RNA circularization strategies in vivo and in vitro.pdf

24542465Nucleic Acids Research,2015,Vol.43,No.4Published online 6 February 2015doi:10.1093/nar/gkv045SURVEY AND SUMMARYRNA circularization strategiesin vivoandin vitroSonja Petkovic and Sabine M uller*Institut f ur Biochemie,Ernst Moritz Arndt Universit at Greifswald,Felix-Hausdorff-Str.4,17487 Greifswald,GermanyReceived March 26,2014;Revised January 07,2015;Accepted January 12,2015ABSTRACTIn the plenitude of naturally occurring RNAs,circularRNAs(circRNAs)and their biological role were un-derestimated for years.However,circRNAs are ubiq-uitous in all domains of life,including eukaryotes,archaea,bacteria and viruses,where they can ful-fill diverse biological functions.Some of those func-tions,as for example playing a role in the life cy-cle of viral and viroid genomes or in the maturationof tRNA genes,have been elucidated;other putativefunctions still remain elusive.Due to the resistanceto exonucleases,circRNAs are promising tools forin vivoapplication as aptamers,trans-cleaving ri-bozymes or siRNAs.How are circRNAs generatedinvivoand what approaches do exist to produce ring-shaped RNAsin vitro?In this review we illustrate theoccurrence and mechanisms of RNA circularizationinvivo,surveymethodsforthegenerationofcircRNAin vitroand provide appropriate protocols.INTRODUCTIONCircular RNAs are found in all kingdoms of life,appearingfor example as genomes of viroidal plant pathogens(1)andof the hepatitis delta virus(HDV)(2),or as spliced tRNAand rRNA introns and as rRNA processing intermediatesin archaea(3,4).Furthermore,circRNAs are formed in thelife cycle of bacterial and eukaryal group I introns,wherethey were suggested to play a role in intron mobility by re-verse splicing(57).However,circRNAs were consideredextremely rare in nature for decades,and in particular ineukaryotes,circRNAs were seen as minor RNA structuralvariants attributed to transcriptional noise(8).This viewhas dramatically changed,as a number of recent reportshave convincingly demonstrated that circRNAs in eukary-otes are highly abundant and evolutionary conserved.Ap-parently,thousands of human transcripts are expressed ascircular isoforms of their linear counterparts(913).Thefunctions of circRNAs in eukaryotes still remain more orless elusive,although it has been suggested that circRNAsmay act as transcription regulators(10)or as competing en-dogenous RNAs to bind miRNAs(RNA sponges)or RNAbinding proteins(protein sponges),and thus to modulatetheirlocalfreeconcentration(1115).Moreover,itwassug-gested that circRNAs might encode proteins with func-tions distinct from those of their canonical linear counter-parts.At least in vitro translation of circRNAs was demon-strated(16).Very interestingly,circRNAs were described asbiomarkers of aging in Drosophila(17)and as putative dis-ease biomarkers in human saliva(18).However,the largenumber of detected circRNAs is also critically discussed.There is some agreement that artifacts of RT-PCR detec-tion may add to the large number of circRNAs.A recent re-port raises serious doubts regarding the biological functionof most circRNAs.Based on the results of a computationalapproach for identification and analysis of circRNAs,theauthors suggest that apart from CDR1as,which indeed ap-pears to function as miR-7 sponge(11,12),a large majorityof circRNAs might be just inconsequential side products ofpre-mRNAsplicing(13).Nevertheless,asmentionedabove,there is also good agreement that circRNAs may fulfill im-portant functions,although not much is known yet.What might be the advantage of RNA being in a cir-cular form rather than in the traditional linear one?Themost obvious reason is certainly stability.Since exonucle-ases are ubiquitously spread,circRNAs are advantageousin terms of being protected against degradation.The cova-lentlyclosedringstructuremaybenotonlyfavorableforen-dogenous RNAs,but should also be beneficial to the appli-cation of RNAs for example as antisense-RNAs,aptamers,ribozymes or siRNAs(1923).What are the strategies for production of circRNAs?Invivo,circRNAs usually result from splicing events,eitheras exonic circRNA from circularization of exons or as in-tronic circRNA,such as,for example,circular tRNA andrRNA introns produced from archaeal splicing.CircularRNAs appear in viruses and viroids and as viroid-like satel-lite RNAs.In vitro,RNA circularization involves the in-*To whom correspondence should be addressed.Tel:+49 3834 8622843;Fax:+49 3834 864471;Email:smuelleruni-greifswald.dePresentaddress:SonjaPetkovic,Institutf urMolekulareMedizin,UKS-H,CampusL ubeck,Universit atzuL ubeck,RatzeburgerAllee160,23538L ubeck,Germany.C?The Author(s)2015.Published by Oxford University Press on behalf of Nucleic Acids Research.This is an Open Access article distributed under the terms of the Creative Commons Attribution License(http:/creativecommons.org/licenses/by/4.0/),whichpermits unrestricted reuse,distribution,and reproduction in any medium,provided the original work is properly cited.Nucleic Acids Research,2015,Vol.43,No.4 2455tramolecular formation of a 3?,5?