Regulation of circRNA biogenesis

The advent of high throughput technologies has revealed that mammalian genomes are pervasively transcribed, most for long noncoding RNAs (lncRNAs, at least 200 nt long). Thousands of lncRNAs from intergenic regions (large intergenic noncoding RNA, lincRNA) have been uncovered by massive deep sequencing from the repertoire of polyadenylated (poly(A)+) RNAs, together with multiple chromatin landscapes. These lncRNAs are messenger RNA (mRNA)-like, with linear signatures of 5′ mG caps and 3′ poly(A)+ tails. Unexpectedly, mammalian transcriptomes are even more complex with the expression of RNAs without polyadenylated tails (poly(A)– RNAs) [1], leading to the identification of new lncRNA formats, such as circular RNAs. Due to the covalently close structure and without 3′ poly(A) tails, circular RNAs failed to be analyzed in most transcriptome analyses mainly for polyadenylated RNAs. By taking advantage of deep sequencing from nonpolyadenylated RNA population [1], thousands of circular RNAs were identified to be widely expressed in human cell lines. There are at least two different types of circular RNAs processed from pre-RNA splicing: one type is derived from spliced introns (circular intronic RNAs) [2] and the other type is from back-spliced exons (exonic circular RNAs) [3]. Circular intronic RNAs (ciRNAs) are produced from introns that fail to be debranched after splicing, but covalently circularized with 2′,5′-phosphodiester bond between a splice donor site and a branch point site. The formation of ciRNAs can be reconstituted in expression vectors with the requirement of consensus motifs flanking 2′,5′-phosphodiester bonds. Importantly, ciRNAs were shown to play an important cis-regulatory role in local gene expression [2]. Exonic circular RNAs (circRNAs) are produced from back-spliced circularization [3]. Unlike (normal) RNA splicing that joins an upstream splice donor site with a downstream splice acceptor site, leading to a linear RNA transcript (Figure 1A), back splicing joins a downstream splice donor site reversely with an upstream splice acceptor site, yielding a circular RNA transcript with 3′,5′-phosphodiester bond at the joint site (Figure 1B). In last decades, only a handful of circRNAs were identified and indicated as byproducts of splicing errors with no function. Until recently, the genome-wide profiling of

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.

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

CircSTK40 contributes to recurrent implantation failure via modulating the HSP90/AKT/FOXO1 axis

Artificial Circular RNA Sponges Targeting MicroRNAs as a Novel Tool in Molecular Biology

Preeclampsia (PE) is a multiple organ and system disease that seriously threatens the safety of the mother and infant during pregnancy, and has a profound impact on the morbidity and mortality of the mother and new babies. Presently, there are no remedies for cure of PE as to the mechanisms of PE are still unclear, and the only way to eliminate the symptoms is to deliver the placenta. Thus, new therapeutic targets for PE are urgently needed. Approximately 95% of human transcripts are thought to be non-coding RNAs, and the roles of them are to be increasingly recognized of great importance in various biological processes. Circular RNAs (circRNAs) are a class of non-coding RNAs, with no 5' caps and 3' polyadenylated tails, commonly produced by back-splicing of exons. The structure of circRNAs makes them more stable than their counterparts. Increasing evidence shows that circRNAs are involved in the pathogenesis of PE, but the biogenesis, functions, and mechanisms of circRNAs in PE are poorly understood. In the present review, we mainly summarize the biogenesis, functions, and possible mechanisms of circRNAs in the development and progression of PE, as well as opportunities and challenges in the treatment and prevention of PE.

Circular RNAs are abundant endogenous non-coding RNA with no 5′ cap and 3′ polyadenylation tail that modify liner mRNAs and have no terminal structures. Our knowledge of the biogenesis of circular RNAs has been expanded, and circular RNAs were shown to be key regulators of various diseases, especially cancers. Head and neck cancers are the sixth most popular cancers worldwide, and the overall survival rates remain unsatisfactory. Recent studies have indicated that circular RNAs are involved in the tumorigenesis, progression, invasion and chemosensitivity of head and neck cancers and that some circular RNAs could serve as diagnostic and prognostic biomarkers. In this study, we summarize research advances in the regulation of circular RNA biogenesis, their characteristics and functions, the involvement of circular RNAs in the pathophysiology of head and neck cancers and their potential clinical utilization, as well as the likely directions of future studies.

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.

CIRCexplorer3: A CLEAR Pipeline for Direct Comparison of Circular and Linear RNA Expression

We recently reported that circular RNA is efficiently translated by a rolling circle amplification (RCA) mechanism in a cell-free Escherichia coli translation system. Recent studies have shown that circular RNAs composed of exonic sequences are abundant in human cells. However, whether these circular RNAs can be translated into proteins within cells remains unclear. In this study, we prepared circular RNAs with an infinite open reading frame and tested their translation in eukaryotic systems. Circular RNAs were translated into long proteins in rabbit reticulocyte lysate in the absence of any particular element for internal ribosome entry, a poly-A tail, or a cap structure. The translation systems in eukaryote can accept much simpler RNA as a template for protein synthesis by cyclisation. Here, we demonstrated that the circular RNA is efficiently translated in living human cells to produce abundant protein product by RCA mechanism. These findings suggest that translation of exonic circular RNAs present in human cells is more probable than previously thought.

