein-coding genes of the rice circRNAs originated recently after divergence of monocotyledonous plants, implying recent origin of the majority of rice circRNAs, a conclusion further supported by the results based on analysis of 4663 full-length circRNAs in Arabidopsis thaliana. Accordingly, we proposed three models to address the origination of different types of circRNAs. Taken together, the results obtained in this study provide new insights for the evolutionary dynamics of plant circRNAs and candidate circRNAs for further functional exploration.

Circular RNAs acting as ceRNAs mediated by miRNAs may be involved in the synthesis of soybean fatty acids.

Soybean oil is composed of fatty acids and glycerol. The content and composition of fatty acids partly determine the quality of soybean seeds. Circular RNAs (circRNAs) are endogenous non-coding RNAs that competitively bind to microRNAs (miRNAs) through miRNA recognition elements, thereby acting as sponges to regulate the expression of target genes. Although circRNAs have been identified previously in soybean, only their expression has been investigated without exploration of the competitive endogenous RNAs (ceRNAs) network of circRNAs-miRNAs-mRNAs. In this study, circRNAs in immature pods of a low linolenic acid soybean Mutant 72' (MT72) and the wild-type control 'Jinong 18' (JN18) were systematically identified and analyzed at 30 and 40days after flowering using high-throughput sequencing technology. We identified 6377 circRNAs, of which 114 were differentially expressed. Gene ontology and KEGG pathway analyses of targeted mRNAs in the ceRNAs network indicated that the differentially expressed circRNAs may be involved in fatty acid transport, suggesting that circRNAs may play a post-transcriptional regulatory role in soybean oil synthesis. This study provides a foundation for future exploration of the function of circRNAs in soybean and presents novel insights to guide further studies of plant circRNAs.

PCirc: random forest-based plant circRNA identification software

BackgroundCircular RNA (circRNA) is a novel type of RNA with a closed-loop structure. Increasing numbers of circRNAs are being identified in plants and animals, and recent studies have shown that circRNAs play an important role in gene regulation. Therefore, identifying circRNAs from increasing amounts of RNA-seq data is very important. However, traditional circRNA recognition methods have limitations. In recent years, emerging machine learning techniques have provided a good approach for the identification of circRNAs in animals. However, using these features to identify plant circRNAs is infeasible because the characteristics of plant circRNA sequences are different from those of animal circRNAs. For example, plants are extremely rich in splicing signals and transposable elements, and their sequence conservation in rice, for example is far less than that in mammals. To solve these problems and better identify circRNAs in plants, it is urgent to develop circRNA recognition software using machine learning based on the characteristics of plant circRNAs.ResultsIn this study, we built a software program named PCirc using a machine learning method to predict plant circRNAs from RNA-seq data. First, we extracted different features, including open reading frames, numbers of k-mers, and splicing junction sequence coding, from rice circRNA and lncRNA data. Second, we trained a machine learning model by the random forest algorithm with tenfold cross-validation in the training set. Third, we evaluated our classification according to accuracy, precision, and F1 score, and all scores on the model test data were above 0.99. Fourth, we tested our model by other plant tests, and obtained good results, with accuracy scores above 0.8. Finally, we packaged the machine learning model built and the programming script used into a locally run circular RNA prediction software, Pcirc (https://github.com/Lilab-SNNU/Pcirc).ConclusionBased on rice circRNA and lncRNA data, a machine learning model for plant circRNA recognition was constructed in this study using random forest algorithm, and the model can also be applied to plant circRNA recognition such as Arabidopsis thaliana and maize. At the same time, after the completion of model construction, the machine learning model constructed and the programming scripts used in this study are packaged into a localized circRNA prediction software Pcirc, which is convenient for plant circRNA researchers to use.

Identification and Characterization of Plant Circular RNAs.

Recent studies have reported that circular RNAs (circRNAs) are a newly discovered type of ubiquitous, abundant and stable noncoding RNAs (ncRNAs) that play important roles in various biological processes in eukaryotic organisms. However, the biological functions of circRNAs in plants remain largely unknown and need further studies. Identification of plant circRNAs from plant circRNA database or sequencing analysis is a first step to investigate their functions. Here, we provide a series of protocols for circRNA identification including circular forms, composition features and location even in plant tissues which are rich in polysaccharides and polyphenols and difficult to extract RNAs.

Experimental Strategies for Studying the Function of Plant CircRNAs.

Circular RNAs (circRNAs) represent a novel group of noncoding RNA whose functions are little known in plants. Genetic manipulation is necessary for studying the biological function of specific circRNA. Here, we describe strategies to study the function of plant circRNAs including artificial microRNA-mediated circRNA knockdown, gain-of-function study, full-length circRNA identification, and circRNA-protein interaction. These methods can be applied to functional characterization of circRNAs in plants and would be promising to facilitate the research on plant circRNAs.

Expression Landscape of circRNAs in Arabidopsis thaliana Seedlings and Adult Tissues.

