The Biogenesis, Functions, and Challenges of Circular RNAs

Covalently closed circular RNAs (circRNAs) are produced by precursor mRNA back-splicing of exons of thousands of genes in eukaryotes. circRNAs are generally expressed at low levels and often exhibit cell-type-specific and tissue-specific patterns. Recent studies have shown that their biogenesis requires spliceosomal machinery and can be modulated by both cis complementary sequences and protein factors. The functions of most circRNAs remain largely unexplored, but known functions include sequestration of microRNAs or proteins, modulation of transcription and interference with splicing, and even translation to produce polypeptides. However, challenges exist at multiple levels to understanding of the regulation of circRNAs because of their circular conformation and sequence overlap with linear mRNA counterparts. In this review, we survey the recent progress on circRNA biogenesis and function and discuss technical obstacles in circRNA studies. Covalently closed circular RNAs (circRNAs) are produced by precursor mRNA back-splicing of exons of thousands of genes in eukaryotes. circRNAs are generally expressed at low levels and often exhibit cell-type-specific and tissue-specific patterns. Recent studies have shown that their biogenesis requires spliceosomal machinery and can be modulated by both cis complementary sequences and protein factors. The functions of most circRNAs remain largely unexplored, but known functions include sequestration of microRNAs or proteins, modulation of transcription and interference with splicing, and even translation to produce polypeptides. However, challenges exist at multiple levels to understanding of the regulation of circRNAs because of their circular conformation and sequence overlap with linear mRNA counterparts. In this review, we survey the recent progress on circRNA biogenesis and function and discuss technical obstacles in circRNA studies. A variety of circular RNAs have been reported to be generated by distinct mechanisms. For example, they were identified as circular RNA genomes in plant viroids (Sanger et al., 1976Sanger H.L. Klotz G. Riesner D. Gross H.J. Kleinschmidt A.K. Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures.Proc. Natl. Acad. Sci. USA. 1976; 73: 3852-3856Crossref PubMed Scopus (1191) Google Scholar) and hepatitis delta virus (Kos et al., 1986Kos A. Dijkema R. Arnberg A.C. van der Meide P.H. Schellekens H. The hepatitis delta (delta) virus possesses a circular RNA.Nature. 1986; 323: 558-560Crossref PubMed Scopus (437) Google Scholar). Housekeeping noncoding RNAs, including small nucleolar RNAs (snoRNAs) and RNase P RNA, were found in circular formats in archaea (Danan et al., 2012Danan M. Schwartz S. Edelheit S. Sorek R. Transcriptome-wide discovery of circular RNAs in Archaea.Nucleic Acids Res. 2012; 40: 3131-3142Crossref PubMed Scopus (381) Google Scholar). Circular RNA intermediates can also be generated during rRNA processing (Danan et al., 2012Danan M. Schwartz S. Edelheit S. Sorek R. Transcriptome-wide discovery of circular RNAs in Archaea.Nucleic Acids Res. 2012; 40: 3131-3142Crossref PubMed Scopus (381) Google Scholar, Tang et al., 2002Tang T.H. Rozhdestvensky T.S. d’Orval B.C. Bortolin M.L. Huber H. Charpentier B. Branlant C. Bachellerie J.P. Brosius J. Hüttenhofer A. RNomics in Archaea reveals a further link between splicing of archaeal introns and rRNA processing.Nucleic Acids Res. 2002; 30: 921-930Crossref PubMed Scopus (101) Google Scholar) or permuted tRNAs with rearranged segments in archaea and algae (Soma et al., 2007Soma A. Onodera A. Sugahara J. Kanai A. Yachie N. Tomita M. Kawamura F. Sekine Y. Permuted tRNA genes expressed via a circular RNA intermediate in Cyanidioschyzon merolae.Science. 