Nanopore-Mediated Sequencing of Circular RNA.

Circular RNAs (circRNAs) constitute a group of RNAs defined by a covalent bond between the 5' and 3' end formed by a unique back-splicing event. Most circRNAs are composed of more than one exon, which are spliced together in a linear fashion. This protocol describes methods to sequence full-length circRNA across the back-splicing junction, allowing unambiguous characterization of circRNA-specific exon-intron structures by long-read sequencing (LRS). Two different sequencing approaches are provided: (1) Global circRNA sequencing (the circNick-LRS strategy) relying on circRNA enrichment from total RNA followed by total circRNA long-read sequencing, and (2) targeted circRNA sequencing (the circPanel-LRS strategy) where a preselected panel of circRNA are sequenced without prior circRNA enrichment. Both methods were originally described in Karim et al. (Rahimi et al., Nat Commun 12: 4825, 2021) where they were applied to characterize the exon-intron structure of >10.000 circRNAs in mouse and human brains.

A Circular RNA Expressed from the FAT3 Locus Regulates Neural Development.

Circular RNAs (circRNAs) are key regulators of cellular processes, are abundant in the nervous system, and have putative regulatory roles during neural differentiation. However, the knowledge about circRNA functions in brain development is limited. Here, using RNA-sequencing, we show that circRNA levels increased substantially over the course of differentiation of human embryonic stem cells into rostral and caudal neural progenitor cells (NPCs), including three of the most abundant circRNAs, ciRS-7, circRMST, and circFAT3. Knockdown of circFAT3 during early neural differentiation resulted in minor transcriptional alterations in bulk RNA analysis. However, single-cell transcriptomics of 30 and 90 days differentiated cerebral organoids deficient in circFAT3 showed a loss of telencephalic radial glial cells and mature cortical neurons, respectively. Furthermore, non-telencephalic NPCs in cerebral organoids showed changes in the expression of genes involved in neural differentiation and migration, including FAT4, ERBB4, UNC5C, and DCC. In vivo depletion of circFat3 in mouse prefrontal cortex using in utero electroporation led to alterations in the positioning of the electroporated cells within the neocortex. Overall, these findings suggest a conserved role for circFAT3 in neural development involving the formation of anterior cell types, neuronal differentiation, or migration.

A circular RNA generated from an intron of the insulin gene controls insulin secretion.

Fine-tuning of insulin release from pancreatic ß-cells is essential to maintain blood glucose homeostasis. Here, we report that insulin secretion is regulated by a circular RNA containing the lariat sequence of the second intron of the insulin gene. Silencing of this intronic circular RNA in pancreatic islets leads to a decrease in the expression of key components of the secretory machinery of ß-cells, resulting in impaired glucose- or KCl-induced insulin release and calcium signaling. The effect of the circular RNA is exerted at the transcriptional level and involves an interaction with the RNA-binding protein TAR DNA-binding protein 43 kDa (TDP-43). The level of this circularized intron is reduced in the islets of rodent diabetes models and of type 2 diabetic patients, possibly explaining their impaired secretory capacity. The study of this and other circular RNAs helps understanding ß-cell dysfunction under diabetes conditions, and the etiology of this common metabolic disorder.

High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. It is one of the most prevalent chronic inflammatory skin conditions in adults worldwide, with a considerable negative impact on quality of life. Circular RNAs (circRNAs) are a recently identified type of non-coding RNA with diverse cellular functions related to their exceptional stability. In particular, some circRNAs can bind and regulate microRNAs (miRNAs), a group of RNAs that play a role in the pathogenesis of psoriasis. The aim of this study was to characterize the circRNAome in psoriasis and to assess potential correlations to miRNA expression patterns. METHODS: We used high-throughput RNA-sequencing (RNA-seq), NanoString nCounter technology and RNA chromogenic in situ hybridization (CISH) to profile the circRNA expression in paired lesional and non-lesional psoriatic skin from patients with psoriasis vulgaris. In addition, 799 miRNAs were profiled using NanoString nCounter technology and laser capture microdissection was used to study the dermis and epidermis separately. RESULTS: We found a substantial down-regulation of circRNA expression in lesional skin compared to non-lesional skin. We observed that this mainly applies to the epidermis by analyzing laser capture microdissected tissues. We also found that the majority of the circRNAs were downregulated independently of their corresponding linear host genes. The observed downregulation of circRNAs in psoriasis was neither due to altered expression levels of factors known to affect circRNA biogenesis, nor because lesional skin contained an increased number of inflammatory cells such as lymphocytes. Finally, we observed that the overall differences in available miRNA binding sites on the circRNAs between lesional and non-lesional skin did not correlate with differences in miRNA expression patterns. CONCLUSIONS: We have performed the first genome-wide circRNA profiling of paired lesional and non-lesional skin from patients with psoriasis and revealed that circRNAs are much less abundant in the lesional samples. Whether this is a cause or a consequence of the disease remains to be revealed, however, we found no evidence that the loss of miRNA binding sites on the circRNAs could explain differences in miRNA expression between lesional and non-lesional skin.

