Short intronic repeat sequences facilitate circular RNA production

Noncoding circular RNAs are widespread in the tree of life. Particularly, intron-containing circular RNAs which apparently upregulate their parental gene expression. Entamoeba histolytica, the causative agent of dysentery and liver abscesses in humans, codes for several noncoding RNAs, including circular ribosomal RNAs, but no intron containing circular RNAs have been described to date. Divergent RT-PCR and diverse molecular approaches, allowed us to detect bona fide full-length intronic circular RNA (flicRNA) molecules. Self-splicing reactions, RNA polymerase II inhibition with Actinomycin D, and second step of splicing-inhibition with boric acid showed that the production of flicRX13 (one of the flicRNAs found in this work, and our test model) depends on mRNA synthesis and pre-mRNA processing instead of self-splicing. To explore the cues and factors involved in flicRX13 biogenesis in vivo, splicing assays were carried out in amoeba transformants where splicing factors and Dbr1 (intron lariat debranching enzyme 1) were silenced or overexpressed, or where Rabx13 wild-type and mutant 5′ss (splice site) and branch site minigene constructs were overexpressed. Whereas SF1 (splicing factor 1) is not involved, the U2 auxiliary splicing factor, Dbr1, and the GU-rich 5′ss are involved in postsplicing flicRX13 biogenesis, probably by Dbr1 stalling, in a similar fashion to the formation of ciRNAs (circular intronic RNAs), but with distinctive 5′-3′ss ligation points. Different from the reported functions of ciRNAs, the 5′ss GU-rich element of flicRX13 possibly interacts with transcription machinery to silence its own gene in cis. Furthermore, introns of E. histolytica virulence-related genes are also processed as flicRNAs.

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.

Neurofibrillary tangles formed by the microtubule protein tau (MAPT) are the pathologic hallmark of tauopathies, including Alzheimer's disease and ∼1/2 of frontotemporal lobar degeneration (FTLD-Tau) cases. We reported that MAPT forms circular RNAs through back-splicing of exon 12 to either exon 10 or exon 7 (BBA Dis 1864:2753; PMID: 29729314). The MAPT circRNAs require primate-specific Alu elements to form, which might explain their absence in mice. Both MAPT circRNAs lack stop codons and could form multimers of microtubule binding repeats upon translation. In cell culture studies, we used transfected reporter genes that express tau circRNAs with a 3xFlag tag. We tested the effect of FTLD-Tau mutants (K317M and V337M), junction-specific siRNAs and adenosine deaminase acting on RNA (ADAR) enzymes on circRNA translation, evaluated by RNAse protection, Western blots and validated by mass spectrometry. Circular MAPT constructs were readily translated and regulated by endogenous factors including ADAR and customized siRNAs. The circ12->7 RNA contained a single natural occurring start codon. The circRNA is formed and translated when exons 7 and 12 were flanked by authentic tau or heterologous Alu elements. The circ12->10 RNA lacks a natural start codon, but introduction of the FTLD-Tau mutations K317M and V337M induced translation of the circular RNA. The presence of ADAR activity strongly promoted translation of both circRNAs, even in the absence of start codons, possibly by changing an AUA to an AUI start codon (I-inosine). Despite the lack of a stop codon, translation of the circular MAPT RNA products stopped at defined sites, indicating that circular RNAs use stop signals distinct from canonical ones. Our data indicate that MAPT circular RNAs are translated into proteins that could cause tau aggregations relevant to tauopathies. As most FTLD-tau causing mutations are in the regions near the circularizing part of the gene, they could act through regulating circular RNA formation and/or translation. ADAR-dependent upregulation of circTau translations caused by inflammation and/or brain injury could trigger tau pathology via this mechanism. Finally, tau circRNA can be selectively removed using siRNAs. We are assessing the impact of MAPT circRNAs on proteinaceous aggregations.

