2.1.1 Circular RNA in nature
Most studied RNAs are linear molecules, but circular RNAs (circRNAs) have been found in all domains of life (Guo et al., 2014; Danan et al., 2012; Lasda and Parker, 2014). Although they were previously considered oddities, experimental artefacts or products of spurious splicing, circRNAs have now been shown to be common (Jeck et al., 2013) and to play important regulatory roles (Memczak et al., 2013; Hansen et al., 2013; Ye et al., 2015). However, they remain relatively understudied and the reasons for their circularity remain unclear. circRNAs are found in viroids, small virus like particles composed of a single infectious non-coding circular RNA genome, and that have been suggested to be relics of pre-cellular life forms (Diener, 1989; Flores et al., 2014). Hepatitis delta virus, a satellite virus of hepatitis B virus, also has a circular genome (Macnaughton et al., 2002). In both cases, the circular RNA serves as a template for rolling circle replication, resulting in a concatamer that undergoes enzyme-mediated or autocatalytic cleavage. This process produces monomers which are then circularised to produce more virus or viroid genomes. circRNAs have also been found in some bacteria (Molina-Sánchez et al., 2006) and in many archaea
(Danan et al., 2012), where they exist as by-products of intron splicing, or in certain species as circular versions of snoRNAs and of the ribozyme RNase P (Lasda and Parker, 2014). In human cells, circular RNAs were recently shown to be more common than previously thought (Jeck et al., 2013). This has spurred much interest, especially due to the role they may play in disease (Chen, 2016a; Guarnerio et al., 2016). In humans, some circRNAs have been shown to function as miRNA ’sponges’ that can suppress the activity of miRNAs, by binding to them whilst being resistant to miRNA mediated degradation (Hansen et al., 2013). However, this role as a regulator of miRNAs may be not be generalisable to other expressed circular RNAs (Guo et al., 2014), and whether many circular products have an important function or whether most are merely by- products of spurious splicing reactions remains unclear. How circularity affects RNA behaviour in general remains unexplained (Barrett and Salzman, 2016), and what roles circRNAs can play as well as the reasons for their unusual topology remains an open question.
2.1.2 Advantages of circularisation
One major change brought by circularity is the absence of free ends, which render circRNAs resistant to exonuclease degradation (Jeck et al., 2013). As some endonucle- ases are known to cleave close to RNA ends, they are also affected by circularisation. For example, inE. coli, the rapid RNA turnover – the average RNA half-life is about 5 minutes (Selinger et al., 2003; Chen et al., 2015) – is mostly due to degradation by RNase E (Mackie, 2000). Although an endonuclease, it has higher affinity for RNAs with monophosphorylated 5’ ends. Dephosphorylation of newly transcribed tri-phosphorylated RNA is undertaken by the RppH protein, which tags it for subse- quent degradation by RNase E (Deana et al., 2008). As a result, RNA circularisation can result in stabilisation (Mackie, 2000). circRNA transcripts have also been found to have an increased half-life compared to their corresponding linear transcripts in human cells (Enuka et al., 2015). Aside from increased resistance to nucleases, the 20
2.1. Introduction
increased thermal stability that results from the joining of the ends has also been put forward as a possible advantage of having circular RNA. This might be important in thermophilic archaea, where several types of circular RNAs have been found (Danan et al., 2012).
2.1.3 RNA circularisation
RNA circles can be generated either through RNA back-splicing, when the acceptor splice site is situated upstream of the donor splice site (Zhang et al., 2016), or by liga- tion of RNA ends by an RNA ligase (Petkovic and Müller, 2015). A relatively simple way of generating artificial circular RNAs is to use self-splicing group I intron ribozymes, through the reorganisation of their sequence by a circular permutation (Puttaraju and Been, 1992). In this case the dispensable P6 loop of a T4 bacteriophagetdgene, which contains a self-splicing group 1 intron, is removed, and the exons are joined together (see fig. 2.3). This results in a back-splicing reaction and the circularisation of the exons. Additional sequences can be inserted between the two exons of the permuted intron-exon (PIE) ribozyme, which results in their inclusion within the processed circRNA (Perriman and Ares, 1998; Umekage and Kikuchi, 2009).
2.1.4 Methods for detection of circularity
There are a number of methods used to detect circular RNA, reviewed by Jeck and Sharpless (2014, also see fig. 2.1). Divergent primer RT-PCR can detect circRNAs, as the splicing reaction reorders the primer binding sites into convergent orientations (see fig.2.1A). 2D gel electrophoresis and gel trapping are biochemical methods for detection of circular topology. The former separates the RNA in denaturing conditions, with different acrylamide concentration in each dimension. The migration speed changes linearly with acrylamide concentration, but the slope of this linear change is different for linear and circular RNAs. Linear RNAs thus appear along a diagonal on the 2D gel, whereas circular RNAs appear outside of it (fig. 2.1B). Gel trapping relies
on the mixing of RNA with melted agarose, which traps circular molecules during polymerisation. Linear molecules migrate normally, but circular molecules stay in the trap (fig. 2.1C). Exonucleases such as RNase R can also be used to degrade linear RNA, and have been used in combination with deep sequencing for high throughput circRNA detection (Jeck et al., 2013, see fig. 2.1D.).
A
5’ 3’
PCR with divergent primers Product if sequences reordered
B
1D 2D 7 .5 % P AM 5% PAM Linear RNAs along diagonal Circular RNAC
RNA loaded in melted agarose Cross-linking traps nonlinear RNA Circular RNA stays trapped Linear RNA migratesD
Exonuclease digestion Control RNA seq, comparisonFigure 2.1 – Methods for detecting circRNA.A: Divergent primer PCR. Reorganisation of exons after ligation replaces primers in convergent orientation, allowing PCR am- plification. B. 2D polyacrylamide (PAM) gel electrophoresis. Linear RNAs migrate
along a diagonal. CircRNAs appear outside as they migrate more slowly in highly cross linked gels. C. Gel trap elecrophoresis. Non-linear RNAs are trapped by cross
linking.D. Exonuclease such as RNase R selectively degrade linear RNA. They can be
combined with next generation sequencing for high throughput circRNA detection. Figure adapted from Jeck and Sharpless (2014).