-phosphodiester bond,re-quiring close proximity of the 3?-and 5?-terminus of the lin-ear precursor.Here we review pathways of circRNA forma-tion in vivo and strategies for RNA circularization in vitro.Furthermore,we provide a collection of protocols to beused for the purpose of RNA circularization(Supplemen-tary data).CIRCULARIZATIONIN VIVOFormation of exonic circRNAsIn eukaryotic cells the spliceosome acts to remove intronsfrom primary transcripts in a two-step mechanism.In thefirststep,the2?-OHgroupofadefinedadenosinewithintheintron(branch point adenosine)attacks the 5?-splice site,generating a free 3?-OH group at the 5?-exon and the lariatintermediate.The second step involves nucleophilic attackofthegenerated3?-OHgroupontothe3?-splicesite,produc-ing the final products:an excised lariat intron and a linearRNA composed of the two combined exons(Figure 1a).In addition,exonic circRNAs may result from spliceo-somal action.They were first observed in 1991(24).Sincethen,thousands of endogenous circRNAs have been iden-tified in mammalian cells,some of them highly abundantand evolutionary conserved(for recent reviews see(2527).The detailed mechanism of circRNA biogenesis hasremained elusive.Currently,two major mechanisms involv-ing the canonical spliceosome are discussed:(i)direct back-splicing and(ii)exon skipping(Figure 1b and c).What is referred to as direct backsplicing(25)was his-torically termed mis-splicing by exon shuffling or exonscrambling,where exons are spliced in non-canonical or-der(8).However,taking into account that circRNAs maybe generated by purpose rather than resulting from mis-splicing events,backsplicingis a more appropriate name.Mechanistically,directbacksplicinginvolvesjoiningofthe3?-tail of an expected downstream exon to the 5?-head ofan exon that is normally upstream.The downstream splicedonor pairs with an unspliced upstream splice acceptor.Asa result,the exon becomes circularized(8)(Figure 1b).Thesecond mechanism involves creation of a lariat containingan exon produced from exon skipping.This lariat subse-quently undergoes internal splicing,thereby removing theintron and generating a circRNA(28,29)(Figure 1c).Bothmechanismsareplausibleinvivo,althoughdirectbacksplic-ing is favored as the more frequently used pathway(25).In addition,it cannot be ruled out that multiple mecha-nisms are involved in exonic circRNA formation.Recentfindings indicate that exon circularization is dependent onflanking intronic sequences(3034).RNARNA interac-tions across flanking introns compete with pairing withinindividual introns and thus determine the efficiency of exoncircularization(3034).Furthermore,it was demonstratedthatcircularizationandlinearsplicingcompeteagainsteachother,assigning circRNAs a functional role in gene regula-tion(15,34).In order to produce a desired circRNA in vivo,overex-pression vectors have been designed which include the exonto be circularized and partial sequences of the flanking in-trons to produce pairing,but missing additional upstreamand downstream exonic sequences.Upon delivery in mam-malian cells,these vectors were shown to successfully pro-duce circRNAs(11,12).Formation of intronic circRNAsGroup II self-splicing introns generate a branched lariat-intermediate and a lariat-intron using the same chemistryasthespliceosomedescribedabove(Figure1a).Inaddition,there is also evidence for a mechanism,where group II in-trons are excised as RNA circles,although circularizationoccursbyformationofa2?,5?-phosphodiesterbond(35,36).Circleformationrequirespriorreleaseofthe3?-exon,forex-ample by a trans-splicing mechanism.The terminal 2?-OHgroup of the intron attacks the 5?-exonintron junction(5?-splice site),thus generating the circularized intron and the5?-exon(Figure 2a).In contrast to the spliceosome and group II introns,group I introns self-splice by first recruiting guanosine(exoG)as an external nucleophile that initiates splicing bynucleophilic attack onto the 5?-splice site and thereby be-comes attached to the 5?-end of the intron.Upon secondtransesterification,exons are ligated,and a linear catalyticintron is released(reviewed in(37).Interestingly,the ex-cised linear intron can undergo circularization by nucle-ophilic attack of the 3?-terminal guanosine onto a phos-phodiester bond near the 5?-end of the intron(38).The 5?-terminal sequence is released and the intron is circularized(Figure 2b).The choice of the circularization site dependson pairing of the three nucleotides preceding the cleavedphosphate to a specific binding site within the intron,thusdefining the phosphate to be attacked.As a result,a vari-ety of truncated intron circles are formed,which,however,appear to be short-lived in vivo(39).In addition to truncated circles also formation of fulllength intron circles was observed.This pathway is initiatedby hydrolytic cleavage at the 3?-splice site followed by nu-cleophilic attack of?G onto the 5?