Prenatal alcohol exposure induces circular and linear long non-coding RNAs, and protein-coding genes linked to proinflammatory neuroimmune function, promoting nerve injury-induced allodynia following morphine treatment

Circular RNAs (circRNAs) belong to a recently re-discovered species of RNA that emerge during RNA maturation through a process called back-splicing. A downstream 5’ splice site is linked to an upstream 3’ splice site to form a circular transcript instead of a canonical linear transcript. Recent advances in next-generation sequencing (NGS) have brought circRNAs back into the focus of many scientists. Since then, several studies reported that circRNAs are differentially expressed across tissue types and developmental stages, implying that they are actively regulated and not merely a by-product of splicing. Though functional studies have shown that some circRNAs could act as miRNA-sponges, the function of most circRNAs remains unknown. To expand our understanding of possible roles of circular RNAs, we propose a new pipeline that could fully characterizes candidate circRNA structure from RNAseq data – FUCHS: FUll CHaracterization of circular RNA using RNA- Sequencing. Currently, most computational prediction pipelines use back-spliced reads to identify circular RNAs. FUCHS extends this concept by considering all RNA-seq information from long reads (typically > 150 bp) to learn more about the exon coverage, the number of double break point fragments, the different circular isoforms arising from one host-gene, and the alternatively spliced exons within the same circRNA boundaries. This new knowledge will enable the user to carry out differential-motif enrichment and miRNA-seed analysis to determine potential regulators during circRNA biogenesis. FUCHS is an easy-to-use Python based pipeline that contributes a new aspect to the circRNA research. The pipeline is available as git repository: https://github.com/dieterich-lab/FUCHS

Cryptic Splice Sites and Cryptic Splicing

Circular RNAs (circRNAs) constitute a class of covalently circular non-coding RNA molecules formed by 5′ and 3′ end back-splicing. The rapid development of bioinformatics and large-scale sequencing has led to the identification of functional circRNAs. Despite an overall upward trend, studies focusing on the roles of circRNAs in immune diseases remain relatively scarce. In the present study, we obtained a differential circRNA expression profile based on microarray analysis of peripheral blood mononuclear cells (PBMCs) in children with type 1 diabetes mellitus (T1DM). We characterized one differentially expressed circRNA back-spliced from the MYB Proto-Oncogene Like 2 (MYBL2) gene in patients with T1DM, termed as hsa_circ_0060450. Subsequent assays revealed that hsa_circ_0060450 can serve as the sponge of miR-199a-5p, release its target gene, Src homology 2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2), encoded by the tyrosine-protein phosphatase non-receptor type 11 gene (PTPN11), and further suppress the JAK-STAT signaling pathway triggered by type I interferon (IFN-I) to inhibit macrophage-mediated inflammation, which indicates the important roles of circRNAs in T1DM and represents a promising therapeutic molecule in the treatment of T1DM.

Background: Circular RNAs (circRNAs) are covalently closed circular noncoding RNAs that are expressed in various life forms. CircRNAs have many characteristics, such as structural stability and tissue-specific expression that contribute to their role as a microRNA (sponge in gene regulation. Summary: Recent evidence suggests that circRNAs play an important role in the pathogenesis of cerebrovascular diseases (CVDs); however, the exact mechanism remains controversial. CircRNAs that are related to CVDs have great clinical significance. Key Messages: The present review provides an overview of the general biology of circRNAs, their relevant regulatory mechanisms, and their role in the pathophysiology of CVDs.

Circular RNAs (circRNA) are a recently described class of RNA molecules that have attracted substantial attention as new components of disease mechanisms and as potential biomarkers in multiple diseases, including cancer. CircRNAs are often highly conserved and exhibit developmental stage- and disease-specific expression. Several studies have reported circRNA expression patterns that are associated with specific cancer types and with patient prognosis. Here, we overview the active registered clinical trials that investigate the value of circRNAs as cancer biomarkers and discuss the potential of circRNAs in clinical cancer care. Taken together, circRNAs are actively being investigated as diagnostic, predictive, and prognostic biomarkers, and their potential to serve as therapeutic intervention points motivates ongoing translational and clinical research.

