The invasion of circRNAs

KEYWORDS:

• biogenesis
• circRNA
• disease
• RNA
• RNA splicing
• RNomics
• small and large non-coding RNAs
• splicing/pre-mRNA processing

Single-stranded covalently closed circular RNA (circRNA) molecules come in many disguises, with different biogenesis mechanisms and functions. They were initially discovered in 1976 by Sanger et al. constituting the rod-like genome of viroids in plants.¹ In 1981 it was demonstrated that some self-splicing RNA reactions produce circular intron RNA as their final product² and in 1986 a circRNA was reported to constitute the genome of human Hepatitis Delta virus.³ In 1988 circRNA entered the scene also in Archaea where introns in some instances are enzymatically ligated to act as circular mRNA and function as transposons.⁴ In the early nineties- the first glimpse of another fascinating world of circRNA appeared, namely a circRNA derived from the SRY locus in mouse testis.⁵ Already from this study a route for its biosynthesis was predicted: A 5′ splice site would redirect its splicing from the cognate downstream 3′ splice site to an upstream 3´splice site (termed back-splicing) and thereby produce a 5′-3′ ligated circRNA. This spawned a large interest into circRNA, and several laboratories showed examples of exons that seemingly produced circRNAs within mammalian cells and tissues during the following years. However, presumably due to the lack of global transcriptome analyses back then, circRNAs failed to spark a general interest from the RNA community, until 2011 where the Sharpless laboratory and we almost simultaneously published the discovery of some highly expressed circRNAs, ciRS-7/cdr1as and cANRIL,^6,7 putting circRNA back into the field of non-coding RNA research. The Sharpless and Brown laboratories effectively affirmed the relevance and impact of circRNAs by performing global analyses of circRNA expression^8,9 – revealing a vast number of circRNA molecules previously overlooked and, shortly thereafter, the Rajewsky laboratory and we characterized the ciRS-7/cdr1as as a miR-7 sponge showing functional relevance of a circRNA and, importantly, resolving a reason for the circular topology, namely resistance toward miRNA-induced target destabilization.^10,11 Since then, prominent research laboratories throughout the world have worked intensively on documenting and describing circRNA expression across organisms, tissues, and cell-lines all pointing toward circRNAs as a widespread and dynamic class of RNA, seemingly expressed in all splicing competent organisms. Typically, circRNAs are composed of well-defined exons from protein-coding gene loci, however, the complexity of back-splicing events in some genes are astonishing and add significantly to the diversity of RNAs produced from alternative linear splicing events. Some genes are spliced into a large number of different circRNAs predicted to contain from 1 up to more than 70 exons (e.g. circRNA isoforms derived from the Titin locus), often with the individual isoform being tightly regulated across development time points and cell types. Neuronal tissue seems to express the highest diversity of circRNA, in particular during developmental processes, but the picture is still far from complete. Notably, cancer cells generally exhibit deregulated expression of specific circRNA, some of which are suggested to play vital roles in cancer development and to serve as promising biomarkers.

The molecular function of circRNAs is just beginning to be unraveled. When the SRY circRNA was discovered it was suggested that the back-splicing would compete with production of normal mRNA and hence protein expression, but investigations of more recently discovered circRNAs have provided examples of more active functions, such as miRNA- and protein sponging. The circular nature entails a very long half-life in cells and even a small rate of back-splicing can lead to significant steady-state levels of circRNA, a prerequisite for effective sponging. Recently several studies expanded the repertoire of circRNA functionalities by showing that some circRNAs can act as templates for translation and, therefore, may not be non-coding after all. The translational potential of circRNAs as well as the importance of the resulting peptide will be an interesting field of research to follow over the coming years.

Another question addressed by several groups is to understand what distinguishes the back-splicing event from forward splicing and how is it regulated. In some instances inverted repeats in the adjacent introns appear to be the main driver, but many of the most highly expressed circRNA lack obvious repeats. Whether these all are driven by back-reaching events involving for instance RNA binding proteins still remains to be established. Also, the understanding of how circRNA, in the absence of 5′-cap and poly-A tail, is exported to the cytoplasm, which is its dominating location, remains to be investigated.

The elucidation of circRNA-mediated gene regulation will inevitably place circRNAs as potential targets for therapeutics. The back-splicing junction constitutes a unique circRNA signature that can be specifically targeted by RNA interference or other antisense technologies, ideally without disturbing the linear mRNA expression. Also, intronic inverted repeats and protein binding sites may be targeted in the nucleus and thereby block circRNA production. If malfunction of the cell is caused by deficient circRNA production, several circRNA generating vector systems can be designed for restoration of specific circRNAs. Building on this, artificial circRNAs are readily designed that can sponge deleterious miRNA or proteins or direct the assembly of regulatory complexes in the cell. CircRNA may, with its superior stability, become the preferred artificial RNA engineering platform in cells.

In this special edition book on circRNA, we have collected an array of review papers written by the forefront experts in the field of circRNA – spanning from circRNA in Archaea and plants to circRNA in human diseases as well as functional aspects and clinical prospects of circRNAs within our cells. In 13 articles, this book comprises an exceptional overview of circRNA biology as of today.

This book would not be possible without the hard-working efforts of all the contributors. Thus, we would like thank and acknowledge all the circRNA experts who have contributed to this volume with highly competent manuscripts. We hope that readers of this edition would obtain a comprehensive insight into current state-of-the-art of circRNA biology and challenges, and from here take part in pushing this exiting research field into the future by addressing and answering important questions regarding the functional aspects and therapeutic potential of circRNAs.