Circular (circ)RNA is a special type of endogenous RNA consisting of a covalently closed loop structure without 5‘ to 3‘ polarity and a polyadenylated tail. Accumulating evidence suggests that circRNAs play important roles in the development and progression of human cancers. However, the role of circRNAs in the progression of hepatocellular carcinoma (HCC) is largely unknown. This was addressed in the present study using high-throughput sequencing to identify aberrantly expressed circRNAs in HCC patient tissue and cell lines. We found that circ-baculoviral IAP repeat-containing (BIRC)6 was upregulated in HCC tissue samples and cells; this was associated with the overall survival of HCC patients. circ-BIRC6 knockdown reduced HCC cell proliferation, migration, and invasion and enhanced their apoptosis. Additionally, circ-BIRC6 overexpression negatively regulated the expression of microRNA miR-3918, which was identified as an inhibitor of B cell lymphoma (Bcl)2. The tumor-suppressive effect of circ-BIRC6 deletion was abrogated by inhibiting miR-3918. These results indicate that circ-BIRC6 functions as a competing endogenous RNA that regulates Bcl2 expression by sponging miR-3918, and may serve as a prognostic biomarker and therapeutic target for the treatment of HCC.

Circular RNAs (circRNAs)3 bear a unique topological feature of circularity among the RNA species. CircRNAs, together with other RNA species without protein-coding potential, are grouped as noncoding RNAs (1). They are single-stranded, covalently closed circular RNA molecules and are hypothesized to be mainly generated through back-splicing of exons from precursor messenger RNAs. CircRNAs were once considered to be aberrant splicing by-products that were only present in minute amounts in cells. With specific molecular assay design (enrichment of circRNAs through depletion of linear RNAs in the sequence library), advances in sequencing technologies, and specialized bioinformatics algorithms for identification of back-spliced junctions, global profiling of circRNAs (2) has now become feasible and circRNAs are shown to be widespread in diverse cell types. Researchers have been trying to understand the biological function of circRNAs. These molecules might be indirectly involved in gene regulation as competing endogenous RNAs or microRNA sponges. The potential of this RNA species as a disease biomarker is to be explored. There are a few biological properties of circRNAs that make them suitable for biomarker development. First, the circular topology and lack of open ends of circRNAs confer certain resistance to exoribonuclease-mediated RNA degradation. CircRNAs have been shown to be more stable than their linear counterparts (1). Second, as mentioned above, some circRNAs are abundant in the various human cell types (3). Third, the circRNA expression patterns are diverse among different cell types and could exhibit tissue specificity. In a recent large-scale study involving circRNA profiling of over 800 tumor tissue samples of 17 different cancer types (4), hundreds of circRNAs were suggested to be tissue-specific and/or cancer-specific. The researchers proposed that the tissue specificity of these circRNAs could be attributed to the tissue specificity of the “parental” gene. Previous studies have demonstrated the presence of cell-free circRNAs in …

Circular RNA forms had been described in all domains of life. Such RNAs were shown to have diverse biological functions, including roles in the life cycle of viral and viroid genomes, and in maturation of permuted tRNA genes. Despite their potentially important biological roles, discovery of circular RNAs has so far been mostly serendipitous. We have developed circRNA-seq, a combined experimental/computational approach that enriches for circular RNAs and allows profiling their prevalence in a whole-genome, unbiased manner. Application of this approach to the archaeon Sulfolobus solfataricus P2 revealed multiple circular transcripts, a subset of which was further validated independently. The identified circular RNAs included expected forms, such as excised tRNA introns and rRNA processing intermediates, but were also enriched with non-coding RNAs, including C/D box RNAs and RNase P, as well as circular RNAs of unknown function. Many of the identified circles were conserved in Sulfolobus acidocaldarius, further supporting their functional significance. Our results suggest that circular RNAs, and particularly circular non-coding RNAs, are more prevalent in archaea than previously recognized, and might have yet unidentified biological roles. Our study establishes a specific and sensitive approach for identification of circular RNAs using RNA-seq, and can readily be applied to other organisms.

BackgroundWhile RNA is often created from linear splicing during transcription, recent studies have found that non-canonical splicing sometimes occurs. Non-canonical splicing joins 3’ and 5’ and forms the so-called circular RNA. It is now believed that circular RNA plays important biological roles such as affecting susceptibility of some diseases. During the past several years, multiple experimental methods have been developed to enrich circular RNA while degrade linear RNA. Although several useful software tools for circular RNA detection have been developed as well, these tools are based on reads mapping may miss many circular RNA. Also, existing tools are slow for large data due to their dependence on reads mapping.MethodIn this paper, we present a new computational approach, named CircMarker, based on k-mers rather than reads mapping for circular RNA detection. CircMarker takes advantage of transcriptome annotation files to create the k-mer table for circular RNA detection.ResultsEmpirical results show that CircMarker outperforms existing tools in circular RNA detection on accuracy and efficiency in many simulated and real datasets.ConclusionsWe develop a new circular RNA detection method called CircMarker based on k-mer analysis. Our results on both simulation data and real data demonstrate that CircMarker runs much faster and can find more circular RNA with higher consensus-based sensitivity and high accuracy ratio compared with existing tools.

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.