Circular RNA Profiling by Illumina Sequencing via Template-Dependent Multiple Displacement Amplification

Circular RNAs (circRNAs) are newly discovered incipient non-coding RNAs with potential roles in disease progression in living organisms. Significant reports, since their inception, highlight the abundance and putative functional roles of circRNAs in every organism checked for, like O. sativa, Arabidopsis, human, and mouse. CircRNA expression is generally less than their linear mRNA counterparts which fairly explains the competitive edge of canonical splicing over non-canonical splicing. However, existing methods may not be sensitive enough for the discovery of low-level expressed circRNAs. By combining template-dependent multiple displacement amplification (tdMDA), Illumina sequencing, and bioinformatics tools, we have developed an experimental protocol that is able to detect 1,875 novel and known circRNAs from O. sativa. The same method also revealed 9,242 putative circRNAs in less than 40 million reads for the first time from the Nicotiana benthamiana whose genome has not been fully annotated. Supported by the PCR-based validation and Sanger sequencing of selective circRNAs, our method represents a valuable tool in profiling circRNAs from the organisms with or without genome annotation.

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Circular RNA profiling reveals abundant and diverse circRNAs of SARS-CoV-2, SARS-CoV and MERS-CoV origin

10.1101/2020.12.07.415422 ◽

2020 ◽

Author(s):

Shaomin Yang ◽

Hong Zhou ◽

Ruth Cruz-Cosme ◽

Mingde Liu ◽

Jiayu Xu ◽

...

Keyword(s):

De Novo ◽

Splice Junction ◽

Circular Rna ◽

Circular Rnas ◽

Sequencing Data ◽

Rna Profiling ◽

Systematic Strategy ◽

Coronavirus Infection ◽

Abundance And Diversity ◽

First Time

ABSTRACTCircular RNAs (circRNAs) encoded by DNA genomes have been identified across host and pathogen species as parts of the transcriptome. Accumulating evidences indicate that circRNAs play critical roles in autoimmune diseases and viral pathogenesis. Here we report that RNA viruses of the Betacoronavirus genus of Coronaviridae, SARS-CoV-2, SARS-CoV and MERS-CoV, encode a novel type of circRNAs. Through de novo circRNA analyses of publicly available coronavirus-infection related deep RNA-Sequencing data, we identified 351, 224 and 2,764 circRNAs derived from SARS-CoV-2, SARS-CoV and MERS-CoV, respectively, and characterized two major back-splice events shared by these viruses. Coronavirus-derived circRNAs are more abundant and longer compared to host genome-derived circRNAs. Using a systematic strategy to amplify and identify back-splice junction sequences, we experimentally identified over 100 viral circRNAs from SARS-CoV-2 infected Vero E6 cells. This collection of circRNAs provided the first line of evidence for the abundance and diversity of coronavirus-derived circRNAs and suggested possible mechanisms driving circRNA biogenesis from RNA genomes. Our findings highlight circRNAs as an important component of the coronavirus transcriptome.SummaryWe report for the first time that abundant and diverse circRNAs are generated by SARS-CoV-2, SARS-CoV and MERS-CoV and represent a novel type of circRNAs that differ from circRNAs encoded by DNA genomes.

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A novel circular RNA, circXPO1, promotes lung adenocarcinoma progression by interacting with IGF2BP1

Cell Death and Disease ◽

10.1038/s41419-020-03237-8 ◽

2020 ◽

Vol 11 (12) ◽

Author(s):

Qi Huang ◽

Haifa Guo ◽

Shaodong Wang ◽

Yi Ma ◽

Haiming Chen ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Therapeutic Target ◽