2007; 318: 450-453Crossref PubMed Scopus (78) Google Scholar). RNA intermediates escaped from intron lariat debranching can form circular RNAs as well (Box 1; Gardner et al., 2012Gardner E.J. Nizami Z.F. Talbot Jr., C.C. Gall J.G. Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis.Genes Dev. 2012; 26: 2550-2559Crossref PubMed Scopus (94) Google Scholar, Kopczynski and Muskavitch, 1992Kopczynski C.C. Muskavitch M.A. Introns excised from the Delta primary transcript are localized near sites of Delta transcription.J. Cell Biol. 1992; 119: 503-512Crossref PubMed Scopus (64) Google Scholar, Qian et al., 1992Qian L. Vu M.N. Carter M. Wilkinson M.F. A spliced intron accumulates as a lariat in the nucleus of T cells.Nucleic Acids Res. 1992; 20: 5345-5350Crossref PubMed Scopus (64) Google Scholar, Talhouarne and Gall, 2014Talhouarne G.J. Gall J.G. Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.RNA. 2014; 20: 1476-1487Crossref PubMed Scopus (84) Google Scholar, Zhang et al., 2013Zhang Y. Zhang X.O. Chen T. Xiang J.F. Yin Q.F. Xing Y.H. Zhu S. Yang L. Chen L.L. Circular intronic long noncoding RNAs.Mol. Cell. 2013; 51: 792-806Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar). Despite these different forms of circular RNAs, most currently studied circular RNAs (circRNAs) are produced from precursor mRNA (pre-mRNA) back-splicing of exons in which a downstream 5′ splice site (ss) is joined with an upstream 3′ ss, and the resulting RNA circle is ligated by a 3′-5′ phosphodiester bond at the junction site (Figure 1A; reviews by Chen, 2016Chen L.L. The biogenesis and emerging roles of circular RNAs.Nat. Rev. Mol. Cell Biol. 2016; 17: 205-211Crossref PubMed Scopus (928) Google Scholar, Lasda and Parker, 2014Lasda E. Parker R. Circular RNAs: diversity of form and function.RNA. 2014; 20: 1829-1842Crossref PubMed Scopus (768) Google Scholar, Wilusz, 2018Wilusz J.E. A 360° view of circular RNAs: From biogenesis to functions. Wiley Interdiscip.Rev. RNA. 2018; 9: e1478PubMed Google Scholar).Box 1Circular Intronic RNAsCircular intronic RNAs (ciRNAs) represent another class of circular RNA molecules. They are derived from lariat introns of Pol II transcripts and depend on a consensus RNA motif containing a 7-nt GU-rich motif near the 5′ splice site and an 11-nt C-rich motif at the branchpoint site to escape debranching. The resulting RNA circles are covalently ligated by 2′ 5′-phosphodiester bonds at the joining sites and lack the 3′ linear sequences from the 3′ end of the introns to the branchpoint sites (Zhang et al., 2013Zhang Y. Zhang X.O. Chen T. Xiang J.F. Yin Q.F. Xing Y.H. Zhu S. Yang L. Chen L.L. Circular intronic long noncoding RNAs.Mol. Cell. 2013; 51: 792-806Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar). Interestingly, stable lariat intronic RNAs (named sisRNAs) were also found in oocytes of Xenopus tropicalis (Gardner et al., 2012Gardner E.J. Nizami Z.F. Talbot Jr., C.C. Gall J.G. Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis.Genes Dev. 2012; 26: 2550-2559Crossref PubMed Scopus (94) Google Scholar, Talhouarne and Gall, 2014Talhouarne G.J. Gall J.G. Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.RNA. 2014; 20: 1476-1487Crossref PubMed Scopus (84) Google Scholar), and maternally inherited sisRNAs could trigger expression of their host genes via a positive feedback loop during embryogenesis (Tay and Pek, 2017Tay M.L. Pek J.W. Maternally Inherited Stable Intronic Sequence RNA Triggers a Self-Reinforcing Feedback Loop during Development.Curr. Biol. 2017; 27: 1062-1067Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In human cells, ciRNAs have little enrichment for miRNA target sites but, rather, largely accumulate in