The biogenesis, biology and characterization of circular RNAs.

SummaryCircular RNAs (circRNAs) have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, the underlying regulatory mechanism of circRNAs biogenesis remains largely unknown. Here, we integrated in-depth multi-omics data including epigenome, transcriptome, and non-coding RNA and identified candidate circRNAs in six cellular contexts. Next, circRNAs were divided into two classes (high versus low) with different expression levels. Machine learning models were constructed that predicted circRNA expression levels based on 11 different histone modifications and host gene expression. We found that the models achieve great accuracy in predicting high versus low expressed circRNAs. Furthermore, the expression levels of host genes of circRNAs, H3k36me3, H3k79me2, and H4k20me1 contributed greatly to the classification models in six cellular contexts. In summary, all these results suggest that epigenetic modifications, particularly histone modifications, can effectively predict expression levels of circRNAs.

Emerging roles of circular RNAs in systemic lupus erythematosus

The biogenesis, biological functions and modification of Circular RNAs

Circular RNAs (circRNAs) are covalently closed, endogenous biomolecules in eukaryotes with tissue-specific and cell-specific expression patterns, whose biogenesis is regulated by specific cis-acting elements and trans-acting factors. Some circRNAs are abundant and evolutionarily conserved, and many circRNAs exert important biological functions by acting as microRNA or protein inhibitors ('sponges'), by regulating protein function or by being translated themselves. Furthermore, circRNAs have been implicated in diseases such as diabetes mellitus, neurological disorders, cardiovascular diseases and cancer. Although the circular nature of these transcripts makes their detection, quantification and functional characterization challenging, recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of state-of-the-art approaches for their identification, and novel approaches to functional characterization are emerging. PMID: 31395983

Biogenesis and Functions of Circular RNAs Come into Focus.

Identification and characterization of circular RNAs in mammary gland tissue from sheep at peak lactation and during the nonlactating period

Long and Repeat-Rich Intronic Sequences Favor Circular RNA Formation under Conditions of Reduced Spliceosome Activity.

Identification, biogenesis, function, and mechanism of action of circular RNAs in plants

Long non-coding RNAs (lncRNAs), including linear lncRNAs and circular RNAs (circRNAs), exhibit a surprising range of structures. Linear lncRNAs and circRNAs are generated by different pathways. Linear lncRNAs perform functions that depend on their specific sequences, transcription, and DNA elements of their gene loci. In some cases, linear lncRNAs contain a short open reading frame encoding a peptide. circRNAs are covalently closed RNAs with tissue-specific and cell-specific expression patterns that have recently been extensively investigated. Pioneering work focusing on their biogenesis and functional characterization indicates that circRNAs regulate cell development via multiple mechanisms and play critical roles in the immune system. Furthermore, circRNAs in exosomes function on target cells. As with linear lncRNAs, specific circRNAs can also be translated. In this review, we summarize current understanding and highlight the diverse structure, regulation, and function of linear lncRNAs and circRNAs.

Recent advances on genome-wide profiling and characterization of circular RNAs have suggested their versatile roles in diverse biological processes, yet systematic elucidation of their molecular characteristics and functional mechanisms remains challenging. Here, we introduce CIRCpedia v3 (https://bits.fudan.edu.cn/circpediav3), an expanded repository to annotate both circular RNAs from back-splicing of exons (circRNAs) and circular RNAs from intron lariats (ciRNAs) by profiling 2413 sequencing datasets across 20 species. Building upon the previous version of CIRCpedia, this release identifies >2 million circular RNAs and introduces transformative advances to facilitate circular RNA research: (i) community-recommended nomenclature with enhanced molecular profiling, enabling quantitative comparison of circular/linear isoform dynamics; (ii) an interactive platform with real-time comparative analyses of circRNAs and visualizations; and (iii) integrated toolkits to identify base-editable sites, predict circRNA subcellular localization, detect circRNA degradation signals for stability optimization, predict m6A modification sites, assess circRNA coding potential, and design divergent polymerase chain reaction primers and small interfering RNAs (siRNAs). By integrating insights from cross-species expression, molecular characterization, and functional predictions, CIRCpedia v3 empowers researchers to prioritize context-specific circular RNA candidates in biological or disease conditions and to accelerate mechanistic discovery and therapeutic development.

It was long assumed that eukaryotic precursor mRNAs (pre-mRNAs) are almost always spliced to generate a linear mRNA that is subsequently translated to produce a protein. However, it is now clear that thousands of protein-coding genes can be non-canonically spliced to produce circular noncoding RNAs, some of which are expressed at much higher levels than their associated linear mRNAs. How then does the splicing machinery decide whether to generate a linear mRNA or a circular RNA? Recent work has revealed that intronic repetitive elements, including sequences derived from transposons, are critical regulators of this decision. In most cases, circular RNA biogenesis appears to be initiated when complementary sequences from 2 different introns base pair to one another. This brings the splice sites from the intervening exon(s) into close proximity and facilitates the backsplicing event that generates the circular RNA. As many pre-mRNAs contain multiple intronic repeats, distinct circular transcripts can be produced depending on which repeats base pair to one another. Intronic repeats are thus critical regulatory sequences that control the functional output of their host genes, and potentially cause the functions of protein-coding genes to be highly divergent across species.