Noncoding Rnas ◽

Xenograft Model ◽

Gene Copy Number ◽

Circular Rna ◽

Gene Copy ◽

Circular Rnas ◽

Functional Roles ◽

Tumor Tissues

AbstractStudies have demonstrated that noncoding RNAs play important roles in various types of cancer; however, noncoding RNAs derived from regions of genomic alterations have rarely been explored, especially for circular RNAs (circRNA). Previously, we found several circRNAs were upregulated in lung adenocarcinoma (LUAD) tumor tissues by RNA sequencing. Here, we characterized a novel circRNA, circXPO1, in LUAD, which is derived from a well-established cancer therapeutic target, XPO1. circXPO1, is formed by back-splicing of exon 3 and exon 4 of XPO1 gene. circXPO1 was highly expressed in LUAD tissues compared with paired adjacent non-tumor tissues, and high circXPO1 expression correlated with worse overall survival. circXPO1 expression was positively correlated with the XPO1 gene copy number. Mechanically, circXPO1 could bind with IGF2BP1 and enhance CTNNB1 mRNA stability, and subsequently promote LUAD progression. In a LUAD patient-derived xenograft model, intratumoural injection of cholesterol-conjugated siRNA specifically targeting circXPO1 efficiently suppressed tumor growth. To summary, these results suggest that circXPO1 is critical for LUAD progression and may serve as a biomarker for poor prognosis and a therapeutic target. On the other hand, the functional roles of noncoding transcripts derived from coding genes should be re-evaluated.

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ciRS-7 exonic sequence is embedded in a long non-coding RNA locus

10.1101/169508 ◽

2017 ◽

Cited By ~ 1

Author(s):

Steven P. Barrett ◽

Kevin R. Parker ◽

Caroline Horn ◽

Miguel Mata ◽

Julia Salzman

Keyword(s):

Circular Rnas ◽

Biological Regulation ◽

Functional Roles ◽

Rna Transcripts ◽

Non Coding Rna ◽

Genome Wide ◽

And Function ◽

Long Non Coding Rna ◽

Human And Mouse ◽

Exonic Sequence

AbstractciRS-7 is an intensely studied, highly expressed and conserved circRNA. Essentially nothing is known about its biogenesis, including the location of its promoter. A prevailing assumption has been that ciRS-7 is an exceptional circRNA because it is transcribed from a locus lacking any mature linear RNA transcripts of the same sense. Our interest in the biogenesis of ciRS-7 led us to develop an algorithm to define its promoter. This approach predicted that the human ciRS-7 promoter coincides with that of the long non-coding RNA, LINC00632. We validated this prediction using multiple orthogonal experimental assays. We also used computational approaches and experimental validation to establish that ciRS-7 exonic sequence is embedded in linear transcripts that are flanked by cryptic exons in both human and mouse. Together, this experimental and computational evidence generate a new view of regulation in this locus: (a) ciRS-7 is like other circRNAs, as it is spliced into linear transcripts; (b) expression of ciRS-7 is primarily determined by the chromatin state of LINC00632 promoters; (c) transcription and splicing factors sufficient for ciRS-7 biogenesis are expressed in cells that lack detectable ciRS-7 expression. These findings have significant implications for the study of the regulation and function of ciRS-7, and the analytic framework we developed to jointly analyze RNA-seq and ChlP-seq data reveal the potential for genome-wide discovery of important biological regulation missed in current reference annotations.Author SummarycircRNAs were recently discovered to be a significant product of ‘host’ gene expression programs but little is known about their transcriptional regulation. Here, we have studied the expression of a well-known circRNA named ciRS-7. ciRS-7 has an unusual function for a circRNA; it is believed to be a miRNA sponge. Previously, ciRS-7 was thought to be transcribed from a locus lacking any mature linear isoforms, unlike all other circular RNAs known to be expressed in human cells. However, we have found this to be false; using a combination of bioinformatic and experimental genetic approaches, in both human and mouse, we discovered that linear transcripts containing the ciRS-7 exonic sequence, linking it to upstream genes. This suggests the potential for additional functional roles of this important locus and provides critical information to begin study on the biogenesis of ciRS-7.

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Circular RNA Profiling and Bioinformatic Modeling Identify Its Regulatory Role in Hepatic Steatosis

BioMed Research International ◽

10.1155/2017/5936171 ◽

2017 ◽

Vol 2017 ◽

pp. 1-13 ◽

Cited By ~ 17

Author(s):

Xing-Ya Guo ◽

Chong-Xin He ◽

Yu-Qin Wang ◽

Chao Sun ◽

Guang-Ming Li ◽

...

