Recently, thousands of circular RNAs (circRNAs) have been discovered in various tissues and cell types from human, mouse, fruit fly and nematodes. However, expression of circRNAs across mammalian brain development has never been examined. Here we profile the expression of circRNA in five brain tissues at up to six time-points during fetal porcine development, constituting the first report of circRNA in the brain development of a large animal. An unbiased analysis reveals a highly complex regulation pattern of thousands of circular RNAs, with a distinct spatio-temporal expression profile. The amount and complexity of circRNA expression was most pronounced in cortex at day 60 of gestation. At this time-point we find 4634 unique circRNAs expressed from 2195 genes out of a total of 13,854 expressed genes. Approximately 20 % of the porcine splice sites involved in circRNA production are functionally conserved between mouse and human. Furthermore, we observe that “hot-spot” genes produce multiple circRNA isoforms, which are often differentially expressed across porcine brain development. A global comparison of porcine circRNAs reveals that introns flanking circularized exons are longer than average and more frequently contain proximal complementary SINEs, which potentially can facilitate base pairing between the flanking introns. Finally, we report the first use of RNase R treatment in combination with in situ hybridization to show dynamic subcellular localization of circRNA during development. These data demonstrate that circRNAs are highly abundant and dynamically expressed in a spatio-temporal manner in porcine fetal brain, suggesting important functions during mammalian brain development.

CircRNAs are novel members of the non-coding RNA family. For several decades circRNAs have been known to exist, however only recently the widespread abundance has become appreciated. Annotation of circRNAs depends on sequencing reads spanning the backsplice junction and therefore map as non-linear reads in the genome. Several pipelines have been developed to specifically identify these non-linear reads and consequently predict the landscape of circRNAs based on deep sequencing datasets. Here, we use common RNAseq datasets to scrutinize and compare the output from five different algorithms; circRNA_finder, find_circ, CIRCexplorer, CIRI, and MapSplice and evaluate the levels of bona fide and false positive circRNAs based on RNase R resistance. By this approach, we observe surprisingly dramatic differences between the algorithms specifically regarding the highly expressed circRNAs and the circRNAs derived from proximal splice sites. Collectively, this study emphasizes that circRNA annotation should be handled with care and that several algorithms should ideally be combined to achieve reliable predictions.

Not Too Much, Not Too Little: The microRNA miR128 is expressed in brain neurons of the mouse. Lek Tan et al. (p. 1254 ) now find that miR128 is crucial to stable brain function. Mice deficient in miR128 developed hyperactivity and were susceptible to fatal seizures, whereas overexpression of miR128 correlated with reduced motor activity and reduced susceptibility to proconvulsive drugs. Experiments using ex vivo–isolated adult brain tissues suggested that miR-128 controlled motor activity by governing the signaling network that determines the intrinsic excitability and signal responsiveness of neurons.

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 β-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 β-cells functions. 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, β-cell proliferation, and apoptosis. 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 β-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 β-cell genes, such as Slc2a2, Akt1, and Mtpn. Our findings point to circular RNAs as novel regulators of β-cell activities and suggest an involvement of this novel class of non-coding RNAs in β-cell dysfunction under diabetic conditions.

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.

Cocaine is a highly addictive drug that exerts its effects by increasing the levels of released dopamine in the striatum, followed by stable changes in gene transcription, mRNA translation, and metabolism within medium spiny neurons in the striatum. The multiple changes in gene and protein expression associated with cocaine addiction suggest the existence of a mechanism that facilitates a coordinated cellular response to cocaine. Here, we provide evidence for a key role of miRNAs in cocaine addiction. We show that Argonaute 2 (Ago2), which plays an important role in miRNA generation and execution of miRNA-mediated gene silencing, is involved in regulation of cocaine addiction. Deficiency of Ago2 in dopamine 2 receptor (Drd2)–expressing neurons greatly reduces the motivation to self-administer cocaine in mice. We identified a distinct group of miRNAs that is specifically regulated by Ago2 in the striatum. Comparison of miRNAs affected by Ago2 deficiency with miRNAs that are enriched and/or up-regulated in Drd2-neurons in response to cocaine identified a set of miRNAs that are likely to play a role in cocaine addiction.