-splice site(40).The finalproductsareacircularfull-lengthintronandnon-ligatedex-ons(Figure 2c).For the Tetrahymena intron,full-length in-tron circles were reported to be minor and barely detectablein vivo(40).However,for more complex nuclear group I in-trons like the one from Didymium iridis,full-length circu-lar introns are formed as major product at splicing condi-tions in vitro(41,42),and are also easily detectable in vivo(43).Here,truncated circular introns were not observed.The ability of forming full-length intron circles seems to bea general feature of all types of nuclear group I introns.Thefunction of truncated and full-length circular introns is notknown,but it was hypothesized that these structures couldplay a role in intron mobility(5).Formation of circular RNAs in archaeaIn archaea,circRNAs were mainly observed in tRNA andrRNA introns and in rRNA processing intermediates.Ar-chaeal introns are cleaved from precursor-RNAs by the as-sistance of a protein,the archaeal splicing endonuclease,and are subsequently ligated by an RNA ligase to form cir-cRNA(3,4).The splicing endonuclease recognizes a bulge-helix-bulge motif,composed of a 4-base pair stem flanked2456 Nucleic Acids Research,2015,Vol.43,No.4Figure 1.Regular linear splicing(a)and two models for the formation of exonic circRNAs(b,c).(a)Upon folding,the branch point adenosine(bpA)attacks the 5?-splice site,delivering the 5?-exon with free 3?-OH group and the lariat intermediate with the intron still linked to the 3?-exon.Nucleophilicattackofthe3?-OHgroupofthe5?-exonontothe3?-splicesiteleadstoligationofthetwoexons,andtoreleaseoftheintronaslariat.(b)Directbacksplicing.Two unspliced introns interact by complementary base pairing,thereby juxtaposing the branch point of the 5?-intron and the 3?-intronexon junction(3?-splice donor)for nucleophilic attack and cleavage.Then,the 3?-splice donor attacks the 5?-intronexon junction(5?-splice acceptor)joining the two intronsand releasing the circularized exon.(c)Exon skipping.Through skipping of an exon,an exon containing lariat is created following the normal mechanismof splicing.Backsplicing then occurs as described above,but within the lariat.As a result,the intron lariat is released and a circular RNA is produced.Figure 2.Formation of intronic circRNAs.(a)Group II intron mediated circRNA formation.Circle formation requires prior release of the 3?-exon.Theterminal 2?-OH group of the intron attacks the 5?-splice site,creating a circular RNA by 2?,5?-phosphodiester formation.(b)Group I intron supportedregular splicing.An exogenous guanosine(exoG)bound in the intron structure serves as nucleophile attacking the 5?-splice site.Upon first transesterifi-cation,the 5?-exon is cut off and exoG becomes linked to the intron.The terminal 3?-OH group of the 5?-exon then attacks the 3?-splice site,the ligatedexons and a linear intron are released.Eventually the linear intron is circularized by nucleophilic attack of 2?-OH group of the terminal guanosine(?G)onto a phosphodiester bond close to the 3?-end and release of a short 3?-tail.Note that in this case a 2?-5?-phosphodiester bridge closes the circle.(c)Priorhydrolysis of exon 2 allows circle formation by direct nucleophilic attack of?G onto the 5?-splice site.by two 3-nucleotide bulges(4446).Cleavage occurs at aspecific site in the bulge regions,generating characteristicfragments with terminal 5?-hydroxyl group and 2?,3?-cyclicphosphate(47).Circularizationproceedsbynucleophilicat-tack of the 5?-OH group onto the 2?,3?-cyclic phosphate ofthe same molecule forming a 3?,5?-phosphodiester bridge(3,4).In addition to tRNA and rRNA introns,archaeal 16Sand 23S rRNAs were found to be excised as circular inter-mediates during rRNA maturation.Both are spliced outof a single RNA precursor before being further processedto the mature rRNAs(48).Archaeal circular RNAs pre-sumably have diverse biological functions,many of themyetunidentified.Arecenttranscriptome-widesearchforcir-cular RNAs in archaea discovered,in addition to excisedNucleic Acids Research,2015,Vol.43,No.4 2457tRNA and rRNA introns,a number of enriched circularnon-coding RNAs,including C/D box RNAs and RNaseP(49).Circular RNAs in viroids,viroid-like satellite RNAs and inthe HDVViroid genomes,viroid-like satellite RNAs and the humanHDV genome are circular RNAs.They replicate througha rolling-circle mechanism,using the circular template ofone or both polarities for reiterative transcription,fol-lowed by cleavage of the produced oligomeric RNA intomonomeric species and ligation to a circular RNA(1,5051)(Figure 3).In some species,cleavage is mediated byhost enzymes,while in others cis-acting ribozymes(ham-merhead ribozyme(HHR),hairpin ribozyme(HPR),HDVribozyme)perform this reaction.Intramolecular ligationappears to be catalyzed mainly by host enzymes,althoughinvitroself-ligationwasalsoobserved(52).Mechanistically,there is some variety among the different members of viroidfamilies,satellite RNAs and the HDV virus.Cleavage pro-duces 5?-phosphate a