Keyword(s):

Hepatic Steatosis ◽

Metabolic Pathways ◽

Target Prediction ◽

Principal Component ◽

Circular Rna ◽

Circular Rnas ◽

Rna Profiling ◽

Pathway Annotation ◽

Wide Range ◽

Chain Acyl

Circular RNAs (circRNAs) exhibit a wide range of physiological and pathological activities. To uncover their role in hepatic steatosis, we investigated the expression profile of circRNAs in HepG2-based hepatic steatosis induced by high-fat stimulation. Differentially expressed circRNAs were subjected to validation using QPCR and functional analyses using principal component analysis, hierarchical clustering, target prediction, gene ontology (GO), and pathway annotation, respectively. Bioinformatic integration established the circRNA-miRNA-mRNA regulatory network so as to identify the mechanisms underlying circRNAs’ metabolic effect. Here we reported that hepatic steatosis was associated with a total of 357 circRNAs. Enrichment of transcription-related GOs, especially GO: 0006355, GO: 004589, GO: 0045944, GO: 0045892, and GO: 0000122, demonstrated their specific actions in transcriptional regulation. Lipin 1 (LPIN1) was recognized to mediate the transcriptional regulatory effect of circRNAs on metabolic pathways. circRNA-miRNA-mRNA network further identified the signaling cascade of circRNA_021412/miR-1972/LPIN1, which was characterized by decreased level of circRNA_021412 and miR-1972-based inhibition of LPIN1. LPIN1-induced downregulation of long chain acyl-CoA synthetases (ACSLs) expression finally resulted in the hepatosteatosis. These findings identify circRNAs to be important regulators of hepatic steatosis. Transcription-dependent modulation of metabolic pathways may underlie their effects, partially by the circRNA_021412/miR-1972/LPIN1 signaling.

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Nanopore sequencing of brain-derived full-length circRNAs reveals circRNA-specific exon usage, intron retention and microexons

Nature Communications ◽

10.1038/s41467-021-24975-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Karim Rahimi ◽

Morten T. Venø ◽

Daniel M. Dupont ◽

Jørgen Kjems

Keyword(s):

Intron Retention ◽

Circular Rna ◽

Nanopore Sequencing ◽

Nonsense Mediated Decay ◽

Functional Roles ◽

Cellular Processes ◽

Long Read ◽

Exon Usage ◽

Non Coding Rnas ◽

Human And Mouse

AbstractCircular RNA (circRNA) is a class of covalently joined non-coding RNAs with functional roles in a wide variety of cellular processes. Their composition shows extensive overlap with exons found in linear mRNAs making it difficult to delineate their composition using short-read RNA sequencing, particularly for long and multi-exonic circRNAs. Here, we use long-read nanopore sequencing of nicked circRNAs (circNick-LRS) and characterize a total of 18,266 and 39,623 circRNAs in human and mouse brain, respectively. We further develop an approach for targeted long-read sequencing of a panel of circRNAs (circPanel-LRS), eliminating the need for prior circRNA enrichment and find >30 circRNA isoforms on average per targeted locus. Our data show that circRNAs exhibit a large number of splicing events such as novel exons, intron retention and microexons that preferentially occur in circRNAs. We propose that altered exon usage in circRNAs may reflect resistance to nonsense-mediated decay in the absence of translation.

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Circular RNA profiling reveals that circular RNAs from ANXA2 can be used as new biomarkers for multiple sclerosis

Human Molecular Genetics ◽

10.1093/hmg/ddx243 ◽

2017 ◽

Vol 26 (18) ◽

pp. 3564-3572 ◽

Cited By ~ 45

Author(s):

Leire Iparraguirre ◽

Maider Muñoz-Culla ◽

Iñigo Prada-Luengo ◽

Tamara Castillo-Triviño ◽

Javier Olascoaga ◽

...