Circular RNAs as novel regulators of ß-cell functions in normal and disease conditions.

OBJECTIVE: There is strong evidence for an involvement of different classes of non-coding RNAs, including microRNAs and long non-coding RNAs, in the regulation of ß-cell activities and in diabetes development. Circular RNAs were recently discovered to constitute a substantial fraction of the mammalian transcriptome but the contribution of these non-coding RNAs in physiological and disease processes remains largely unknown. The goal of this study was to identify the circular RNAs expressed in pancreatic islets and to elucidate their possible role in the control of ß-cells functions. METHODS: We used a microarray approach to identify circular RNAs expressed in human islets and searched their orthologues in RNA sequencing data from mouse islets. We then measured the level of four selected circular RNAs in the islets of different Type 1 and Type 2 diabetes models and analyzed the role of these circular transcripts in the regulation of insulin secretion, ß-cell proliferation, and apoptosis. RESULTS: We identified thousands of circular RNAs expressed in human pancreatic islets, 497 of which were conserved in mouse islets. The level of two of these circular transcripts, circHIPK3 and ciRS-7/CDR1as, was found to be reduced in the islets of diabetic db/db mice. Mimicking this decrease in the islets of wild type animals resulted in impaired insulin secretion, reduced ß-cell proliferation, and survival. ciRS-7/CDR1as has been previously proposed to function by blocking miR-7. Transcriptomic analysis revealed that circHIPK3 acts by sequestering a group of microRNAs, including miR-124-3p and miR-338-3p, and by regulating the expression of key ß-cell genes, such as Slc2a2, Akt1, and Mtpn. CONCLUSIONS: Our findings point to circular RNAs as novel regulators of ß-cell activities and suggest an involvement of this novel class of non-coding RNAs in ß-cell dysfunction under diabetic conditions.

Circular RNAs are abundantly expressed and upregulated during human epidermal stem cell differentiation.

The expression patterns of endogenous circular RNA (circRNA) molecules during epidermal stem cell (EpSC) differentiation have not previously been explored. Here, we show that circRNAs are abundantly expressed in EpSCs and that their expression change dramatically during differentiation in a coordinated manner. Overall, circRNAs are expressed at higher levels in the differentiated cells, and many upregulated circRNAs are derived from developmental genes, including four different circRNAs from DLG1. The observed changes in circRNA expression were largely independent of host gene expression, and circRNAs independently upregulated upon differentiation are more prone to AGO2 binding and have more predicted miRNA binding sites compared to stably expressed circRNAs. In particular, upregulated circRNAs from the HECTD1 and ZNF91 genes have exceptionally high numbers of AGO2 binding sites and predicted miRNA target sites, and circZNF91 contains 24 target sites for miR-23b-3p, which is known to play important roles in keratinocyte differentiation. We also observed that upregulated circRNAs are less likely to be flanked by homologues inverted Alu repeats compared to stably expressed circRNAs. This coincide with DHX9 being upregulated in the differentiated keratinocytes. Finally, none of the circRNAs upregulated upon differentiation were also upregulated upon DNMT3A or DNMT3B knockdown, making it unlikely that epigenetic mechanisms are governing the observed circRNA expression changes. Together, we provide a map of circRNA expression in EpSCs and their differentiated counterparts and shed light on potential function and regulation of differentially expressed circRNAs.