the nucleus to regulate gene transcription in cis by promoting Pol II transcription of their parental genes through unknown mechanisms (Zhang et al., 2013Zhang Y. Zhang X.O. Chen T. Xiang J.F. Yin Q.F. Xing Y.H. Zhu S. Yang L. Chen L.L. Circular intronic long noncoding RNAs.Mol. Cell. 2013; 51: 792-806Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar). Circular intronic RNAs (ciRNAs) represent another class of circular RNA molecules. They are derived from lariat introns of Pol II transcripts and depend on a consensus RNA motif containing a 7-nt GU-rich motif near the 5′ splice site and an 11-nt C-rich motif at the branchpoint site to escape debranching. The resulting RNA circles are covalently ligated by 2′ 5′-phosphodiester bonds at the joining sites and lack the 3′ linear sequences from the 3′ end of the introns to the branchpoint sites (Zhang et al., 2013Zhang Y. Zhang X.O. Chen T. Xiang J.F. Yin Q.F. Xing Y.H. Zhu S. Yang L. Chen L.L. Circular intronic long noncoding RNAs.Mol. Cell. 2013; 51: 792-806Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar). Interestingly, stable lariat intronic RNAs (named sisRNAs) were also found in oocytes of Xenopus tropicalis (Gardner et al., 2012Gardner E.J. Nizami Z.F. Talbot Jr., C.C. Gall J.G. Stable intronic sequence RNA (sisRNA), a new class of noncoding RNA from the oocyte nucleus of Xenopus tropicalis.Genes Dev. 2012; 26: 2550-2559Crossref PubMed Scopus (94) Google Scholar, Talhouarne and Gall, 2014Talhouarne G.J. Gall J.G. Lariat intronic RNAs in the cytoplasm of Xenopus tropicalis oocytes.RNA. 2014; 20: 1476-1487Crossref PubMed Scopus (84) Google Scholar), and maternally inherited sisRNAs could trigger expression of their host genes via a positive feedback loop during embryogenesis (Tay and Pek, 2017Tay M.L. Pek J.W. Maternally Inherited Stable Intronic Sequence RNA Triggers a Self-Reinforcing Feedback Loop during Development.Curr. Biol. 2017; 27: 1062-1067Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). In human cells, ciRNAs have little enrichment for miRNA target sites but, rather, largely accumulate in the nucleus to regulate gene transcription in cis by promoting Pol II transcription of their parental genes through unknown mechanisms (Zhang et al., 2013Zhang Y. Zhang X.O. Chen T. Xiang J.F. Yin Q.F. Xing Y.H. Zhu S. Yang L. Chen L.L. Circular intronic long noncoding RNAs.Mol. Cell. 2013; 51: 792-806Abstract Full Text Full Text PDF PubMed Scopus (1337) Google Scholar). circRNAs were discovered more than 25 years ago. Only a handful of circRNAs were found at that time, and they were often considered aberrant splicing byproducts with little functional potential (Capel et al., 1993Capel B. Swain A. Nicolis S. Hacker A. Walter M. Koopman P. Goodfellow P. Lovell-Badge R. Circular transcripts of the testis-determining gene Sry in adult mouse testis.Cell. 1993; 73: 1019-1030Abstract Full Text PDF PubMed Scopus (763) Google Scholar, Cocquerelle et al., 1992Cocquerelle C. Daubersies P. Majérus M.A. Kerckaert J.P. Bailleul B. Splicing with inverted order of exons occurs proximal to large introns.EMBO J. 1992; 11: 1095-1098Crossref PubMed Scopus (205) Google Scholar, Cocquerelle et al., 1993Cocquerelle C. Mascrez B. Hétuin D. Bailleul B. Mis-splicing yields circular RNA molecules.FASEB J. 1993; 7: 155-160Crossref PubMed Scopus (718) Google Scholar, Nigro et al., 1991Nigro J.M. Cho K.R. Fearon E.R. Kern S.E. Ruppert J.M. Oliner J.D. Kinzler K.W. Vogelstein B. Scrambled exons.Cell. 1991; 64: 607-613Abstract Full Text PDF PubMed Scopus (649) Google Scholar, Pasman et al., 1996Pasman Z. Been M.D. Garcia-Blanco M.A. Exon circularization in mammalian nuclear extracts.RNA. 1996; 2: 603-610PubMed Google Scholar). Because of their non-linear