Keyword(s):

Multiple Sclerosis ◽

Circular Rna ◽

Circular Rnas ◽

Rna Profiling ◽

New Biomarkers

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Circular RNA Circ-0002570 Accelerates Cancer Progression by Regulating VCAN via MiR-587 in Gastric Cancer

Frontiers in Oncology ◽

10.3389/fonc.2021.733745 ◽

2021 ◽

Vol 11 ◽

Author(s):

Lei Yang ◽

Yong-ning Zhou ◽

Miao-miao Zeng ◽

Nan Zhou ◽

Bin-sheng Wang ◽

...

Keyword(s):

Gastric Cancer ◽

Cancer Progression ◽

Circular Rna ◽

Luciferase Reporter ◽

Migration And Invasion ◽

Circular Rnas ◽

Functional Roles ◽

Reporter Assays

BackgroundCircular RNAs (circRNAs) are closely associated with the occurrences and progress of gastric cancer (GC). We aimed to delve into the function and pathological mechanism of Circular RNA-0002570 (circ-0002570) in GC progression.MethodsCircRNAs differentially expressed in GC were screened using bioinformatics technology. The expression of circ-0002570 was detected in GC specimens and cells via qRT-PCR, and the prognostic values of circ-0002570 were determined. The functional roles of circ-0002570 on proliferation, migration, and invasion in GC cells were explored in vitro and in vivo. Interaction of circ-0002570, miR-587, and VCAN was confirmed by dual-luciferase reporter assays, Western blotting, and rescue experiments.ResultsCirc-0002570 expression was distinctly increased in GC tissues compared to adjacent normal specimens, and GC patients with higher circ-0002570 expressions displayed a short survival. Functionally, knockdown of circ-0002570 resulted in the inhibition of cell proliferation, migration, and invasion, and suppressed tumor growth in vivo. Mechanistically, miR-587 was sponged by circ-0002570. VCAN expression in NSCLC was directly inhibited by miR-587. Overexpression of circ-0002570 prevented VCAN from miR-587-mediated degradation and thus facilitated GC progression.ConclusionThe circ-0002570-miR-587-VCAN regulatory pathway promoted the progression of GC. Our findings provided potential new targets for the diagnosis and therapy of GC.

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A Novel Circular RNA hsa_circ_20403 Predicts Poor Prognosis and Promotes Progression in Osteosarcoma

10.21203/rs.3.rs-61032/v1 ◽

2020 ◽

Author(s):

Rui Li ◽

Renzan Zhang ◽

Zhengpeng Liu ◽

Yilong Zhang

Keyword(s):

Biological Function ◽

Bone Cancer ◽

Circular Rna ◽

Migration And Invasion ◽

Circular Rnas ◽

Illumina Hiseq ◽

Promote Cell Proliferation ◽

Cancer Initiation ◽

Tissue Specimens ◽

First Time

Abstract Objective Osteosarcoma (OS) is the most frequent bone cancer in humans. Growing evidence suggests that circular RNAs (circRNAs) exert essential roles in regulating cancer initiation and metastasis. Some research were used Illumina HiSeq to screens differentially expressed of circRNAs in OS and found that hsa_circ_20403 is highly expressed in OS. Methods In this study, we investigated the clinical application value and biological function of hsa_circ_20403 in OS. We were performed to verified the biological functions by CCK-8, flow cytometry, and transwell experiments. Results We found that the expression of circ_20403 is enhanced in both OS tissue specimens and cells. In addition, its expression is closely related to clinic opathological characteristics and prognosis. More importantly, circ_20403 was overexpressed and knocked down in OS cells. We found that circ_20403 can promote cell proliferation, migration and invasion, which can regulate the proportion of G1 and S phase cells. Conclusion Taken together, our study for the first time revealed hsa_circ_20403 as a carcinogen to promote the progression of OS. Our study suggested that hsa_circ_20403 can be used to predict the prognosis of OS and it might be a potential target for OS therapy.

