What Happens at Or After Transcription: Insights Into Circrna Biogenesis and Function

What happens at or after transcription: Insights into circRNA biogenesis and function

Chuan Huang and Ge Shan* School of Life Sciences & CAS Key Laboratory of Brain Function and Disease; University of Science and Technology of China; Hefei, Anhui Province, China

ircular RNAs (circRNAs) are a backsplicing takes place in the nucleus. Clarge family of noncoding RNAs In linear splicing, most introns in the (ncRNAs) found in metazoans. System- pre-mRNA are removed before transcrip- atic studies of circRNAs have just begun. tion is finished.9 We asked whether back- Here, we discuss circRNA biogenesis and splicing was also cotranscriptional? Two functions with a focus on studies indicat- lines of evidence were presented to sup- ing great diversification of circRNAs. We port this idea in Drosophila (Fig. 1).10 highlight the recent identification of a First, a substantial number of circRNAs special subtype of circRNAs, called were found as nascent chromatin-bound EIciRNAs, and their role in transcrip- RNAs. Secondly, a mutant RNA poly- tional regulation. New insights on RNA- merase II (RNAP II) with slower elonga- RNA interaction and other features asso- tion rate in favor of cotranscriptional ciated with circRNA biology are also splicing increased the ratio of linear to discussed. circRNAs for individual genes. These results indicated that most fly circRNAs Circular RNAs (circRNAs) are a might be generated cotranscriptionally, recently identified large family of noncod- 1-4 and linear splicing and backsplicing ing RNAs (ncRNAs) in metazoan. might compete with each other at the Thousands of circRNAs, as alternative cotranscriptional level.8 The argument transcripts from exonic backsplicing of for the posttranscriptional biogenesis of coding genes (exonic circRNAs), have circRNAs was brought forward with the been found in mammalian cells as well as observation that a stable 30 end of pre- in model organisms such as Drosophila 4-6 mRNA was in favor of circRNA produc- Keywords: biogenesis, circular RNA, melanogaster and Caenorhabditis elegans. tion in mammalian cells when tested exon-intron circular RNA, microRNA circRNAs in eukaryotic cells may include with an overexpressing plasmid sponge, transcriptional regulation a variety of molecules, such as plant (Fig. 1).11 Future studies are required to viroids, the genome of some ssRNA Abbreviations: AMO, antisense morpho- resolve these 2 somewhat conflicting viruses, the loop of intronic lariats, and so lino; circRNA, circular RNA; CLIP, findings, although a second possibility on. As the majority of circRNAs identified cross-linked immunoprecipitation; could also be considered. in animal cells are composed of exonic dsRNA, double-stranded RNA; EIciRNA, Itispossiblethatsome(maybeeven circRNAs, in this point-of-view we will exon-intron circular RNA; FISH, fluores- the majority of) circRNAs are generated focus on exonic circRNAs; therefore, cent in situ hybridization; ncRNA, cotranscriptionally, while the biogenesis when we use the term ‘circRNAs’ we are noncoding RNA; QKI, quaking; MBL, of the others may be posttranscrip- referring to exonic circRNAs, unless oth- muscleblind; RNAP II, RNA polymerase tional. In eukaryotic cells, most of the erwise specified.7,8 II; snRNP, small nuclear ribonucleic canonical linear splicing happens proteins; ssRNA, single-stranded RNA. cotranscriptionally, although some introns are still removed posttranscrip- *Correspondence to: Ge Shan; Email: shange@ustc. 9,12 edu.cn Cotranscriptional or tionally. Cotranscriptional splicing efficiency is actually higher in Drosoph- Submitted: 05/19/2015 Posttranscriptional Biogenesis of circRNAs ila than that in mice.13 It is also possi- Revised: 06/27/2015 ble that circRNAs could be divided into Accepted: 06/30/2015 Inthesamewayasregularlinearsplic- 2 subclasses, according to their co- or http://dx.doi.org/10.1080/21541264.2015.1071301 ing, the generation of circRNAs by post-transcriptional biogenesis. www.tandfonline.com Transcription 61 circRNAs function as microRNA sponges? The answer to this question may be nega- tive when we consider that only a few out of the thousands of circRNAs in human cells harbor multiple predicted binding sites for individual microRNAs.18 How- ever, Drosophila circRNAs do harbor over a thousand computational miRNA binding sites conserved across the genus,5 indi- cating a possible divergence in circRNA functions along different branches of the animal kingdom.