RNA Splicing Factor Mutations Drive Aberrant Canonical and Cryptic Circular RNA Biogenesis in Leukemia

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: A new star of noncoding RNAs

High-throughput RNA sequencing (RNA-seq) and dedicated bioinformatics pipelines have synergized to identify an expansive repertoire of unique circular RNAs (circRNAs), exceeding 100,000 variants. While the vast majority of these circRNAs comprise canonical exonic and intronic sequences, microexons (MEs)—which occur in 30% of functional mRNA transcripts—have been entirely overlooked. CircRNAs which contain these known MEs (ME-circRNAs) could be identified with commonly utilized circRNA prediction pipelines, CIRCexplorer2 and CIRI2, but were not previously recognized as ME-circRNAs. In addition, when employing a bespoke bioinformatics pipeline for identifying RNA chimeras, called Hyb, we could also identify over 2000 ME-circRNAs which contain novel MEs at their backsplice junctions, that are uncalled by either CIRCexplorer2 or CIRI2. Analysis of circRNA-seq datasets from gliomas of varying clinical grades compared with matched control tissue has shown circRNAs have potential as prognostic markers for stratifying tumor from healthy tissue. Furthermore, the abundance of microexon-containing circRNAs (ME-circRNAs) between tumor and normal tissues is correlated with the expression of a splicing associated factor, Serine/arginine repetitive matrix 4 (SRRM4). Overexpressing SRRM4, known for regulating ME inclusion in mRNAs critical for neural differentiation, in human HEK293 cells resulted in the biogenesis of over 2000 novel ME-circRNAs, including ME-circEIF4G3, and changes in the abundance of many canonical circRNAs, including circSETDB2 and circLRBA. This shows SRRM4, in which its expression is correlated with poor prognosis in gliomas, acts as a bona fide circRNA biogenesis factor. Given the known roles of MEs and circRNAs in oncogenesis, the identification of these previously unrecognized ME-circRNAs further increases the complexity and functional purview of this non-coding RNA family.

Proto-splice site model of intron origin

The CC10 gene encodes the Clara cell 10-kDa protein, which is expressed in airway epithelial cells. Quantification of CC10 gene expression in freshly isolated human proximal airway epithelial cells demonstrated very high mRNA levels, approximately fivefold greater than gamma-actin mRNA, and in situ hybridization localized CC10 mRNA to nonciliated airway epithelial cells. Sequence analysis demonstrated that the human CC10 gene is comprised of three short exons separated by a long first and short second intron, and with a 5' flanking region typical of a regulated gene. Three Alu repeats were observed in intron 1 and one in intron 2. Two polymorphic regions within the introns were identified. First, a microsatellite was localized 5' to the third Alu repeat in intron 1 with a variable number of 4- and 5-base pair (bp) repeats and a heterozygosity of 0.71. Second, in 3% of the 168 chromosomes examined, there was the insertion of a human-specific Alu repeat in intron 2, 45 bp 3' to the exon 2-intron junction. In three Centre d'Etude du Polymorphisme Humain families, meiotic breakpoint analysis using these two polymorphic loci localized the CC10 gene to 11 p12-q13 between markers D11S16 and D11S97, a region recently linked to atopy and to the beta-subunit of the high-affinity immunoglobulin E receptor. The observations in the present study of high-level expression of the CC10 gene in the epithelium of conducting airways and a subchromosomal localization of the gene to a region potentially linked to inflammatory airway disease, together with the reported anti-inflammatory and immune-modulating properties of the protein, suggest the CC10 gene product may be important in modulating inflammation within the airways. If so, the highly heterozygous microsatellite described in the present study should facilitate analysis of a possible linkage of the CC10 gene with an inherited susceptibility to asthma.