Circular RNAs—The Road Less Traveled

REVIEW published: 10 January 2020 doi: 10.3389/fmolb.2019.00146 Circular RNAs—The Road Less Traveled Ashirbad Guria 1† , Priyanka Sharma 2† , Sankar Natesan 2* and Gopal Pandi 1* 1 Department of Plant Biotechnology, School of Biotechnology, Madurai Kamaraj University, Madurai, India, 2 Department of Genetic Engineering, School of Biotechnology, Madurai Kamaraj University, Madurai, India Edited by: Amaresh Chandra Panda, Institute of Life Sciences (ILS), India Reviewed by: Kotb Abdelmohsen, National Institute on Aging (NIA), United States Alfredo Berzal-Herranz, Instituto de Parasitología y Biomedicina López-Neyra (IPBLN), Spain *Correspondence: Sankar Natesan Gopal Pandi † These authors have contributed Circular RNAs are the most recent addition in the non-coding RNA family, which has started to gain recognition after a decade of obscurity. The first couple of reports that emerged at the beginning of this decade and the amount of evidence that has accumulated thereafter has, however, encouraged RNA researchers to navigate further in the quest for the exploration of circular RNAs. The joining of 5′ and 3′ ends of RNA molecules through backsplicing forms circular RNAs during co-transcriptional or post-transcriptional processes. These molecules are capable of effectively sponging microRNAs, thereby regulating the cellular processes, as evidenced by numerous animal and plant systems. Preliminary studies have shown that circular RNA has an imperative role in transcriptional regulation and protein translation, and it also has significant therapeutic potential. The high stability of circular RNA is rendered by its closed ends; they are nevertheless prone to degradation by circulating endonucleases in serum or exosomes or by microRNA-mediated cleavage due to their high complementarity. However, the identification of circular RNAs involves diverse methodologies and the delineation of its possible role and mechanism in the regulation of cellular and molecular architecture has provided a new direction for the continuous research into circular RNA. In this review, we discuss the possible mechanism of circular RNA biogenesis, its structure, properties, degradation, and the growing amount of evidence regarding the detection methods and its role in animal and plant systems. Keywords: circRNA, biogenesis, long non-coding RNA, miRNA sponging, backsplicing equally to this work Specialty section: This article was submitted to Protein and RNA Networks, a section of the journal Frontiers in Molecular Biosciences Received: 27 September 2019 Accepted: 03 December 2019 Published: 10 January 2020 Citation: Guria A, Sharma P, Natesan S and Pandi G (2020) Circular RNAs—The Road Less Traveled. Front. Mol. Biosci. 6:146. doi: 10.3389/fmolb.2019.00146 INTRODUCTION Circular RNAs (CircRNAs) have recently spread into the non-coding RNA world. The circRNAs are formed by the covalent circularization of a 3′ downstream donor and the 5′ upstream acceptor in an alternate form of pre-mRNA splicing by a process called backsplicing (Szabo and Salzman, 2016). However, the mechanisms of biogenesis, nuclear export, degradation, and the functional significance of circRNAs, remain unclear or exist as proposed theories. Mounting evidence on the presence of circRNAs in all the organisms tested so far shows that the circRNAs are an integral part of living systems (Salzman et al., 2012, 2013; Memczak et al., 2013; Zhang et al., 2013; Zhang X.-O. et al., 2016; Zhang Y. et al., 2016; Ashwal-Fluss et al., 2014; Starke et al., 2015; Pamudurti et al., 2017; Tan et al., 2017; Yang et al., 2017). Despite this, our understanding of their structural and functional aspects is limited. In this review, we have made an attempt to highlight the promising discoveries that have been made in the field of circRNAs in the recent past. Frontiers in Molecular Biosciences | www.frontiersin.org 1 January 2020 | Volume 6 | Article 146 Guria et al. CircRNAs in Animals and Plants HISTORY models of circRNA biogenesis are by direct backsplicing and exon skipping or by lariat intermediate formation (Chen and Yang, 2015) (Figure 1). Both models give rise to circRNAs and linear RNAs from the flanking regions, which raises further questions regarding the frequency of occurrence of one model over another. The exon-skipped linear RNA is either degraded (Egecioglu et al., 2012; Bitton et al., 2015) or results in a truncated protein that is different from the native protein. Recent studies have led to the discovery of many essential cis and trans factors that have a positive or negative regulatory effect on circRNA biogenesis (Figure 1). CircRNA production requires the joint involvement of spliceosomal machinery and the natural splice sites (Starke et al., 2015) through a co-transcriptional mechanism (Ashwal-Fluss et al., 2014; Huang and Shan, 2015). Hence, competition might occur between the canonical splicing and backsplicing mechanisms in the same sequence to form linear mRNA or circRNA, respectively (Ashwal-Fluss et al., 2014; Chen and Yang, 2015). The presence of roughly 1% of circRNAs among mRNAs reveals that canonical splicing is more prominent than backsplicing (Salzman et al., 2013). However, post-transcriptional regulation of circRNA biogenesis is also reported in Fused in Sarcoma (FUS) gene-depleted motor neurons in-vitro (Errichelli et al., 2017). Mutations in natural splice sites from 5′ GU to 5′ CA decreases circRNA production (Ashwal-Fluss et al., 2014). In-vitro studies using single exon minigenes show that, when both the 5′ and 3′ splice sites are mutated, the spliceosomal machinery is inclined toward the next cryptic splice site, which leads to an increase or decrease in the circumference of the circle (Figure 2). It may ultimately result in weakening of the circularization efficiency. On the other hand, it has also been validated that any sequence can be circularized if the last three nucleotides in the 5′ and 3′ spliceosomal recognition sites remain unchanged (Starke et al., 2015). Conversely, most of the plant circRNAs are joined by non-canonical splice sites (Ye et al., 2017; Chu et al., 2018a,b; Guria et al., 2019); the probable reason for this could be the flexibility in binding of the spliceosome machinery. Due to high complementarity, the microRNA (miRNA)-mediated cleavage of circRNAs is possibly another striking reason for the lower number of circRNAs in plants, as shown in Vitis vinifera L. (Gao et al., 2019). Moreover, the identification of miRNA binding and cleavage sites in circRNA, either by rapid amplification of cDNA ends (RACE) or degradome sequencing, is difficult due to lack of a 5′ cap and 3′ poly-A tail. This is compelling evidence, and there might yet be other unidentified mechanisms involved in the biogenesis of circRNA in plants (Chu et al., 2018a,b). Overall, the biogenesis of circRNA is regulated by spliceosomes and the recognition of both the canonical and non-canonical splice junctions. This (...truncated)