EIciRNA as a Special Subtype of circRNA

Recently, a distinct subtype of circR- NAs was found in mammalian cells in our lab.15 This novel subclass of circRNAs associated with RNAP II to regulate transcription in the nucleus.15 We speculated that some of these ncRNAs might regulate transcription; therefore, we initially Figure 1. circRNAs are generated co- or post-transcriptionally, and their biogenesis may be medi- attempted to identify RNAP II-associated ated by complementary sequences (purple and yellow boxes) in the flanking introns. EIciRNAs asso- ncRNAs via cross-linked immunoprecipi- ciate with U1 snRNPs to regulate gene transcription in cis. U1 snRNPs also play roles in preventing tation (CLIP) and identified >100 circR- premature polyadenylation and in determining transcriptional direction. The majority of circRNAs is NAs.15 Further analysis revealed that these composed exclusively of exonic sequences and localize to the cytoplasm; 2 of them were are shown circRNAs were composed of both exonic to function as microRNA sponges. and intronic sequences of coding genes. We termed these special circRNAs exon- Sequences and Proteins Furthermore, circRNAs show expres- intron circular RNAs (EIciRNAs). The Mediating the Biogenesis sion profiles of cell specificity often inde- intron retention makes EIciRNAs distinct of circRNAs pendently from the expression patterns of from most other circRNAs, which are their parent genes, which points to a regu- composed exclusively of exonic sequences. Flanking long introns and complemen- lated biogenesis of circRNAs that is cell- EIciRNAs also tend to have flanking long tary repeats such as Alu elements in flank- and gene-specific.1,2,5,14,15,18-20 It is introns as well as flanking complementary ing introns are positively associated with highly possible that circRNAs are diversi- sequences, just like the other circRNAs, the biogenesis of circRNAs6,11,14-16, sug- fied in various ways in their biogenesis. although other not yet identified features gesting 2 potential features of circRNA may lay in the flanking sequences of biogenesis. Long (maybe also hard-to- EIciRNAs. FISH experiments showed splice) introns could serve as marks for the circRNAs as microRNA Sponges that EIciRNAs localized predominantly in regions of circularization, and then the 2 the nucleus, consistently with their associ- backsplicing sites might be brought Once formed, what, if any, are the ation with RNAP II. Their cellular locali- together by the flanking complementary physiological roles of these non-canonical zation, again, differentiates EIciRNAs sequences (Fig. 1). On the other hand, transcripts? A role in posttranscriptional from the other circRNAs. not all circRNAs are associated with long regulation was demonstrated for 2 circR- flanking introns or intronic repeats, and NAs, circCDR1as and circSry.1,2 Both the exonic sequences involved in back- circRNAs function as microRNA sponges, EIciRNAs Regulate Transcription splicing may also be crucial for the circu- containing multiple copies of microRNA via RNA-RNA Interactions larization of some circRNAs.11,15 RNA binding sites. The canonical functional binding proteins such as ADAR, QKI, site of microRNAs is the cytoplasm, and, Since EIciRNAs associate with RNAP and MBL, known to be involved in RNA interestingly, most circRNAs also localize II and localize almost exclusively to the editing or alternative splicing, are associ- in the cytoplasm.2,3 A natural question to nucleus, we speculated that these circR- ated with the biogenesis of specific ask following these findings is: could it be NAs might play roles in transcriptional circRNAs.6,11,17 possible that many or even the majority of regulation. Indeed, knockdown of

62 Transcription Volume 6 Issue 4 EIciRNAs with either siRNAs or RNa- of U1 snRNA. Recent studies have dem- The discovery of EIciRNAs and the 2 seH-based antisense oligonucleotides onstrated that U1 snRNAs play roles in functions so far associated with circR- (ASO) decreased the transcription of their preventing premature mRNA polyadeny- NAs—microRNA sponges and transcrip- parental genes. With multiple lines of evi- lation and in determining transcriptional tional regulators—as well as the already dence, we showed that EIciRNAs direction of divergent promoters.23-25 The discussed disputes around circRNA bio- enhanced the transcription of their paren- number of U1 snRNA molecules is genesis, strongly indicate that circRNAs tal genes in cis, although the possibility of »100-fold higher than that of other are a family of RNAs with great diversity.8 trans effects of these EIciRNAs on the snRNAs of the mammalian spliceosome.25 Just like linear RNAs, it is possible that expression of other genes could not be Thus, it is highly possible that most or at the only common feature of circRNAs is excluded.15 least a large proportion of U1 snRNAs that they are all circular. How