In central nervous system, axons fail to regenerate after injury while in peripheral nervous system, axons retain certain regenerative ability. Dorsal root ganglion (DRG) neuron has an ascending central axon branch and a descending peripheral axon branch stemming from one single axon and serves as a suitable model for the comparison of growth competence following central and peripheral axon injuries. Molecular alterations underpin different injury responses of DRG branches have been investigated from many aspects, such as coding gene expression, chromatin accessibility, and histone acetylation. However, changes of circular RNAs are poorly characterized. In the present study, we comprehensively investigate circular RNA expressions in DRGs after rat central and peripheral axon injuries using sequencing analysis and identify a total of 33 differentially expressed circular RNAs after central branch injury as well as 55 differentially expressed circular RNAs after peripheral branch injury. Functional enrichment of host genes of differentially expressed circular RNAs demonstrate the participation of Hippo signaling pathway and Notch signaling pathway after both central and peripheral axon injuries. Circular RNA changes after central axon injury are also linked with apoptosis and cellular junction while changes after peripheral axon injury are associated with metabolism and PTEN-related pathways. Altogether, the present study offers a systematic evaluation of alterations of circular RNAs in rat DRGs following injuries to the central and peripheral axon branches and contributes to the deciphering of essential biological activities and mechanisms behind successful nerve regeneration.

ABSTRACTPre-mRNAs from thousands of eukaryotic genes can be non-canonically spliced to generate circular RNAs, some of which accumulate to higher levels than their associated linear mRNA. Recent work has revealed widespread mechanisms that dictate whether the spliceosome generates a linear or circular RNA. For most genes, circular RNA biogenesis via backsplicing is far less efficient than canonical splicing, but circular RNAs can accumulate due to their long half-lives. Backsplicing is often initiated when complementary sequences from different introns base pair and bring the intervening splice sites close together. This process is further regulated by the combinatorial action of RNA binding proteins, which allow circular RNAs to be expressed in unique patterns. Some genes do not require complementary sequences to generate RNA circles and instead take advantage of exon skipping events. It is still unclear what most mature circular RNAs do, but future investigations into their functions will be facilitated by recently described methods to modulate circular RNA levels.

Circular RNA (circRNA) is an endogenous biomolecule in eukaryotes. It has tissue- and cell-specific expression patterns and can act as a microRNA sponge or competitive endogenous RNA. Although circRNA has been found in several species in recent years, the expression profiles in fish gonad are still not fully understood. We detected the expression of circRNA in the ovary, testis, and sex-changed gonad of tilapia by high-throughput deep sequencing, and circRNA-specific computing tools. A total of 20,607 circRNAs were obtained, of which 141 were differentially expressed in the testis and ovary. Among these circRNAs, 135 circRNAs were upregulated and 6 circRNAs were downregulated in female fish. In addition, GO annotation and KEGG pathway analysis of the host genes of circRNAs indicated that these host genes were mainly involved in adherens junction, androgen production, and reproductive development, such as ZP3, PLC, delta 4a, ARHGEF10, and HSD17b3. It is worth noting that we found that circRNAs in tilapia gonads have abundant miRNA-binding sites. Among them, 935 circRNAs have a regulatory effect on miR-212, 856 circRNAs have a regulatory effect on miR-200b-3p, and 529 circRNAs have a regulatory effect on miR-200b-5p. Thus, our findings provide a new evidence for circRNA–miRNA networks in the gonads in tilapia.

Circular RNAs (circRNAs) are an abundant class of non-coding RNAs that are formed by a backsplice event resulting in formation of covalently closed circular RNA molecules. The efficiency of backsplicing is inferior to linear splicing, however, due to their long half-lives circRNAs can accumulate to high levels in cells. Functional characterization of a few circRNAs has shown that they can act as endogenous microRNA sponges and transcriptional regulators; one example is CDR1as/ciRS-7, which has been shown to act as a sponge for miR-7. However, the functions of most circRNAs are still unknown. To study the potential role of circRNAs in cancer, we identified exonic circRNA candidates by analysing ENCODE RNAseq data from a panel of cancer cell lines. We used chimeric alignment detection implemented in the STAR aligner and subsequent filtering of output files to identify chimeric alignments consistent with circRNA backsplice sites. The circular nature of the identified putative circRNAs was validated by testing their resistance to RNase R digestion, and sequences surrounding the backsplice sites were used as target recognition sequences for designing LNA-modified gapmer antisense oligonucleotides (ASOs) to specifically knock down the circular isoforms of the RNA transcripts. Data from ongoing studies on knockdown of several abundant circRNAs in cancer cell lines, assessment of the specificity of backsplice-targeting ASOs relative to the linear mRNA counterparts, and analyses to identify biological effects of modulating circRNA levels in cultured cells will be presented. Citation Format: Charlotte A. Thrue, Andreas Petri, Marianne B. Løvendorf, Karen Dybkær, Dimitrios Papaioannou, Ramiro Garzon, Sakari Kauppinen. Identification and characterization of circular RNAs in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3485. doi:10.1158/1538-7445.AM2017-3485