Research Article

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function

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Science  22 Sep 2017:
Vol. 357, Issue 6357, eaam8526
DOI: 10.1126/science.aam8526

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Cutting out circular RNAs

Circular RNAs are widespread, but their functions have been controversial. Piwecka et al. used CRISPR-Cas9 technology to remove the locus encoding the circular RNA Cdr1as from the mouse genome. Single-cell electrophysiological measurements in excitatory neurons revealed an increase in spontaneous vesicle release from the knockout mice and depression in the synaptic response with two consecutive stimuli, indicating that Cdr1as deficiency leads to dysfunction of excitatory synaptic transmission. Small RNA sequencing of several major regions of the brain showed that expression of two microRNAs, miR-7 and miR-671, that bind to Cdr1as decreased and increased, respectively. These results, along with expression analyses, suggest that neuronal Cdr1as stabilizes or transports miR-7, which in turn represses genes that are early responders to different stimuli.

Science, this issue p. eaam8526

Structured Abstract

INTRODUCTION

Recently, a special class of RNAs has excited researchers and triggered hundreds of now-published studies. Known as circular RNAs (circRNAs), these RNAs are produced by regular transcription from genomic DNA, but the two ends of the (usually) exonic transcripts are covalently closed, probably in most cases by noncanonical splice reactions. Most circRNAs are expressed in the cytoplasm and are unusually stable, suggesting that they may have functions that diverge from those of canonical messenger RNAs (mRNAs) or long noncoding RNAs (lncRNAs).

CircRNAs tend to be weakly expressed, but there are exceptions in animal brains. For example, in the mouse brain, a few hundred circRNAs are highly expressed, often with developmentally specific expression patterns that are conserved in the human brain. We previously proposed that circRNAs may, at least sometimes, serve as regulatory RNAs. A circRNA discovered by the Kjems laboratory, CDR1as, caught our attention because it was covered with >70 binding sites for the microRNA (miRNA) miR-7. Our data suggested that CDR1as might serve to alter the free concentration of miR-7. But what really is the function of CDR1as?

RATIONALE

We first determined which miRNAs specifically bind Cdr1as in postmortem human and mouse brains and characterized Cdr1as expression patterns. Once we had that information, we removed Cdr1as from the mouse genome to study the molecular and behavioral consequences.

RESULTS

We show that Cdr1as is, in the human brain, directly and massively bound by miR-7 and miR-671. In fact, Cdr1as is one of the most common transcripts targeted by miRNAs out of all brain mRNAs or lncRNAs. The expression of miRNAs was generally unperturbed in Cdr1as knockout (KO) mice, with the exception of the two miRNAs that directly interact with Cdr1as, miR-7 and miR-671, which were respectively down-regulated and up-regulated. This perturbation was posttranscriptional, consistent with a model in which Cdr1as interacts with these miRNAs in the cytoplasm. We show that Cdr1as is highly expressed (hundreds of copies within neurons) in somas and neurites, but not in glial cells.

The expression of many immediate early genes (IEGs), which are markers of neuronal activity, was consistently up-regulated in KO animals. For example, c-Fos and a few other miR-7 targets were up-regulated, suggesting that IEG up-regulation can in part be explained by miR-7 down-regulation and that Cdr1as modulates neuronal activity. Cdr1as KO mice showed a strong deficit in prepulse inhibition of the startle response, a sensorimotor gating phenotype that is impaired in several human neuropsychiatric disorders. Electrophysiological measurements indicated an increase in spontaneous vesicle release in Cdr1as KO neurons, suggesting that Cdr1as plays a role in regulating synaptic transmission.

CONCLUSION

Mechanistically, our data indicate that Cdr1as regulates miR-7 stability or transport in neurons, whereas miR-671 regulates Cdr1as levels. Functionally, our data suggest that Cdr1as and its direct interactions with miRNAs are important for sensorimotor gating and synaptic transmission. More generally, because the brain is an organ with exceptionally high and diverse expression of circRNAs, our data suggest the existence of a previously unknown layer of biological functions carried out by circRNAs.

Cdr1as is a brain-enriched circular RNA, expressed in hundreds of copies within neurons and essential for maintaining normal brain function.

Genetic ablation of the Cdr1as locus in mice led to deregulation of miR-7 and miR-671 in the brain, up-regulation of immediate early genes, synaptic malfunctions, and a deficit in prepulse inhibition of the startle reflex, a behavioral phenotype associated with neuropsychiatric disorders.

Abstract

Hundreds of circular RNAs (circRNAs) are highly abundant in the mammalian brain, often with conserved expression. Here we show that the circRNA Cdr1as is massively bound by the microRNAs (miRNAs) miR-7 and miR-671 in human and mouse brains. When the Cdr1as locus was removed from the mouse genome, knockout animals displayed impaired sensorimotor gating—a deficit in the ability to filter out unnecessary information—which is associated with neuropsychiatric disorders. Electrophysiological recordings revealed dysfunctional synaptic transmission. Expression of miR-7 and miR-671 was specifically and posttranscriptionally misregulated in all brain regions analyzed. Expression of immediate early genes such as Fos, a direct miR-7 target, was enhanced in Cdr1as-deficient brains, providing a possible molecular link to the behavioral phenotype. Our data indicate an in vivo loss-of-function circRNA phenotype and suggest that interactions between Cdr1as and miRNAs are important for normal brain function.

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