do EIciRNAs promote the play roles in mechanisms other than splic- For circRNAs to function as micro- expression of their parental genes? We ing. U1 snRNA may even be a Swiss RNA sponges the number of molecule for found that U1 snRNPs associated with Army knife-like molecule involved in individual circRNAs needs to be high EIciRNAs at their parental gene promoter. multiple regulatory events. enough; however, most circRNAs are not Indeed, RNA-DNA double FISH assay abundant. Low circRNA copy numbers showed the colocalization of EIciRNAs lead to the argument that the majority of and U1 snRNAs. It may be even possible Further Insights on these molecules may be piggyback prod- that the global distribution of U1 snRNAs Transcriptional Regulation ucts of pre-mRNA splicing and/or noise is associated, to some degree, with the dis- and circRNAs of gene expression,18 although their tribution of EIciRNAs in the nucleus. restricted cellular localization and cell spe- Further supporting the idea that U1 Transcription is a stringently con- cific expression profiles argue strongly snRNP mediate the cis effect of EIciR- trolled process that requires many regula- against this view. Copy numbers of EIciR- NAs, we found that blocking U1 snRNA tory factors. Several ncRNAs participate NAs do not need to be high for their cis with antisense morpholino (AMO) spe- in transcriptional regulation by using effect in the nucleus. Copy number and cific for U1 snRNA abolished the effect of diverse mechanisms.26 First, ncRNAs such expression profiles are all related to their EIciRNA on the regulation of their paren- as B2 RNA could directly target RNAP II biogenesis and the completely unexplored tal genes at multiple levels. Therefore, the to exert regulatory functions.27,28 A sec- subject of circRNA degradation. The cel- interaction between U1 snRNA and ond class of ncRNAs, such as EBER2 lular localization of individual circRNAs EIciRNA is indispensable for their role in RNA, NEAT2 RNA, and NRSE RNA, may also even be determined together by transcriptional regulation. Furthermore, influence the status of transcriptional acti- biogenesis, transportation, and degrada- we suspected that EIciRNAs interacted vators or repressors.29-31 A third class of tion. The only thing known about with U1 snRNA via the 50 splicing site of ncRNAs includes U1 snRNA and 7SK circRNA degradation is that circRNAs are their retained introns, which is the U1 RNA and associate with factors participat- presumably more stable than other snRNA binding site in canonical splicing. ing in transcriptional initiation, elonga- ncRNAs due to their inaccessibility to Indeed, sterically blocking these sites with tion, or termination.22,32 EIciRNAs seem exonucleases such as RNase R.35,36 Also, AMO decreased the interaction between to utilize a novel mechanism of RNA- whether and how circRNAs are actively EIciRNAs and U1 snRNP, and further RNA interaction for their roles in tran- transported within eukaryotic cells are interactions among RNAP II, U1 snRNP, scriptional regulation. questions that require further EIciRNAs, and their parental gene pro- A major way of regulation by RNA is investigation. moter. This blocking eventually resulted via RNA-RNA interactions.33,34 This is ncRNAs participate in numerous bio- in EIciRNA’s inability to regulate parent nicely exemplified by the series of RNA- logical events through various mecha- gene transcription.15 RNA-mediated events between snRNAs nisms, although many ncRNAs remain and pre-mRNAs, which lead to the poorly understood. This is particularly removal of eukaryotic introns.9,12,21 A true about circRNAs. The identification Multiple Roles of U1 snRNP plethora of ncRNAs also execute their of certain circRNAs playing microRNA physiological roles through specific RNA- sponge roles, the specific features associ- It seems that U1 snRNA plays a central RNA recognition and binding.34 RNA- ated with circRNA biogenesis, and the role in mediating the effect of EIciRNAs. RNA interaction is essential for the 2 func- role of EIciRNAs in transcriptional regu- In eukaryotic cells, U1 snRNA is initially tions so far associated with circRNAs—as lation may provide only a glimpse of the identified as a component of the spliceo- microRNA sponges in the cytoplasm and tip of the iceberg that is circRNA biology. some.21 U1 snRNA also associates with as transcriptional regulators in the nucleus. TFIIH to mediate transcription initiation It is reasonable to speculate that circRNAs for some specific genes.22 The enhance- may play roles by interacting with a pleth- ment of transcription by EIciRNAs ora of RNAs. Also, intramolecular RNA- Disclosure of Potential Conflicts of Interest through interaction with U1 snRNP may RNA binding is involved in circRNA bio- No potential conflicts of interest were also be one of the regulatory mechanisms genesis, as already discussed. disclosed.