RNA-seq is currently the only method that can provide a comprehensive landscape of circular RNA (circRNAs) in the whole organism and its particular organs. Recent years have brought an increasing number of RNA-seq-based reports on plant circRNAs. Notably, the picture they revealed is questionable and depends on the applied circRNA identification and quantification techniques. In consequence, little is known about the biogenesis and functions of circRNAs in plants. In this work, we tested two experimental and six bioinformatics procedures of circRNA analysis to determine the optimal approach for studying the profiles of circRNAs in Arabidopsis thaliana. Then using the optimized strategy, we determined the accumulation of circular and corresponding linear transcripts in plant seedlings and organs. We observed that only a small fraction of circRNAs was reproducibly generated. Among them, two groups of circRNAs were discovered: ubiquitous and organ-specific. The highest number of circRNAs with significantly increased accumulation in comparison to other organs/seedlings was found in roots. The circRNAs in seedlings, leaves and flowers originated mainly from genes involved in photosynthesis and the response to stimulus. The levels of circular and linear transcripts were not correlated. Although RNase R treatment enriches the analyzed RNA samples in circular transcripts, it may also have a negative impact on the stability of some of the circRNAs. We also showed that the normalization of NGS data by the library size is not proper for circRNAs quantification. Alternatively, we proposed four other normalization types whose accuracy was confirmed by ddPCR. Moreover, we provided a comprehensive characterization of circRNAs in A. thaliana organs and in seedlings. Our analyses revealed that plant circRNAs are formed in both stochastic and controlled processes. The latter are less frequent and likely engage circRNA-specific mechanisms. Only a few circRNAs were organ-specific. The lack of correlation between the accumulation of linear and circular transcripts indicated that their biogenesis depends on different mechanisms.

Systematic Identification and Analysis of Light-Responsive Circular RNA and Co-expression Networks in Lettuce (Lactuca sativa).

Circular RNA (circRNA) is a covalently-closed single-stranded RNA molecule that plays an important role in transcriptional regulation of gene expression in a variety of species. Light intensity is a pivotal environmental factor affecting plant growth and development. However, little is known regarding photoresponsive plant circRNAs. Here, we aimed to investigate the expression and function of circRNAs in lettuce leaves in response to different light intensity treatments. We performed RNA sequencing (RNA-Seq) on leaves of lettuce (Lactuca sativa) to determine circRNA expression profiles and reverse-transcription polymerase chain reaction (PCR) to validate the candidate circRNA molecules. We then combined bioinformatics approach to explore the function of the parental genes of circRNA, including network, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathway analysis. We identified 1650 circRNAs in lettuce, of which 1508 (86.40%) were derived from exons. Using real-time PCR, we characterized 10 validated differentially expressed circRNAs and their parental genes, all of which showed expression patterns consistent with RNA-Seq data. Interestingly, the expression of circRNA was, in some cases, inversely correlated with the expression of the parental gene. Furthermore, analysis of the circRNA–microRNA–mRNA network suggests that circRNAs may be involved in plant hormone signaling and chlorophyll metabolism during photoreactivity. These findings provide an essential reference basis for studying circRNAs’ biological mechanisms in light-treated plants.

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.

Characterization and Function of Circular RNAs in Plants.

CircRNAs are covalently closed-loop single-stranded RNA molecules ubiquitously expressing in eukaryotes. As an important member of the endogenous ncRNA family, circRNAs are associated with diverse biological processes and can regulate transcription, modulate alternative splicing, and interact with miRNAs or proteins. Compared to abundant advances in animals, studies of circRNAs in plants are rapidly emerging. The databases and analysis tools for plant circRNAs are constantly being developed. Large numbers of circRNAs have been identified and characterized in plants and proved to play regulatory roles in plant growth, development, and stress responses. Here, we review the biogenesis, characteristics, bioinformatics resources, and biological functions of plant circRNAs, and summarize the distinct circularization features and differentially expression patterns comparison with animal-related results.

Identification, characterization, and functional prediction of circular RNAs in maize.

Circular RNAs (circRNAs) are a new type of intracellular regulator that have been widely identified in animals and plants by high-throughput sequencing. However, there are still few functional studies on circRNAs in plants. To better understand maize circRNAs and their potential functions, we identified 1199 circRNAs in maize from RiboMinus RNA-Seq transcriptome data, and found distinct features of splicing site selection bias, longer flanking introns, and miniature inverted-repeat transposable element (MITE) insertions in flanking introns in maize circRNAs compared to other plant circRNAs. In total, 31 and 36 orthologous circRNAs were identified in rice and maize, respectively, but the orthologous parental genes could not produce orthologous circRNAs, mostly because of long-sequence insertions/deletions at flanking introns and approximately 24.3% of them contained MITE sequences. The majority of maize circRNAs showed high diversity of expression under different treatments and/or in different genetic backgrounds, implying that circRNAs could be involved in various regulatory networks. Twenty-six ecircRNAs were predicted to contain one or more target mimics, and 229 circRNAs had high coding potential, indicating that circRNAs could perform peptide-encoding functions in plants. These results will broaden understanding of the roles of circRNAs in plants and support further functional work on maize.

GreenCircRNA: a database for plant circRNAs that act as miRNA decoys.

Circular RNAs (circRNAs) are endogenous non-coding RNAs that form a covalently closed continuous loop, are widely distributed and play important roles in a series of developmental processes. In plants, an increasing number of studies have found that circRNAs can regulate plant metabolism and are involved in plant responses to biotic or abiotic stress. Acting as miRNA decoys is a critical way for circRNAs to perform their functions. Therefore, we developed GreenCircRNA—a database for plant circRNAs acting as miRNA decoys that is dedicated to providing a plant-based platform for detailed exploration of plant circRNAs and their potential decoy functions. This database includes over 210 000 circRNAs from 69 species of plants; the main data sources of circRNAs in this database are NCBI, EMBL-EBI and Phytozome. To investigate the function of circRNAs as competitive endogenous RNAs, the possibility of circRNAs from 38 plants to act as miRNA decoys was predicted. Moreover, we provide basic information for the circRNAs in the database, including their locations, host genes and relative expression levels, as well as full-length sequences, host gene GO (Gene Ontology) numbers and circRNA visualization. GreenCircRNA is the first database for the prediction of circRNAs that act as miRNA decoys and contains the largest number of plant species. Database URL: http://greencirc.cn