Exosome mediated biological functions within skeletal microenvironment

Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.

Circular RNAs (circRNAs) are widely expressed noncoding RNAs. However, their biogenesis and possible functions are poorly understood. Here, by studying circRNAs that we identified in neuronal tissues, we provide evidence that animal circRNAs are generated cotranscriptionally and that their production rate is mainly determined by intronic sequences. We demonstrate that circularization and splicing compete against each other. These mechanisms are tissue specific and conserved in animals. Interestingly, we observed that the second exon of the splicing factor muscleblind (MBL/MBNL1) is circularized in flies and humans. This circRNA (circMbl) and its flanking introns contain conserved muscleblind binding sites, which are strongly and specifically bound by MBL. Modulation of MBL levels strongly affects circMbl biosynthesis, and this effect is dependent on the MBL binding sites. Together, our data suggest that circRNAs can function in gene regulation by competing with linear splicing. Furthermore, we identified muscleblind as a factor involved in circRNA biogenesis. Copyright © 2014 Elsevier Inc. All rights reserved.