www.tandfonline.com Transcription 63 Funding PMID:25242144; http://dx.doi.org/10.1016/j.molcel. polyadenylation signals. Nature 2013; 499:360-3; 15 2014.08.019 PMID:23792564; http://dx.doi.org/10.1038/nature12349 The work described in Li et al., 2015 11. Liang D, Wilusz JE. Short intronic repeat sequences 24. Berg MG, Singh LN, Younis I, Liu Q, Pinto AM, was supported by the National Basic facilitate circular RNA production. Genes Dev 2014; Kaida D, Zhang Z, Cho S, Sherrill-Mix S, Wan L, et al. 28:2233-47; PMID:25281217; http://dx.doi.org/ U1 snRNP determines mRNA length and regulates iso- Research Program of China 10.1101/gad.251926.114 form expression. Cell 2012; 150:53-64; PMID: (2011CBA01103 and 2015CB943000), 12. Bentley DL. Rules of engagement: co-transcriptional 22770214; http://dx.doi.org/10.1016/j.cell.2012.05.029 National Natural Science Foundation of recruitment of pre-mRNA processing factors. Curr 25. Kaida D, Berg MG, Younis I, Kasim M, Singh LN, Opin Cell Biol 2005; 17:251-6; PMID:15901493; Wan L, Dreyfuss G. U1 snRNP protects pre-mRNAs China (31471225, 81171074, and http://dx.doi.org/10.1016/j.ceb.2005.04.006 from premature cleavage and polyadenylation. Nature 91232702), CAS (KJZD-EW-L01-2), and 13. Khodor YL, Menet JS, Tolan M, Rosbash M. Cotran- 2010; 468:664-8; PMID:20881964; http://dx.doi.org/ scriptional splicing efficiency differs dramatically 10.1038/nature09479 the Fundamental Research Funds for the between Drosophila and mouse. RNA 2012; 18:2174- 26. Goodrich JA, Kugel JF. Non-coding-RNA regulators of Central Universities (WK2070000034). 86; PMID:23097425; http://dx.doi.org/10.1261/ RNA polymerase II transcription. Nat Rev Mol Cell rna.034090.112 Biol 2006; 7:612-6; PMID:16723972; http://dx.doi. 14. Jeck WR, Sorrentino JA, Wang K, Slevin MK, Burd org/10.1038/nrm1946 References CE, Liu J, Marzluff WF, Sharpless NE. Circular RNAs 27. Espinoza CA, Allen TA, Hieb AR, Kugel JF, Goodrich 1. Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO. are abundant, conserved, and associated with ALU JA. B2 RNA binds directly to RNA polymerase II to Circular RNAs are the predominant transcript isoform repeats. RNA 2013; 19:141-57; PMID:23249747; repress transcript synthesis. Nat Struct Mol Biol 2004; from hundreds of human genes in diverse cell types. http://dx.doi.org/10.1261/rna.035667.112 11:822-9; PMID:15300239; http://dx.doi.org/ PLoS One 2012; 7:e30733; PMID:22319583; http:// 15. Li Z, Huang C, Bao C, Chen L, Lin M, Wang X, 10.1038/nsmb812 dx.doi.org/10.1371/journal.pone.0030733 Zhong G, Yu B, Hu W, Dai L, et al. Exon-intron circu- 28. Allen TA, Von Kaenel S, Goodrich JA, Kugel JF. The 2. Memczak S, Jens M, Elefsinioti A, Torti F, Krueger J, lar RNAs regulate transcription in the nucleus. Nat SINE-encoded mouse B2 RNA represses mRNA tran- Rybak A, Maier L, Mackowiak SD, Gregersen