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Comprehensive Circular RNA Profiling Reveals the Regulatory Role of the CircRNA-0067835/miR-155 Pathway in Temporal Lobe Epilepsy

Cellular Physiology and Biochemistry ◽

10.1159/000495589 ◽

2018 ◽

Vol 51 (3) ◽

pp. 1399-1409 ◽

Cited By ~ 17

Author(s):

Guo-Hua Gong ◽

Feng-Mao An ◽

Yu Wang ◽

Ming Bian ◽

Di Wang ◽

...

Keyword(s):

Temporal Lobe Epilepsy ◽

Temporal Lobe ◽

Circular Rna ◽

Fine Tuning ◽

Luciferase Reporter ◽

Circular Rnas ◽

G1 Arrest ◽

Rna Profiling

Background/Aims: Temporal lobe epilepsy (TLE) is the most common form of adult localization-related epilepsy that is accompanied by progressive etiopathology and high incidences of drug resistance. Circular RNAs (circRNAs) play important roles in fine-tuning gene expression, however, the expression profile and clinical significance of circRNAs in TLE remains unknown. Methods: Circular RNA microarray was conducted to identify TLE-related circRNAs. CCK8 assays and flow cytometric assays were conducted to clarify the role of circRNA in TLE in vitro. Bioinformatics analysis and in vitro experiments were conducted to clarify the mechanism of circRNA-mediated gene regulation in TLE cell. Results: 586 differentially expressed circRNAs were identified between TLE and the control tissues. The expression of circRNA-0067835 was significantly down-regulated in tissues and plasma from TLE patients. Lower circRNA-0067835 correlated to increased seizure frequency, HS, and higher Engel’s score. Overexpression of circRNA-0067835 observably decreased SH-SY5Y cell proliferation by causing G1 arrest and promoting apoptosis. Bioinformatics online programs predicted that circRNA-0067835 acted as miR-155 sponge to regulate FOXO3a expression, which was validated using luciferase reporter assay. Conclusion: Our experiments showed that circRNA-0067835 regulated refractory epilepsy progression by acting as a sponge of miR-155 to promote FOXO3a expression, indicating that circRNA-0067835 may serve as a potential therapeutic target for patients with TLE.

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Circular RNA expression profile in the spinal cord of morphine tolerated rats and screen of putative key circRNAs

Molecular Brain ◽

10.1186/s13041-019-0498-4 ◽

2019 ◽

Vol 12 (1) ◽

Cited By ~ 2

Author(s):

Yingqi Weng ◽

Jing Wu ◽

Lin Li ◽

Jiali Shao ◽

Zhengyiqi Li ◽

...

Keyword(s):

Spinal Cord ◽

Morphine Tolerance ◽

Circular Rna ◽

Health Concern ◽

Circular Rnas ◽

Morphine Treatment ◽

Kegg Analysis ◽

Non Coding Rna ◽

First Time

Abstract Morphine tolerance developed after repeated or continuous morphine treatment is a global health concern hindering the control of chronic pain. In our previous research, we have reported that the expression of lncRNAs and microRNAs have been greatly modified in the spinal cord of morphine tolerated rats, and the modulating role of miR-873a-5p, miR-219-5p and miR-365 have already been confirmed. However, whether circular RNAs, another essential kind of non-coding RNA, are involved in the pathogenesis of morphine tolerance is still beyond our knowledge. In this study, we conducted microarray analysis for circRNA profile and found a large number of circRNAs changed greatly in the spinal cord by morphine treatment. Among them, we selected nine circRNAs for validation, and seven circRNAs are confirmed. Gene Ontology/Kyoto Encyclopedia of Genes and Genomes (GO/KEGG) analysis were used for functional annotation. Besides, we confirmed the modified expression of seven circRNAs after validation by real-time PCR, selected 3 most prominently modulated ones among them and predicted their downstream miRNA-mRNA network and analyzed their putative function via circRNA-miRNA-mRNA pathway. Finally, we enrolled the differentially expressed mRNAs derived from the identical spinal cord, these validated circRNAs and their putative miRNA targets for ceRNA analysis and screened a promising circRNA-miRNA-mRNA pathway in the development of morphine tolerance. This study, for the first time, provided valuable information on circRNA profile and gave clues for further study on the circRNA mechanism of morphine tolerance.

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