conformation and lack of polyadenylated (poly(A)) tails, circRNAs are rarely seen in the next-generation RNA sequencing (RNA-seq) profiling that is usually enriched for poly(A)+ RNAs. Only profiling with non- poly(A) RNAs or enrichment of circular RNAs with RNase R, which is an enzyme that preferentially digests linear RNAs, has uncovered widespread expression of circRNAs from pre-mRNA back-splicing. For example, over 10,000 circRNAs have been found in metazoans, from worm and fruit fly (Ivanov et al., 2015Ivanov A. Memczak S. Wyler E. Torti F. Porath H.T. Orejuela M.R. Piechotta M. Levanon E.Y. Landthaler M. Dieterich C. Rajewsky N. Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals.Cell Rep. 2015; 10: 170-177Abstract Full Text Full Text PDF PubMed Scopus (600) Google Scholar, Westholm et al., 2014Westholm J.O. Miura P. Olson S. Shenker S. Joseph B. Sanfilippo P. Celniker S.E. Graveley B.R. Lai E.C. Genome-wide analysis of Drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation.Cell Rep. 2014; 9: 1966-1980Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar) to mouse, monkey, and human (Dong et al., 2017Dong R. Ma X.K. Chen L.L. Yang L. 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Recent research into circRNA biogenesis has shown that back-splicing is catalyzed by the canonical spliceosomal machinery and modulated by both intronic complementary sequences (ICSs) and RNA binding proteins (RBPs). Emerging studies have revealed that some circRNAs are implicated in neuronal function, innate immune responses, cell proliferation, and pluripotency. At the molecular level, they are involved in gene expression by titrating microRNAs, sequestering proteins, modulating RNA polymerase II (Pol II) transcription, and interfering with pre-mRNA processing. Furthermore, a few endogenous circRNAs are translatable, and some others can act as sources of pseudogene derivation. Despite these encouraging advances, it is worthwhile noting that the circular conformation and almost complete sequence overlap with their linear mRNA counterparts have made the precise evaluation of circRNA expression and function challenging. In this review, we survey the most recent progress regarding the regulation of circRNA biogenesis and function. We also discuss experimental designs and their challenges in circRNA studies. In general, circRNAs are expressed at low levels (Guo et al., 2014Guo J.U. Agarwal V. Guo H. Bartel D.P. Expanded identification and characterization of mammalian circular RNAs.Genome Biol. 2014; 15: 409Crossref PubMed Scopus (1013) Google Scholar, Jeck et al., 2013Jeck W.R. Sorrentino J.A. Wang K. Slevin M.K. Burd C.E. Liu J. Marzluff W.F. Sharpless N.E. Circular RNAs are abundant, conserved, and associated with ALU repeats.RNA. 2013; 19: 141-157Crossref PubMed Scopus (2467) Google Scholar, Memczak et al., 2013Memczak S. Jens M. Elefsinioti A. Torti F. Krueger J. Rybak A. Maier L. Mackowiak S.D. Gregersen L.H. Munschauer M. et al.Circular RNAs are a large class of animal RNAs with regulatory potency.Nature. 2013; 495: 333-338Crossref PubMed Scopus (4465) Google Scholar, Salzman et al., 2013Salzman J. Chen R.E. Olsen M.N. Wang P.L. Brown P.O. Cell-type specific features of circular RNA expression.PLoS Genet. 2013; 9: e1003777Crossref PubMed Scopus (1239) Google Scholar, Zhang et al., 2014Zhang X.O. Wang H.B. Zhang Y. Lu X. Chen L.L. Yang L. Complementary sequence-mediated exon circularization.Cell. 2014; 159: 134-147Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). However, some circRNAs are more abundant than their linear transcripts, and their expression is independent of related linear isoforms (Conn et al., 2015Conn S.J. Pillman K.A. Toubia J. Conn V.M. Salmanidis M. Phillips C.A. Roslan S. Schreiber A.W. Gregory P.A. Goodall G.J. The RNA binding protein quaking regulates formation of circRNAs.Cell. 2015; 160: 1125-1134Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar, Rybak-Wolf et al., 2015Rybak-Wolf A. Stottmeister C. Glažar P. Jens M. Pino N. Giusti S. Hanan M. Behm M. Bartok O. Ashwal-Fluss R. et al.Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.Mol. Cell. 2015; 58: 870-885Abstract Full Text Full Text PDF PubMed Scopus (1291) Google Scholar, Salzman et al., 2013Salzman J. Chen R.E. Olsen M.N. Wang P.L. Brown P.O. Cell-type specific features of circular RNA expression.PLoS Genet. 2013; 9: e1003777Crossref PubMed Scopus (1239) Google Scholar, You et al., 2015You X. Vlatkovic I. Babic A. Will T. Epstein I. Tushev G. Akbalik G. Wang M. Glock C. Quedenau C. et al.Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity.Nat. Neurosci. 2015; 18: 603-610Crossref PubMed Scopus (663) Google Scholar, Zhang et al., 2016aZhang X.O. Dong R. Zhang Y. Zhang J.L. Luo Z. Zhang J. Chen L.L. Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.Genome Res. 2016; 26: 1277-1287Crossref PubMed Scopus (436) Google Scholar). In addition, the expression patterns of circRNAs are diverse among cell types and tissues in mammals. For example, a significant enrichment of circRNAs was observed in the brain (Rybak-Wolf et al., 2015Rybak-Wolf A. Stottmeister C. Glažar P. Jens M. Pino N. Giusti S. Hanan M. Behm M. Bartok O. Ashwal-Fluss R. et al.Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.Mol. Cell. 2015; 58: 870-885Abstract Full Text Full Text PDF PubMed Scopus (1291) Google Scholar, You et al., 2015You X. Vlatkovic I. Babic A. Will T. Epstein I. Tushev G. Akbalik G. Wang M. Glock C. Quedenau C. et al.Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity.Nat. Neurosci. 2015; 18: 603-610Crossref PubMed Scopus (663) Google Scholar) and during certain biological processes, such as human epithelial-mesenchymal transition (EMT) (Conn et al., 2015Conn S.J. Pillman K.A. Toubia J. Conn V.M. Salmanidis M. Phillips C.A. Roslan S. Schreiber A.W. Gregory P.A. Goodall G.J. The RNA binding protein quaking regulates formation of circRNAs.Cell. 2015; 160: 1125-1134Abstract Full Text Full Text PDF PubMed Scopus (1143) Google Scholar). Comparison of circRNA expression from human and mouse revealed that only a small portion (10%∼20%) of human circRNAs could be observed in parallel mouse samples (Dong et al., 2017Dong R. Ma X.K. Chen L.L. Yang L. Increased complexity of circRNA expression during species evolution.RNA Biol. 2017; 14: 1064-1074Crossref PubMed Scopus (118) Google Scholar, Guo et al., 2014Guo J.U. Agarwal V. Guo H. Bartel D.P. Expanded identification and characterization of mammalian circular RNAs.Genome Biol. 2014; 15: 409Crossref PubMed Scopus (1013) Google Scholar, Rybak-Wolf et al., 2015Rybak-Wolf A. Stottmeister C. Glažar P. Jens M. Pino N. Giusti S. Hanan M. Behm M. Bartok O. Ashwal-Fluss R. et al.Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.Mol. Cell. 2015; 58: 870-885Abstract Full Text Full Text PDF PubMed Scopus (1291) Google Scholar). This is in part due to the predominant contribution of cis ICSs across circRNA-forming exons in circRNA formation (Dong et al., 2017Dong R. Ma X.K. Chen L.L. Yang L. Increased complexity of circRNA expression during species evolution.RNA Biol. 2017; 14: 1064-1074Crossref PubMed Scopus (118) Google Scholar). Of note, back-splicing often requires ICSs residing in introns flanking circularized exons (Jeck et al., 2013Jeck W.R. Sorrentino J.A. Wang K. Slevin M.K. Burd C.E. Liu J. Marzluff W.F. Sharpless N.E. Circular RNAs are abundant, conserved, and associated with ALU repeats.RNA. 2013; 19: 141-157Crossref PubMed Scopus (2467) Google Scholar, Zhang et al., 2014Zhang X.O. Wang H.B. Zhang Y. Lu X. Chen L.L. Yang L. Complementary sequence-mediated exon circularization.Cell. 2014; 159: 134-147Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, Liang and Wilusz, 2014Liang D. Wilusz J.E. Short intronic repeat sequences facilitate circular RNA production.Genes Dev. 2014; 28: 2233-2247Crossref PubMed Scopus (539) Google Scholar) (discussed in detail below). It has been shown that repetitive element sequences, which contribute the most to the formation of ICSs, evolve fast in time, leading to increased complexity of circRNA expression in evolution (Dong et al., 2017Dong R. Ma X.K. Chen L.L. Yang L. Increased complexity of circRNA expression during species evolution.RNA Biol. 2017; 14: 1064-1074Crossref PubMed Scopus (118) Google Scholar, Rybak-Wolf et al., 2015Rybak-Wolf A. Stottmeister C. Glažar P. Jens M. Pino N. Giusti S. Hanan M. Behm M. Bartok O. Ashwal-Fluss R. et al.Circular RNAs in the Mammalian Brain Are Highly Abundant, Conserved, and Dynamically Expressed.Mol. Cell. 2015; 58: 870-885Abstract Full Text Full Text PDF PubMed Scopus (1291) Google Scholar). For instance, the conservation of circRNA-forming exonic sequences in the GCN1L1 locus is high between human and mouse. However, a pair of ICSs exists in human flanking introns but not the corresponding mouse locus (Figure 1B, left). Correspondingly, circGCN1L1 is only detected in humans (Figure 1B, right), although the exonic sequences are conserved between human and mouse (Zhang et al., 2014Zhang X.O. Wang H.B. Zhang Y. Lu X. Chen L.L. Yang L. Complementary sequence-mediated exon circularization.Cell. 2014; 159: 134-147Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). At the individual gene level, it has been found that one gene locus can produce multiple circRNAs (Gao et al., 2016Gao Y. Wang J. Zheng Y. Zhang J. Chen S. Zhao F. Comprehensive identification of internal structure and alternative splicing events in circular RNAs.Nat. Commun. 2016; 7: 12060Crossref PubMed Scopus (160) Google Scholar, Jeck et al., 2013Jeck W.R. Sorrentino J.A. Wang K. Slevin M.K. Burd C.E. Liu J. Marzluff W.F. Sharpless N.E. Circular RNAs are abundant, conserved, and associated with ALU repeats.RNA. 2013; 19: 141-157Crossref PubMed Scopus (2467) Google Scholar, Memczak et al., 2013Memczak S. Jens M. Elefsinioti A. Torti F. Krueger J. Rybak A. Maier L. Mackowiak S.D. Gregersen L.H. Munschauer M. et al.Circular RNAs are a large class of animal RNAs with regulatory potency.Nature. 2013; 495: 333-338Crossref PubMed Scopus (4465) Google Scholar, Salzman et al., 2013Salzman J. Chen R.E. Olsen M.N. Wang P.L. Brown P.O. Cell-type specific features of circular RNA expression.PLoS Genet. 2013; 9: e1003777Crossref PubMed Scopus (1239) Google Scholar, Zhang et al., 2014Zhang X.O. Wang H.B. Zhang Y. Lu X. Chen L.L. Yang L. Complementary sequence-mediated exon circularization.Cell. 2014; 159: 134-147Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar, Zhang et al., 2016aZhang X.O. Dong R. Zhang Y. Zhang J.L. Luo Z. Zhang J. Chen L.L. Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.Genome Res. 2016; 26: 1277-1287Crossref PubMed Scopus (436) Google Scholar) with mechanisms related to alternative back-splicing and alternative splicing site selection. Alternative back-splicing selectively uses different downstream 5′ splice donors or upstream 3′ splice acceptors, leading to alternative 5′ or 3′ back-splicing choices to generate multiple circRNAs from a single gene locus (Figure 1C; Zhang et al., 2016aZhang X.O. Dong R. Zhang Y. Zhang J.L. Luo Z. Zhang J. Chen L.L. Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.Genome Res. 2016; 26: 1277-1287Crossref PubMed Scopus (436) Google Scholar). For example, multiple highly expressed circRNAs from human DNMT3B and XPO1 gene loci are generated through the alternative back-splicing mechanism. Importantly, these alternatively back-spliced circRNAs could be experimentally validated by northern blotting (NB) (Zhang et al., 2014Zhang X.O. Wang H.B. Zhang Y. Lu X. Chen L.L. Yang L. Complementary sequence-mediated exon circularization.Cell. 2014; 159: 134-147Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). Alternative splicing also occurs within circRNAs that contain multiple exons. All four basic types of canonical alternative splicing have been found in circRNAs: cassette exon, intron retention, alternative 5′ splicing, and alternative 3′ splicing (Zhang et al., 2016aZhang X.O. Dong R. Zhang Y. Zhang J.L. Luo Z. Zhang J. Chen L.L. Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.Genome Res. 2016; 26: 1277-1287Crossref PubMed Scopus (436) Google Scholar). It is worthwhile noting that circRNAs produced from a single gene locus in this manner have the same back ss (bss) but contain distinct canonical ss within each circRNA (Figure 1C; Gao et al., 2016Gao Y. Wang J. Zheng Y. Zhang J. Chen S. Zhao F. Comprehensive identification of internal structure and alternative splicing events in circular RNAs.Nat. Commun. 2016; 7: 12060Crossref PubMed Scopus (160) Google Scholar, Zhang et al., 2016aZhang X.O. Dong R. Zhang Y. Zhang J.L. Luo Z. Zhang J. Chen L.L. Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.Genome Res. 2016; 26: 1277-1287Crossref PubMed Scopus (436) Google Scholar). For example, NB assays confirmed that the CAMSAP1 locus produces two major circRNA isoforms with or without a retained intron (Salzman et al., 2013Salzman J. Chen R.E. Olsen M.N. Wang P.L. Brown P.O. Cell-type specific features of circular RNA expression.PLoS Genet. 2013; 9: e1003777Crossref PubMed Scopus (1239) Google Scholar, Zhang et al., 2014Zhang X.O. Wang H.B. Zhang Y. Lu X. Chen L.L. Yang L. Complementary sequence-mediated exon circularization.Cell. 2014; 159: 134-147Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar) and that the human XPO1 locus contains a circRNA-predominant cassette exon (Zhang et al., 2014Zhang X.O. Wang H.B. Zhang Y. Lu X. Chen L.L. Yang L. Complementary sequence-mediated exon circularization.Cell. 2014; 159: 134-147Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). Alternative back-splice selection and/or circRNA-specific alternative splice selection leads to thousands of new exons (previously unannotated in NCBI Reference Sequence Database (RefSeq), University of California Santa Cruz [UCSC] Known Genes, or Ensembl) included within circRNAs. For instance, in the human MED13L locus, several previously unannotated exons were identified in multiple non-poly(A) RNA-seq datasets from various cell lines and could be experimentally validated (Zhang et al., 2016aZhang X.O. Dong R. Zhang Y. Zhang J.L. Luo Z. Zhang J. Chen L.L. Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.Genome Res. 2016; 26: 1277-1287Crossref PubMed Scopus (436) Google Scholar). Among them, three are alternatively back-spliced, and one is a new cassette exon mainly alternatively spliced within circRNAs. In contrast, these novel exons were barely detected in the linear MED13L mRNA (Zhang et al., 2016aZhang X.O. Dong R. Zhang Y. Zhang J.L. Luo Z. Zhang J. Chen L.L. Yang L. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs.Genome Res. 2016; 26: 1277-1287Crossref PubMed Scopus (436) Google Scholar). Although less conserved, these circRNA-predominant novel exons have similar sequence signatures as annotated ones. In this case, it remains unclear how the spliceosome could specifically recognize these exons for circular but not l