LH, Struct Mol Biol 2015; 22:256-64; PMID:25664725; scription in response to heat shock. Nat Struct Mol Munschauer M, et al. Circular RNAs are a large class of http://dx.doi.org/10.1038/nsmb.2959 Biol 2004; 11:816-21; PMID:15300240; http://dx.doi. animal RNAs with regulatory potency. Nature 2013; 16. Starke S, Jost I, Rossbach O, Schneider T, Schreiner S, org/10.1038/nsmb813 495:333-8; PMID:23446348; http://dx.doi.org/ Hung LH, Bindereif A. Exon circularization requires 29. Lee N, Moss WN, Yario TA, Steitz JA. EBV Noncod- 10.1038/nature11928 canonical splice signals. Cell Rep 2015; 10:103-11; ing RNA Binds Nascent RNA to Drive Host PAX5 to 3. Hansen TB, Jensen TI, Clausen BH, Bramsen JB, Fin- PMID:25543144; http://dx.doi.org/10.1016/j.celrep. Viral DNA. Cell 2015; 160: 607-18; PMID:25662012; sen B, Damgaard CK, Kjems J. Natural RNA circles 2014.12.002 http://dx.doi.org/10.1016/j.cell.2015.01.015 function as efficient microRNA sponges. Nature 2013; 17. Conn SJ, Pillman KA, Toubia J, Conn VM, Salmanidis 30. Yang L, Lin C, Liu W, Zhang J, Ohgi KA, Grinstein 495:384-8; PMID:23446346; http://dx.doi.org/ M, Phillips CA, Roslan S, Schreiber AW, Gregory PA, JD, Dorrestein PC, Rosenfeld MG. ncRNA- and Pc2 10.1038/nature11993 Goodall GJ. The RNA Binding Protein Quaking Regu- methylation-dependent gene relocation between nuclear 4. Wang PL, Bao Y, Yee MC, Barrett SP, Hogan GJ, lates Formation of circRNAs. Cell 2015; 160:1125-34; structures mediates gene activation programs. Cell Olsen MN, Dinneny JR, Brown PO, Salzman J. Circu- PMID:25768908; http://dx.doi.org/10.1016/j.cell.2015. 2011; 147: 773-88; PMID:22078878; http://dx.doi. lar RNA is expressed across the eukaryotic tree of life. 02.014 org/10.1016/j.cell.2011.08.054 PLoS One 2014; 9:e90859; PMID:24609083; http:// 18. Guo JU, Agarwal V, Guo H, Bartel DP. Expanded 31. Kuwabara T, Hsieh J, Nakashima K, Taira K, Gage dx.doi.org/10.1371/journal.pone.0090859 identification and characterization of mammalian FH. A small modulatory dsRNA specifies the fate of 5. Westholm JO, Miura P, Olson S, Shenker S, Joseph B, circular RNAs. Genome Biol 2014; 15:409; adult neural stem cells. Cell 2004; 116:779-93; Sanfilippo P, Celniker SE, Graveley BR, Lai EC. PMID:25070500; http://dx.doi.org/10.1186/s13059- PMID:15035981; http://dx.doi.org/10.1016/S0092- Genome-wide analysis of drosophila circular RNAs 014-0409-z 8674(04)00248-X reveals their structural and sequence properties and age- 19. You X, Vlatkovic I, Babic A, Will T, Epstein I, Tushev 32. Yang Z, Zhu Q, Luo K, Zhou Q. The 7SK small dependent neural accumulation. Cell Rep 2014; G, Akbalik G, Wang M, Glock C, Quedenau C, et al. nuclear RNA inhibits the CDK9/cyclin T1 kinase to 9:1966-80; PMID:25544350; http://dx.doi.org/ Neural circular RNAs are derived from synaptic genes control transcription. Nature 2001; 414:317-22; PMID: 10.1016/j.celrep.2014.10.062 and regulated by development and plasticity. Nat Neu- 11713532; http://dx.doi.org/10.1038/35104575 6. Ivanov A, Memczak S, Wyler E, Torti F, Porath HT, rosci 2015; 18:603-10; PMID:25714049; http://dx. 33. Cech TR, Steitz JA.The noncoding RNA revolution- Orejuela MR, Piechotta M, Levanon EY, Landthaler doi.org/10.1038/nn.3975 trashing old rules to forge new ones.Cell 2014; 157:77- M, Dieterich C, et al. Analysis of intron sequences 20. Rybak-Wolf A, Stottmeister C, Glazar P, Jens M, Pino 94; PMID:24679528; http://dx.doi.org/10.1016/j. reveals hallmarks of circular RNA biogenesis in animals. N, Giusti S, Hanan M, Behm M, Bartok O, Ashwal- cell.2014.03.008 Cell Rep 2015; 10:170-7; PMID:25558066; http://dx. Fluss R, et al.Circular RNAs in the Mammalian Brain 34. Guil S, Esteller M. RNA-RNA interactions in gene reg- doi.org/10.1016/j.celrep.2014.12.019 Are Highly Abundant, Conserved, and Dynamically ulation: the coding and noncoding players. Trends Bio- 7. Chen L, Shan G. Circular RNAs remain peculiarly Expressed. Mol Cell 2015; S1097-2765:00218-X; chem Sci 2015; 40:248-56; PMID:25818326; http:// unclear in biogenesis and function. Sci China Life Sci PMID:25921068 dx.doi.org/10.1016/j.tibs.2015.03.001 2015; 58(6):616-8; http://dx.doi.org/10.1007/s11427- 21. Seraphin B, Rosbash M. Identification of functional U1 35. Vincent HA, Deutscher MP. Substrate recognition and 015-4855-y snRNA-pre-mRNA complexes committed to spliceo- catalysis by the exoribonuclease RNase R. J Biol Chem 8. Chen L, Huang C, Wang X, Shan G. Circular RNAs in some assembly and splicing. Cell 1989; 59:349-358; 2006; 281: 29769-75; PMID:16893880; http://dx.doi. eukaryotic cells. Curr Genom 2015; 16(5): 312-8. PMID:2529976; http://dx.doi.org/10.1016/0092-8674 org/10.1074/jbc.M606744200 9. Moore MJ, Proudfoot NJ. Pre-mRNA processing (89)90296-1 36. Suzuki H, Zuo Y, Wang J, Zhang MQ, Malhotra A, reaches back to transcription and ahead to translation. 22. Kwek KY, Murphy S, Furger A, Thomas B, O’Gorman Mayeda A. Characterization of RNase R-digested cellu- Cell 2009; 136:688-700; PMID:19239889; http://dx. W, Kimura H, Proudfoot NJ, Akoulitchev A. U1 lar RNA source that consists of lariat and circular doi.org/10.1016/j.cell.2009.02.001 snRNA associates with TFIIH and regulates transcrip- RNAs from pre-mRNA splicing. Nucleic Acids Res 10. Ashwal-Fluss R, Meyer M, Pamudurti NR, Ivanov A, tional initiation. Nat Struct Biol 2002; 9:800-5; 2006; 34:e63; PMID:16682442; http://dx.doi.org/ Bartok O, Hanan M, Evantal N, Memczak S, Rajewsky PMID:12389039 10.1093/nar/gkl151 N, Kadener S. circRNA biogenesis competes with pre- 23. Almada AE, Wu X, Kriz AJ, Burge CB, Sharp PA. Pro- mRNA splicing. Mol Cell 2014; 56:55-66; moter directionality is controlled by U1 snRNP and

64 Transcription Volume 6 Issue 4